Microbiomes Health and The Environment 1676562923

Microbiomes: Health and the Environment
Microbiomes: Health and the

Environment
DYLAN PARKS
MAVS OPEN PRESS
ARLINGTON
Microbiomes: Health and the Environment by Dylan Parks is licensed under a Creative
Commons Attribution 4.0 International License, except where otherwise noted.
Contents
About the Publisher vii

Mavs Open Press
About This Project ix
Acknowledgments xi
Part I. An Introduction to Microbiomes
1. An Introduction to Microbiomes 3
Part II. Analyzing Microbiomes
2. Analyzing Microbiomes 23
3. Environmental Metagenomics 42
Part III. Human Health and Disease
4. Human Health and Disease 57

5. The Gut Microbiome 63
6. The Oral Microbiome 85
7. The Skin Microbiome 115
8. The Respiratory Microbiome 133
9. The Vaginal Microbiome 153
10. Mental Health and Multi-Microbiome Interactions 180
Part IV. Environmental Microbiomes
11. Environmental Nutrient Cycling and Human 215

Health
12. The Ocean Microbiome and Marine Life 242
13. Soil Microbiomes 289
14. Plant Microbiomes 357
15. Pollution and Bioremediation 406
Part V. Other Microbiome Applications
16. Forensic Microbiomes 479
Part VI. Journal Club
17. Journal Club Articles 543
Part VII. Case Studies
18. Case Study #1 - Human Health 547

19. Case Study #2 - Environment 561
20. Case Study #3 - Synthesis (Create Your Own) 574
Part VIII. Additional Resources
21. The Integrative Human Microbiome Project 577

22. BMC Microbiome Open Collections 616
Bibliography 617
Image Credits 705
Derivative Notes 719

About the Publisher
MAVS OPEN PRESS
About Mavs Open Press
Creation of this resource was supported by Mavs Open Press,

operated by the University of Texas at Arlington Libraries (UTA
Libraries). Mavs Open Press offers no-cost services for UTA faculty,
staff, and students who wish to openly publish their scholarship.
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Bookstore or can be purchased directly from XanEdu, Mavs Open
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About the Publisher | vii

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viii | About the Publisher

About This Project
Overview
Microbiomes: Health and the Environment was created to provide

accessible insight into the novel and complex world of polymicrobial
community interactions. As we push forward into the future of
medicine and environmental health, it is imperative that we learn
from each other, from history, and keep up to date with the latest
advances in research and technology. This book not only provides
content from the latest microbiome studies, but contains
interactive tools, videos, and thought-provoking questions to help
the reader hone and truly understand the respective topic. Though
there is much overlap between themes due to the ubiquitous nature
of microbes, the book is broken down into sections pertaining to
both human health (e.g., gut health and disease, as well as other
organ-specific niches) and aspects of the environment (e.g., nutrient
cycling and climate change, marine health, soil and plant health,
etc.) influenced by microbes. However, the content is designed to
bridge ideas and aspects between these themes to support the
One Health concept: that the health of people, animals, and the
environment are all interconnected. This project will continue to
grow with new findings, and adapt with the ever-changing world of
microorganisms.
Creation Process
This project was completed by thorough review of published

literature from reputable resources and written in a style accessible
About This Project | ix

to both experienced scholars and newcomers to the subject.
Specific excerpts and articles are provided throughout the book
to give the reader first-hand insight into microbiome research.
Supporting content such as images and videos are also implemented
to give the audience a well-rounded and multifaceted view into
each topic. Interactive tools, quizzes, and critical thinking questions
make each section entertaining and informative. A section with case
studies and directions for original synthesis make information in the
book applicable to real world situations.
About the Author
Dr. Dylan Parks is an Assistant Professor of Instruction at the

University of Texas at Arlington in the Biology Department. As a
microbiologist, he has worked in both academia and industry,
serving as laboratory technician at a local brewery. His research
interests include microbiomics, biomedical sciences, fermentation
science, and microbial symbiotic interactions.
x | About This Project

Acknowledgments
UTA CARES Grant Program
Creation of this OER was funded by the UTA CARES Grant Program,
which is sponsored by UTA Libraries. Under the auspices of UTA’s
Coalition for Alternative Resources in Education for Students
(CARES), the grant program supports educators interested in
practicing open education through the adoption of OER and, when
no suitable open resource is available, through the creation of new
OER or the adoption of library-licensed or other free content.
Additionally, the program promotes innovation in teaching and
learning through the exploration of open educational practices,
such as collaborating with students to produce educational content
of value to a wider community. Information about the grant
program and funded projects is available online.
Lead Author/Editor/Project Manager
Dylan Parks, PhD – Assistant Professor of Instruction, The

University of Texas at Arlington
Editors
Stephanie Lucas, MS – Alumni, The University of Texas at Arlington
Acknowledgments | xi
Other Acknowledgments
Cover art design by Westley Harwart
xii | Acknowledgments
PART I
AN INTRODUCTION TO
MICROBIOMES
An Introduction to Microbiomes | 1
2 | An Introduction to Microbiomes
1. An Introduction to
Microbiomes
An Introduction to Microbiomes
Microorganisms represent the fundamentals of life and interact

with almost every facet of it. Yet, for the majority of their existence
they have been largely ignored, or rather unseen by humans. The
unraveling of the complex interactions between all life forms is an
endless duty and seems to generate more questions than answers.
However, the cracks in knowledge produced by peering into the
unknown offers insight into our life and the world around us. These
minute creatures have shaped Earth’s evolution since their dawn
almost three-and-a-half billion years ago, and continuously affect
our environment and health. The importance of the planet’s
collection of microbes is realized more and more each day, and our
unceasing investigation of them will surely unlock secrets of life we
could never imagine.
The study of microbiomes is fairly novel in the context of
understanding and applying their communal existence to ourselves
and surroundings. Though scientists have recognized symbiotic
relationships and traditionally focused on individual microbes and
their interactions with human health, environmental impact,
industrial applications, etc., their respective communities and
influence as a whole in these areas have only just begun to be
elucidated. That is in no small part due to the daunting task of
cataloging the immense and complex interplay between the
multitude of different microorganisms in a given environment,
though rapid advances in technology have begun to ease analysis.
What is a microbiome?
A microbiome can be best described as a collective polymicrobial

community, or ‘microbiota’, and its associated activity with genetic
and physio-chemical constituents in a defined spaciotemporal
habitat (Figure 1). These members of the microbiota include
bacteria, archaea, algae, protozoa, fungi, and viruses (though the
latter is somewhat debated since viruses and their derivatives aren’t
technically living). Within this symbiotic context with a particular
eukaryotic host, the entire entity is termed a ‘holobiont’ and the
aggregate of genetic material termed the ‘hologenome’. Interactions
between these partners may have long occurred, shaping the
evolution of each, whereas others may be novel or transient,
sometimes resulting in prompt change and infectious diseases. The
change in the normal microbiota, or dysbiosis, can result in a variety
of different diseases. As so, their study has been especially
important in the fields of life sciences, human health, and medicine.
Figure 1. A schematic highlighting the composition of the term microbiome

containing both the microbiota (community of microorganisms) and their
“theatre of activity” (structural elements, metabolites/signal molecules, and
the surrounding environmental conditions). (Berg et al., 2020 adapted by
Dylan Parks).
Quick Quiz
An interactive H5P element has been excluded

from this version of the text. You can view it
online here:
https://uta.pressbooks.pub/
microbiomeshealthandtheenvironment/?p=1529#h5p-1
Our fascination with microorganisms began before we even fully

understood them or were even able to see them. Their implications
concerning human health were primarily explored during the
‘golden age of microbiology’ with the work of Louis Pasteur and
Robert Koch. Their experiments and discoveries shed light on not
only the ubiquitous nature of microbes, but their importance in
our everyday lives. Other significant milestones and historical
microbiological development can be viewed in Figure 2. Human
health and infectious diseases were central to the field of
microbiology, though food microbiology, industrial applications,
and microbial ecology became increasingly explored. Over the last
couple centuries, a microbial catalog of knowledge has slowly
grown, but much of these findings were limited to those organisms
that could be cultured and measured. The advent of sequencing
and ‘multi-omics’ technologies has since allowed researchers to
document microorganisms that were previously missed or ignored
with traditional techniques, and with further advances, larger
microbial communities and symbioses can be better understood.
The ‘microbiome’ was first defined in the late 1980s when a group of
microbial ecologists were studying the rhizosphere, which provided
context to better describe these polymicrobial communities
(Whipps et al., 1988). Many other similar definitions have been
published since then with varying specifics on genetic expression,
symbioses, and ecological interactions (Lederberg & McCray, 2001,
Marchesi & Ravel, 2015, Berg et al., 2020). The ‘holobiont’ concept
stems from Adolf Meyer-Abich’s ‘theory of holobiosis’ proposed in
1943 and was independently conceived and popularized in the early
1990s by Lynn Margulis, though it only described the host and a
single symbiont (Margulis, 1991, Baedke et al., 2020). Since then
has been expanded to include the entire microbiota in multiple
symbiotic contexts (Simon et al., 2019). In recent decades there has
been a steady increase in microbiome publications as the subject
has grown in popularity. Along with that, there has been more
analytical breakdown as certain microbiomes are being described
with emphasis on specific members, such as the ‘bacteriome’,
‘archaeome’, ‘mycobiome’, ‘protistome’, and ‘virome’, and these terms
are best used to refer to the distinct contribution of those particular
microbes within the entire microbiome context. In general, though,
most microbiomes are delineated by their specific host or type of
environment, with the human microbiome being the most popular
example.
Figure 2. The history of microbiome research from seventieth century until
our days, highlighting the shift of the paradigm from microbes as unsocial
organisms causing diseases to the holistic view of microorganisms being the
center of the One Health Concept: positively interconnecting all areas of our
lives. (Berg et al., 2020).
The Human Microbiome
It was evident that the human microbiome and its involvement in

a micro and macro scale needed to be characterized. The Human
Microbiome Project (HMP) set out in 2007 with this as one of its
primary goals (Turnbaugh et al., 2007). The program also set out
with initiatives to develop a set of microbial genome sequences,
explain the relationship between disease and microbiome changes
and evaluate the data with multi-omics approaches, develop new
tools and technology for computational analysis, establish a data
analysis and coordinating center and research repositories, as well
as address ethical, social, and legal implications of HMP research
(Human Microbiome Project). The second phase of the HMP
launched in 2014, called the Integrative Human Microbiome Project
(iHMP), having the main mission to completely characterize the
human microbiota with a key focus on human health and disease
using three projects: pregnancy and preterm birth, onset of
inflammatory bowel disease (IBD), and onset of type 2 diabetes (NIH
Human Microbiome Project, The Integrative HMP (iHMP) Research
Network Consortium, 2019). Aside from these, the human
microbiome and disruption of the microbiota has been linked to
several other important conditions and diseases including multiple
sclerosis, diabetes (types 1 and 2), allergies, asthma, autism, and
cancer (Backhed et al., 2012, Hsiao et al., 2013, Petersen and Round,
2014, Trompette et al., 2014, Garrett, 2015, Lloyd-Price et al., 2016).
It makes sense that the human microbiome can have such an
impact on human health and behavior if you consider that we are
essentially a collection of organisms forming a living entity. In a way,
our symbionts may even actually define more of who we are than
just our own unique biological makeup. For instance, the ratio of
microbial cells associated with a human body could equal, if not
exceed (traditional estimates were tenfold), the number of human
cells (Sender et al., 2016). Even more interesting is viewing our
genetic makeup; the human genome contains about 20,000 genes,
but its hologenome contains > 33 million genes brought by its
microbiota (Huttenhower et al., 2012, Lloyd-Price et al., 2016, Simon
et al., 2019). Furthermore, the composition and rate of change of
each person’s microbiota is distinctive from one individual to
another since it is influenced by variables like age, lifestyle, diet,
antibiotics, occupation, environment, etc. (Gilbert et al., 2018). The
genetic wealth and member diversity contributed from the
microbiota has roles in adaptation, survival, development, growth,
and reproduction of the holobiont and can affect fitness in the short
term as well as have long lasting effects concerning the evolution of
both partners (Rosenberg, and Zilber-Rosenberg, 2011).
One or more interactive elements has been excluded

from this version of the text. You can view them online
here: https://uta.pressbooks.pub/
microbiomeshealthandtheenvironment/?p=1529#oembed-1
Co-evolution of the host-microbiota symbiosis can be considered

even more unique when viewing the microbial consortium at
different locations or organs in the host, as their makeup is
governed by and reflects specific physiological processes in those
areas. For example, the bacteria found in the human gut microbiota
are primarily from the phyla Bacteroides and Firmicutes, whereas
Actinobacteria and Proteobacteria command the skin microbiome,
though there is some overlap and it is important to note that there
are differences depending on exact location (e.g. dry vs. moist areas
of the skin) (Grice and Segre, 2011, Jandhyala et al. 2015). Though
there are differences between various microbiota within a
holobiont, they can still influence each other to some degree. In
the case of the gut and skin microbiotas in humans, deemed the
‘gut-skin axis’, there are indications that both the health of the
gastrointestinal (GI) tract and skin, as well as their response to
stressors, are correlated (Levcovich et al., 2013, O’Neill et al., 2016,
Salem et al. 2018). Even more interesting is the effects certain
microbiota can have on germ-free organs like the brain. Studies on
the ‘gut-brain axis’ show that the microbiota in the GI tract, and
in some cases disruption of it, are associated with many mental
illnesses and neurodegenerative disorders including depression,
anxiety, autism, schizophrenia, Parkinson’s disease, and Alzheimer’s
disease (Clapp et al. 2017, Foster et al. 2017, Cryan et al. 2019).
A variety of different ‘axes’ which demonstrate interplay between
microbiota, organs, and locations have been identified in the human
body and much of what is known about their connections is novel
and early in its research.
Environmental Microbiomes
Not only can our microbiome regulate who we are, but those
communities in the surrounding environment can affect us, our
microbiome, and others. Environmental microbiomes can directly
or indirectly affect our health through ecological interactions. For
example, soil microbiomes in the rhizosphere of plant roots and
plant microbiomes of economically important crops have
implications in agriculture, human health, and ecology (Saleem et
al., 2019, Hirt, 2020). Plant growth, health, soil nutrient cycling and
availability, and defense against potential pathogens are dictated
by their own symbionts as well as their microbial neighbors in the
ground, which include a variety of bacteria, protists, viruses, and
network of fungi known as mycorrhizae (Busby et al., 2017, Hannula
et al., 2017, Pratama and van Elsas, 2018, Zhong et al., 2019,). By
better understanding the functional interactions of all the players
in these environmental microbiomes, it may be possible to address
problems like agricultural soil fertility, plant disease, and pollution.
Thus, harnessing better microbiomes either by specifically
engineering a microbial consortia for a targeted area/specimen
or transplanting a natural community could improve sustainable
agricultural practices which are desperately needed to feed the
expanding population of humans while protecting the environment
(Elhady et al., 2018, Arif et al., 2020, Hernandez-Alvarez et al., 2022).
Studying animal-microbiome systems can also give us
information about our environment. For instance, how
anthropogenic-induced changes have impacted things like climate-
change and approaches to maintain homeostasis with various
environmental factors. A promising reservoir of research is the
microbiota of marine animals since most of the planet is covered
in water. Corals, sponges, various fish, and marine mammals have
all been investigated to document the response of their microbial
symbionts to changing environmental factors (Apprill, 2017). Other
animal microbiome models are being used in an analogous means to
understand how the microbiota could potentially influence human
health and fitness. The microbiomes of classic biomedical models
such as the fruit fly, zebrafish, and nematode worm are being
investigated since these organisms are well known and readily
available to work with in many labs (Douglas, 2019). Though, it is
important to consider a variety of animal host-microbiome models
as each could contribute unique insights to beneficial microbial
interactions within the human holobiont. For example, the gut
microbiome of honeybees, the skin microbiome of freshwater
polyps, and the individual interaction of Vibro fischeri and the
Hawaiian bob-tailed squid have provided valuable information to
understanding host-microbiome relationships (Douglas, 2019).
Further research of these and other systems are needed for the
pursuit of technological and medical advances or cultural/societal
changes necessary for overall human and planetary benefit.
Microbiome Analysis
Traditionally, it has been difficult to characterize complete

microbial communities, as most projects require substantial time
and resources. Though, advancements in NGS and ‘omics’
technologies have begun to allow researchers to tackle microbiome
analytics using a variety of approaches including metagenomics,
metatranscriptomics, proteomics, metabolomics, culturomics,
etc.(Integrative HMP (iHMP) Research Network Consortium, 2014,
Bashiardes et al., 2016, Daliri et al., 2017, Janson and Hofmockel,
2018, Lin et al., 2019, Diakite et al., 2020, ). Each technique is selected
and applied depending on the experimental setup and what
questions are being addressed. For instance, does the research care
about the identification of members of the microbiome, how they
are interacting with the host or each other, what macromolecules
are present, what genes are being expressed, what is the functional
potential, etc. These different types of investigations can then be
integrated together in network analyses to establish linkages and
correlations within the microbiome datasets, though statistical
models and analytical tools must be carefully selected to avoid false
outcomes and shortcomings (Jiang et al., 2019). Due to the particular
limitations of these approaches and the unavoidably large datasets
produced by microbiomic studies, it is paramount to continually
develop novel technologies to unearth the knowledge buried in
these microbiomes.
Considering the abundance of microbiomes that no doubt play role

in human health, the extent to which microorganisms sway our lives
is almost impossible to foresee, and the opportunities they present
for improvement to industry, agriculture, environmental and human
health is potentially unlimited.
Check Your Understanding
• What is the difference between a microbiome and

microbiota?
• In what ways does a microbiome influence a
holobiont?
• How can an environmental microbiome indirectly
impact human health?
Media Attributions
• Video 1 – What is the human microbiome? by

The Conversation. Licensed under Creative
Commons: By Attribution 3.0 License
https://creativecommons.org/licenses/by/3.0/
• Video 2 – Human Science (Part I) – The Gut
Brain Axis, Microbiome & the power of Probiotics
by Infognostica. Licensed under Creative
• Figure 1 – Microbiome Schematic by Berg et al.,
2020 adapted by Dylan Parks. Licensed under
Creative Commons: By Attribution 4.0 License
http://creativecommons.org/licenses/by/4.0/
• Figure 2 – History of Microbiome Research by
Berg et al., 2020. Licensed under Creative
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sciadv.aaw0759
PART II
ANALYZING
MICROBIOMES
Analyzing Microbiomes | 21
22 | Analyzing Microbiomes
2. Analyzing Microbiomes
Analyzing Microbiomes
Choosing the correct approach when characterizing a microbiome

is important when considering what questions are trying to be
answered. Who are the players? What is the function? What genes
are being expressed? How do they interact?
Even though when assessing a microbiome all members of the
microbiota should be considered, some insight can be gained by
viewing individual groups, such as the prokaryotic component,
which can be further compartmentalized into the bacteriome and
archaeome. In some cases, it is difficult to completely characterize
all of the members due to inherent biological differences between
these communities, and in other situations, certain groups may be
absent or irrelevant to the study. In so, analyzing these individual
pieces could help reveal their distinct importance and contribution
to the overall big picture.
The type of analysis also depends on the research question. If
the sole interest is to identify all the bacteria in a sample, then a
metagenomic approach may work best. Whereas, if an interest lies
in what the microorganisms are doing, then a metatranscriptomics
or metaproteomics approach would be more appropriate. If there is
an interest in one or a few particular members of the microbiome,
culture-based techniques (culturomics) could be implemented,
which can often catch rare microbes that are missed using
sequencing techniques because of data filtering protocols (Lagier
et al., 2012, Allaband et al., 2019). However, it is usually difficult to
characterize an entire microbiome based on just a few microbes and
achieving multiple isolations is extremely difficult as a ‘one-size-
fits-all’ type media does not exist.
Sample Collection
Depending on the type of research questions and the particular

microbiome of interest, there are different ways to collect microbial
communities that will be subjected to analysis. That is, the approach
to collecting microbes from the human gut is much different than
getting a soil sample. Really, there is no perfect method for any
one microbiome, though some may be more difficult to obtain than
others, and will depend on a variety of factors such as feasibility,
cost, patient acceptance, and downstream analytical methods
(Allaband et al., 2019). It can also be very difficult to capture all
microbes within a microbiome sample, as inevitably some will be
missed or cannot be collected by a particular technique. For
example, when analyzing the gut microbiome, stool collection is
preferred due to its ease and frequent accessibility, but this method
often misses microbes in the small intestine and others which are
mucosally adherent as opposed to excreted (Eckburg et al., 2005,
de Carcer et al., 2010, Allaband et al., 2019). Additionally, collection
and storage materials, storage time, and transport options should
be considered as these can compromise sample integrity and create
problems for various analytical approaches (Costello et al., 2009,
Caporaso et al., 2011, McDonald et al., 2018).
Approaches to Characterization
What is the Difference between Culture-Dependent and Independent

Methods of Microbial Community Characterization?
-Excerpt taken from Findley and Grice, 2014 available
under the Creative Commons CC0 public domain
dedication:
Up until the 1980s, microbiologists routinely relied on

culture-dependent methods for microbial isolation,
identification, and characterization. Colony morphology,
stains (i.e., Gram stain), biochemical characteristics (i.e.,
coagulase test), motility tests, antibiotic resistance profiles,
and other characteristics guided bacterial and/or fungal
identification and taxonomy. However, this approach has
several limitations, including an inability to mimic in vivo
conditions and selection against slow-growing and/or
fastidious organisms. With recent advances in sequencing
technologies and development of bioinformatics tools and
reference databases, researchers are now better equipped
to capture microbial diversity without the biases of
culture-based approaches.
Culture-independent methods of microbial identification

rely on a targeted amplicon strategy, which employs highly
conserved microbe-specific molecular markers and does
not rely on growing isolates in pure culture. The 16S
ribosomal RNA (rRNA) gene is used for bacterial
identification, while fungi and other microeukaryotes are
identified using either the 18S rRNA gene or the Internal
Transcribed Spacer (ITS) region. A complementary
approach to amplicon-based surveys is whole genome
shotgun metagenomics. With this approach, one can
identify the microbiota present and gain insight into the
functional potential of the microbiota in an untargeted
manner.
Within the culture-independent targeted approach, PCR
amplification techniques, like quantitative PCR (qPCR) and reverse
transcriptase PCR (RT-PCR) can be implemented to detect and
quantify specific organisms, genes, or expression values within the
microbiome sample. Untargeted approaches typically give whole
community characterization and include metagenomics,
metatranscriptomics, metaproteomics, and metabolomics which
have a variety of applications depending on the type of study.
Culturomics
Cultivating all the microorganisms within a certain microbiome can

be quite challenging and time-consuming, but nonetheless can
provide important information that would otherwise be missed with
other approaches. Creating media that mimics the original
environment can help to capture organisms that exist at lower
concentrations than that of the detectable threshold of molecular
tools (Lagier et al., 2012). The application of matrix-assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-
TOF MS) has enhanced culture-dependent studies by allowing fast
and reliable identification of bacterial taxa in a cost-effective
manner (Seng et al., 2009, Lagier et al., 2012). Even though there
have been advances in culturomics, the approach still has certain
biases based on the types of microbes that can be easily grown
(e.g. aerobic microbes are much easier to grow than anaerobic ones
in most cases), as well as the selection for those that grow more
rapidly and outcompete the others in a sample (Allaband et al.,
2019). Thusly, it is imperative to close the gap between microbial
richness observed in nature versus what is cultivable in the lab in
order to better understand in vivo microbial functioning within a
microbiome (Sarhan et al., 2019).
Metagenomics and DNA sequencing
Next-generation sequencing (NGS), also known as high-throughput

sequencing (HTS), allows the generation of thousands of sequencing
reads in a cost-effective manner (Nkrumah-Elie, et al., 2018). These
reads can then be bioinformatically processed and annotated to
identify microbial taxa, genes, potential functions, etc. depending
on the sample type and experimental conditions (Rivera-Pinto et al.,
2018). However, differences in wet lab work, sequencing platforms,
processing pipelines, and statistical approaches can affect data
output, which make reproducibility come into question (Gloor et
al., 2016). Usually, information about microbiome compositions are
provided as relative abundances instead of the true abundances
due to sequencing platform imitations. This data is considered an
arbitrary sum and is referred to as compositional data (Jiang et al.,
2019). As sequence generation and pipelines improve and become
standardized, the development and accessibility for analytical tools
can help to streamline and produce consistent and more accurate
results while aggregating data for microbiome characterization and
meta-analysis without advanced expertise (Markowitz et al., 2007,
Dhariwal et al., 2017, Gonzalez et al., 2018, Chong et al., 2020,
Mitchell et al., 2020, Bharti and Grimm, 2021, Chen et al., 2021).
Figure 1. An illustration of targeted amplicon and metagenomic sequencing
approaches. A schematic overview demonstrating diverse sample types along
with commonly utilized sequencing platforms, as well as systematic and
stepwise data processing steps. (Bharti and Grimm, 2021).
Figure 2. Schematic representations of the in vitro models used for

underpinning the microbe–microbe and host–microbe cross talks.
Figure 3. Visualizing the unique challenges of microbiome data. A mock set of
bacterial samples from two populations where each colored shape is a
bacterial taxon. (A) Compositionality. The taxon abundance table depicts the
count of each observed taxon in each sample. When sequencing microbiome
samples, the resulting counts of taxa are not representative of the actual taxa
counts in the sample due to constraints of sequencing. Due to this, relative
abundances are generally used in analysis of microbiome data. The bar plots
illustrate the difference in community representation between raw counts
(top) and relative abundances (bottom). (B) Normalization. Due to the
constraints of sequencing, the overall sequencing depth of a sample can
impact the results. For example, shallow sequencing may miss rare taxa such
as the green taxon V in the example sample A that is present in low
abundance in the community. (C) Sparsity. Microbiome data are often very
sparse, where most observations are zero. This is illustrated by the histogram
of taxa counts for each sample where most counts are zero and there are few
taxa with high counts. This can also be seen in the table for part A, where
many entries are zero. (D) Heterogeneity. The table summarizes the taxonomic
heterogeneity in the mock dataset between the two populations. Each sample
has a unique taxonomic composition, but there are also population specific
signatures. The samples in each population are dominated by a few taxa, and
these dominant taxa are different for the two populations. Additionally, there
are taxa that are highly abundant in one sample and absent from the rest,
such as the purple taxon Y in sample A.

Metatranscriptomics and RNA sequencing
While metagenomic studies have been instrumental in identifying

various microbes and genes in a given sample, it does not
differentiate between viable cells or when those genes are being
expressed (Gosalbes, et al., 2011, Bashiardes et al., 2016).
Metatranscriptomic strategies of microbiome analysis allow
researchers to capture gene activity which can delineate the
functional dynamics of a particular community and it’s interactions
with its host or environment. These RNA-based approaches can
complement metagenomics, as the extent of expression of those
annotated genes can be measured (Franzosa et al., 2014). This is
considerably important concerning certain disease states because
the presence of a pathogen does not always produce deleterious
conditions, but rather it is the functional activity of certain
organisms.
RNA-seq experiments usually begin with isolation of total RNA in
a sample, then selection is based upon whether it is prokaryotic or
eukaryotic in origin, and mRNA must be isolated from other RNA
species (i.e. rRNA and tRNA) (Bashiardes et al., 2016). cDNA is then
synthesized from the mRNA, adapters are ligated to create a library,
amplification and sequencing follows, and the generated reads are
mapped to a reference genome where expression can be measured
(Giannoukos et al., 2012, Bashiardes et al., 2016). These experiments
can be difficult and sensitive, as care must be taken with sample
collection, and avoiding contamination and degradation from
ribonucleases is important to maintain integrity of the samples as
well. Furthermore, metatranscriptomics may not always give the
whole picture of community expression due to the high complexity
of organisms, the delicateness of RNA, the wide range of transcript
expression, and various limitations with current technology (Shakya
et al., 2019).
Metaproteomics
Metaproteomics gives a snapshot of the entire protein content of

a microbiome sample under various conditions, which can further
provide insight into gene activity and metabolic functions. This
strategy identifies peptides by matching tandem mass spectrometry
(MS/MS) data to protein sequence databases, which can be further
assigned to functional groups via annotation databases (Sajulga et
al., 2020). This approach can be convoluted though, as many
peptides match to similar proteins produced by different organisms,
and proteins can be multi-functional making it difficult for
assignment to any one group. Additionally, bioinformatic analysis
of metaproteomes isn’t exactly standardized which makes
reproducibility and comparison of studies problematic
(Schiebenhoefer et al., 2019, Sajulga et al., 2020).
Metabolomics
Metabolomics analyzes the non-protein metabolites that are

produced and/or regulated by a given microbiome, which adds
another angle of characterization to community-host-
environmental interaction and functional dynamics. By
understanding how various microbes process metabolites like
natural products, nutrients, and medications, we can better
determine how they influence their ecosystem through molecular
interactions. Researchers typically obtain these molecules through
gas or liquid chromatography and subsequently analyze them by
mass spectrometry (Allaband et al., 2019, Bauermeister et al., 2021).
Metabolomics strategies can either be targeted or untargeted,
where the former is more sensitive and compares metabolites to a
pre-determined bank with reference standards, however, it cannot
detect novel molecules. Since most metabolites produced by
microbiomes are unknown, an untargeted approach using tandem
mass spectrometry is useful in detecting many small molecules,
though annotation of modified metabolites is challenging (Watrous
et al., 2012, Wang et al., 2016, Allaband et al., 2019). As with other
microbiome analytical approaches, various data analysis tools and
pipelines of the metabolome need to be standardized to a degree
to improve overall research context. Moreover, major advancements
in the known inventory of microorganism-derived metabolites and
their functions are required to enhance metabolomics studies (Lee-
Sarwar et al., 2020, Bauermeister et al., 2021).
Drag and Drop Quiz

online here:
Conclusion and Future Directions
While various approaches to characterizing a microbiome each have

their advantages and drawbacks, network analyses integrating all
the -omics (i.e. metagenomics, metatranscriptomics,
metaproteomics, and metabolomics) could be best to gain a holistic
view of compositional and functional dynamics (Bashiardes et al.,
2016, Jiang et al., 2019). Novel and improved approaches to collect
and analyze various microbiomes will help to minimize loss of
information and provide a better picture of how these communities
interact with and influence their host or environment. Also, the
establishment of more comprehensive publicly available databases,
and protocols for streamlining computational procedures will allow
more consistent and reproducible research for upon which
knowledge can be accumulated.
Check your Understanding
• What is the difference between culture-dependent

and independent methods of microbiome analysis?
• What is the importance of culturomics in the scope
of microbiome analysis?
• How can inaccurate or inconsistent results be
produced from different metagenomic approaches to
microbiome analysis?
• When would a metatranscriptomic analysis be
more appropriate than a metagenomic approach?
How do these approaches complement each other?
• What are the benefits and limitations of
metabolomic analysis?
Media Attributions
• Figure 1 – Targeted amplicon and metagenomic

sequencing approaches to microbiome analysis by
Bharti and Grimm, 2021. Licensed under Creative
• Figure 2 – In Vitro Model Schematic by Ventura
et al., 2020. Licensed under Creative Commons: By
Attribution 4.0 License
• Figure 3 – Example microbiome samples and
challenge of analysis by Jiang et al., 2019. Licensed
under Creative Commons: By Attribution 4.0
License http://creativecommons.org/licenses/
by/4.0/
• Video 1 – Life in the Lab: Working with human
gut microbiota by yourgenome. Licensed under
• Video 2 – New technique explores gut
microbiome one microbe at a time by Research
Square. Licensed under Creative Commons: By
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3. Environmental
Metagenomics
Environmental Metagenomics
Chapter taken from Hozzein, 2020 [1] available under the

Creative Commons Attribution 3.0 License.
1. Introduction
Metagenomics can be defined as the techniques and

procedures that are used for the culture-independent
analysis of the total genomic content of microorganisms
living in a certain environment [2]. It has many useful
applications with very promising potential in both medical
and environmental microbiology. The most common use of
metagenomics in environmental microbiology is studying
the diversity of microbial communities in particular
environments through the analysis of rRNA genes and how
these communities change in response to changes in
physical and chemical properties of these environments [3].
Metagenomics also provides an opportunity to obtain and
42 | Environmental Metagenomics
identify novel enzymes with industrial applications from
extreme environments where unculturable extremophiles
live. In such circumstances, functional metagenomics
enables the isolation of genes coding for extremozymes,
enzymes that are capable of being catalytically active in
extreme conditions, or genes that will allow for better
understanding of the mechanisms that make such
organisms resistant to extreme environmental conditions
[4].
Metagenomics has special importance when it comes to

studying soil microbiology. It is estimated that the number
of distinct microorganisms in 1 gram of soil exceeds the
number of microbial species cultured so far [5]. Therefore,
metagenomics seems to be the ideal culture-independent
technique for unraveling the biodiversity of soil
microorganisms and to study how this biodiversity is
affected with continuously changing conditions.
2. Sequencing technologies and

metagenomics
Recently, taxonomic profiling, characterization, and

analysis of microbial communities are being mostly
performed using different next-generation sequencing
(NGS) platforms. Metagenomic samples are high-
throughput, short-read sequences, and the cost is relatively
decreasing. In addition, these platforms are advantageous,
avoiding the need for cloning of DNA fragments [6].
Recent advances in NGS technologies were developed to

suit various numbers of applications, cost, and capabilities
Environmental Metagenomics | 43
[7]. The most commonly used platforms are the 454 Life
Sciences (Roche) and Illumina systems (Solexa) [8]. The 454
sequencing technology, which was the first commercially
available next-generation technology, is based on the
pyrosequencing technique. It provides high throughput and
relatively cheap analysis [9]. During the sequencing reaction
in this technique, nucleotide incorporation into the growing
chain is detected by the capture of the released
pyrophosphate, which is converted into a light through an
enzymatic reaction. Different nucleotides are sequentially
added into each nucleotide incorporation event; therefore
the light signal can be attributed to a specific nucleotide.
Finally, the light signals are converted into sequence
information. In the 454 pyrosequencer, the DNA fragments
are amplified after being fixed on beads in a water-oil
emulsion [10]. Pyrosequencing has been employed widely
in the analysis of microbial diversity in many environments
including marine environments [11] and different soil
environments [12, 13].
Illumina sequencing technology relies on the use of

fluorescently labeled reversible terminator nucleotides.
Instead of being chemically modified to prevent further DNA
synthesis (dideoxynucleotides) which is the case with
Sanger sequencing, the terminator nucleotides are attached
with blocking group that can be removed from the nitrogen
base in a single step. DNA synthesis takes place on a chip
where primers are attached. After each cycle, the dyes
attached to each nucleotide are excited by laser followed by
scanning of the incorporated bases. In order for the next
synthesis cycle to proceed, the blocking group and the dye
are first removed by a chemical reaction. Illumina
sequencing platform was successfully used to study
microbial diversity in many environments [14, 15, 16].
In addition to the abovementioned technologies, recently

developed sequencing technologies are available and being
employed in metagenomic studies. These include SOLiD
5500 W Series developed by Applied Biosystems, single-
molecule real-time (SMRT) DNA sequencing from Pacific
Biosciences, and Ion Torrent semiconductor sequencing [8].
More innovative technologies are being developed that
could be of great use for metagenomic studies in the near
future. Strand sequencing technologies, currently being
developed by Oxford Nanopore technologies, enable the
sequencing of intact DNA strand that passes through a
protein nanopore [17]. Irys Technology, developed by
BioNano Genomics, represents one of the very promising
new technologies in genomics era [8].
3. The metagenomic approaches
Metagenomics research strategy starts with selecting a

proper ecological or biological environment of interest that
hosts a wide variety of microbial communities which may
have potential biotechnological applications. Environments
that attract metagenomic researchers are mainly those
characterized with extreme conditions or unique
environmental conditions. These include environments with
highly acidic or alkaline pH; high metal concentrations,
pressures, or radiation; and high salinity or extreme
temperatures [4].
Metagenomic analysis starts with isolating genomic DNA
that represents the whole community in the soil sample,
constructing a DNA library from the isolated DNA, and
screening the available library for a target gene. It is
important here to select the DNA extraction method that
will provide enough yield and DNA that represents the
diversity of the whole microbial community in the target
environment. This is still one of the most challenging steps
of metagenomic analysis. The chemical and physical
characteristics of soils are very wide and complex,
depending on the type of the soil examined, that will make it
difficult to develop a reference method for DNA extraction
from soils. Besides, soils contain many substances that are
co-extracted with genomic DNA and harbor inhibitory
effects on the downstream processing of the extracted DNA.
Examples include humic and fulvic acids [18]. Therefore,
optimization and comparison between different extraction
methods are usually required for each type of soil
[19, 20, 21, 22].
A DNA library is then constructed from the genomic DNA

isolated from the target environment. This is performed by
fragmenting the isolated DNA into fragments with
appropriate sizes that would allow for their cloning. This
is performed by either using restriction enzyme digestion
or mechanical shearing. DNA fragments obtained from such
processes are cloned into the proper cloning vector. Plasmid
vectors are used for small DNA fragments, and the libraries
generated are called small-insert genomic libraries. Large
inserts are cloning into cosmid or fosmid vectors which can
hold inserts up to 40 kb in size or BAC vector which can
carry inserts with sizes that exceed 40 Kb [23].
DNA libraries are usually constructed in a microorganism

that is well-studied and is easy to manipulate inside the
laboratory such as Escherichia coli. In case there is a need
for expressing the genes within the DNA inserts in other
microorganisms, shuttle vectors are used to transfer the
libraries into a proper host [24].
Finally, a screening assay is applied to search for a gene

of a particular function, and the gene product is functionally
analyzed. There are two different metagenomic strategies
that are commonly used in research. The first one is focused
on the use of marker genes such as the ribosomal genes 16S
rRNA [25] and 18S rRNA [26] to study the composition of the
microbial community in a certain environment or specific
protein-coding gene with medical or industrial importance
[27, 28, 29]. Such a strategy is called targeted metagenomics.
The second approach is the shotgun metagenomics, in
which a wide coverage of genomic DNA sequences is
achieved using high-throughput next-generation
sequencing to assess the entire taxonomic structure or
functional potential of microbial communities [30].
The most challenging aspect of the screening process in

metagenomics is the analysis of a huge amount of sequence
data that are generated from the constructed library. A wide
range of bioinformatic tools has been developed over the
years to help analyze the metagenomic data and compare it
to available online databases.
Acknowledgments
This work was funded by the Deanship of Scientific

Research at Princess Nourah bint Abdulrahman University,
through the Research Groups Program Grant no.
(RGP-1438-0006).
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PART III
HUMAN HEALTH AND
DISEASE
Human Health and Disease | 55

56 | Human Health and Disease
4. Human Health and Disease
Human Health and Disease
The human microbiome plays a vital role in maintaining

homeostasis of various organ systems and protecting against
infectious agents. It can be subcategorized into local or regional
microbiomes throughout the body, such as the gut, oral, skin, lung,
and vaginal microbiomes. These manicured microecosystems are
highly organized and complex, with each person having their own
distinct makeup and distribution of various microorganisms.
Though, individual microbiomes are unique, there is still capacity to
understand key role players in the community and their potential
for adaptation to improve human health in general.

Figure 1. Possible definitions of a healthy microbiome: composition, function,
dynamics, and ecology. a Early definitions of a “healthy” microbiome
generally focused on sets of taxa that might be expected to be found
prevalently in healthy people. While purely taxonomic cores of any type have
remained elusive, even in relatively narrowly defined populations, each
body-site habitat possesses strong phylogenetic enrichments. Typical genera
(or families in the gut) in healthy populations at different sites are shown
here. b Metagenomic measurements have allowed the functional potential of
the microbiome at different sites to be assessed. These studies have yielded
more consistently shared functional cores of body-wide and niche-specific
pathways that are maintained in health. LPS lipopolysaccharide, PAMP
pathogen-associated molecular pattern. c Ecological assembly patterns
provide another possible definition of a healthy microbiome, because each
host may draw from a “typical” meta-population of potential microbes
through a mix of partially stochastic processes. These processes may include
the order in which microbes colonize their respective human habitat (affected
by geography and early exposures, for example), the prolonged availability of
each microbe in the host’s local environment, and host selection. d The healthy
microbiome can also be characterized in terms of its dynamics, depicted here
in a simplified model as a conceptual energy landscape. The infant
microbiome (yellow point) starts out in an unstable state and gradually
descends towards one of potentially several healthy adult attractor states.
Perturbations (dashed red arrows) can either be resisted (green point) or can
move the microbiome out of the healthy state, after which a resilient
microbiome will return to a healthy state (not necessarily the original healthy
state) or fall into an unhealthy state (red). (Lloyd-Price et al., 2016).

Disruption, or dysbiosis, of the microbiome can cause serious
diseases and allow for opportunistic infections to occur. This
disturbance can be caused by a variety of factors including changes
in diet, exercise, geographical location, age, habits, as well as
medical intervention like antimicrobial chemotherapy. This change
in microbial composition subsequently leads to the development
and exacerbation of a number of diseases impacting essentially
every aspect of human physiology including the digestive,
respiratory, integumentary, central nervous, and reproductive
systems. Alternatively, dysbiosis may be the result of a particular
disease, and in other cases disease-dysbiosis may be bidirectional
(Silverman et al., 2019). The human microbiome can be influenced
by many of the above mentioned factors, but also others like life
partners, pet ownership, and occupation, which do not necessarily
correlate with or contribute to dysbiosis (Kiecolt-Glaser et al., 2019,
Kates et al., 2020).
A major driving feature of many of these elements is the host’s
genetics, which can also directly impact their microbiome (Tabrett
and Horton, 2020). Part of the gut microbiome and even individual
types of bacteria have been shown to be heritable (Kurilshikov et
al., 2017), and it is likely that other areas of the human microbiome,
like the skin microbiome, could be passed from parent to offspring
as well (Si et al., 2015). The host genotype is intimately linked to
its microbiome and has been shown to affect dysbiosis-induced
diseases such as inflammatory bowel disease and atopic dermatitis
(Knights et al., 2013, Dabrowska and Witkiewicz, 2016, Woo and
Sibley, 2020). Host genetics and their microbiome can even
indirectly affect one’s susceptibility to other diseases. For example,
the attractiveness of mosquitos to particular individuals is an
heritable trait which is influenced by the many factors, including
the skin microbiome; therefore altering the potential of contracting
a mosquito-borne pathogen, such as a Plasmodium parasite which
causes malaria (Martinez et al., 2020). It seems there is a
combination between environmental factors, host genetics,
behavior, and others that continually build and adapt the human

microbiome, however, is worthy to note that the former may play
a larger role in shaping particular microbiomes, such as the gut
microbiome (Rothschild et al., 2018).

Media Attributions
• Figure 1 – Possible definitions of a healthy

microbiome: composition, function, dynamics, and
ecology by Lloyd-Price et al., 2016 adapted by
Dylan Parks. Licensed under Creative Commons
Attribution 4.0 International License
• Video 1 – You have the microbiome you deserve
by Research Square. Licensed under Creative
References
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Genetics and Gut Microbiome Composition. Frontiers in
Microbiology, 7, 1357. https://www.frontiersin.org/article/

10.3389/fmicb.2016.01357
2. Kates, A. E., Jarrett, O., Skarlupka, J. H., Sethi, A., Duster, M.,
Watson, L., Suen, G., Poulsen, K., & Safdar, N. (2020). Household
Pet Ownership and the Microbial Diversity of the Human Gut
Microbiota. Frontiers in Cellular and Infection Microbiology, 10,
fcimb.2020.00073
3. Kiecolt-Glaser, J. K., Wilson, S. J., & Madison, A. (2019). Marriage
and Gut (Microbiome) Feelings: Tracing Novel Dyadic Pathways
to Accelerated Aging. Psychosomatic Medicine, 81(8), 704–710.
https://doi.org/10.1097/PSY.0000000000000647
4. Knights, D., Lassen, K. G., & Xavier, R. J. (2013). Advances in
inflammatory bowel disease pathogenesis: linking host
genetics and the microbiome. Gut, 62(10), 1505.
https://doi.org/10.1136/gutjnl-2012-303954
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Host Genetics and Gut Microbiome: Challenges and
Perspectives. Trends in Immunology, 38(9), 633–647.
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(2020) Differential attraction in mosquito–human interactions
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N., Shilo, S., Lador, D., Vila, A. V., Zmora, N., Pevsner-Fischer,
M., Israeli, D., Kosower, N., Malka, G., Wolf, B. C., … Segal, E.
(2018). Environment dominates over host genetics in shaping
human gut microbiota. Nature, 555(7695), 210–215.
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j.coi.2019.08.007
10. Tabrett, A., & Horton, M. W. (2020). The influence of host
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82(1), 222–228. https://doi.org/10.1016/j.jaad.2019.08.078

5. The Gut Microbiome
The Gut Microbiome
The microbiome associated with the human gastrointestinal tract,

termed the gut microbiome, may arguably be the most important
component of the collective human microbiome. It has been shown
to affect numerous other regions of the body and serves a role
in many types of diseases throughout it. This is because many of
the microbial products are absorbed in the alimentary canal and
distributed throughout the body via the cardiovascular system.
Sample procurement to characterize the gut microbiome usually
comes in the form of fecal matter, which readily available and less
invasive. However, there are novel attempts to characterize gut
microbiome constituents by sampling the mucosal-luminal
interface (Yan et al., 2020). DNA and other features of interest can
then be extracted from the microbes to provide information
regarding gut microbiome composition and function.
Composition
The gut microbiome is quite versatile and its composition can vary
widely among individuals with different ethnicities, across
geographic locations, and with age (Yatsunenko et al., 2012, Gaulke
and Sharpton, 2018). The host’s genetics do play a role in the
composition of the gut microbiome, as certain members are
heritable, while other’s abundances are the causal result of
congenital diseases (Xu et al., 2020). Though diet, which is closely
related to the aforementioned variables, may be the single strongest
influencing factor when it comes to structuring this digestive
community and alterations are reflected in both short-term and
The Gut Microbiome | 63

long-term dietary interventions (David et al., 2014, Xu and Knight,
2015). Indeed, the type of diet, such as a high-fat diet, will have a
direct impact on the gut microbiome. More specifically, the unique
macromolecules within the gut can modify microbial abundance
and predicted functions. For example, a certain oil in the diet could
result in higher microbial richness, however, this diversity may not
alone be a predictor of better health outcomes (Abulizi et al., 2019).
Dietary vitamin content and even receptors required for processing
them are in part modulated by the microbiome. For example,
vitamin D deficiency and downregulation of its affiliated receptor
is associated with pathogenesis of various diseases and their
restoration promotes healthy host-microbe homeostasis (Jin et al.,
2015). It is also interesting to note that diet usually changes
throughout the year as certain food items become available or
absent depending on the season. An increased gut prevalence of
Bacteroidetes, which can digest complex plant carbohydrates, could
be explained by a diet consisting heavily of produce during a harvest
season (Davenport et al., 2014). The composition and diversity of
the gut microbiome can even be linked to personality traits. Those
individuals with larger social networks have a more diverse gut
microbiome, and those affected by stress and anxiety show an
altered composition with reduced diversity (Johnson, 2020). The
connection between the gut microbiome and mental states and
behaviors will be discussed more in the section “Mental Health”.
64 | The Gut Microbiome

Video Quiz

online here:
Dysbiosis and Disease
The gastrointestinal mucosal immune system modulates and

responds to the gut microbiome, where the resident members aid in
its development and transient pathogens cause dysfunction, leading
to various diseases (Shi et al., 2017). Inflammatory diseases such
as systemic lupus erythematosus, rheumatoid arthritis, IBD, and
ankylosing spondylitis are implicated in the impaired interaction
between the intestinal microbiota and mucosal immune system
(Arbuckle et al., 2003, Mikuls et al., 2012, Costello et al., 2015).
Microbial dysbiosis within these cases are marked by changes in
composition and diversity of specific groups of organisms (Shi et
al., 2017). So, the gut microbiome can not only serve as an indicator

for such diseases, but may also eventually become a target for
treatment, with maintaining proper homeostasis a major goal.
Gut microbiome dysbiosis, whether it causes a particular disease
or manifests as a result of it, is quickly becoming the focus of many
gastrointestinal illnesses. Specific members of the gut microbiome
(e.g. bacteriome, mycobiome, virome) can even vary and be affected
differently depending on the disease and its severity. For example,
while much of the focus is usually on bacteria in the gut, it is
important to not discount the contributions of other
microorganisms like bacteriophages and fungi. The gut phageome
can vary in diversity, complexion, and in so has been shown to
contribute to diseases like IBD, malnutrition, and AIDS (Norman
et al., 2015, Reyes et al., 2015, Monaco et al., 2016, Shkoporov and
Hill, 2019). Similarly, the gut mycobiome has shown to have roles in
IBD, colorectal cancer, and even neurological diseases (Forbes et al.,
2019, Gu et al., 2019, Qin et al., 2021). Though, it is likely the complex
interactions between all members of the gut microbiome with the
host undoubtedly play a role in various degrees for the progression
of gastrointestinal and other diseases.
Those with Inflammatory Bowel Disease (IBD) experience
substantial fluctuations in the gut microbiome, which is implicated
due to signs and symptoms of the disease state, diet, as well as
increased medication during flare ups (Walters et al., 2014,
Halfvarson et al., 2017). IBD is a blanket term for two disorders,
ulcerative colitis and Crohn’s disease, which are characterized by
chronic inflammation of the gastrointestinal tract and commonly
results in frequent diarrhea, abdominal pain, bloody stool, weight
loss, and fatigue. It is likely that the gut microbiome plays both a
role in the development of these conditions and is affected by the
induced changes. The gut virome component responds to disease-
induced environmental change of IBD patients by shifting from
virulent to temperate bacteriophage core, which subsequently
affects the bacteriome and dysbiosis condition (Clooney et al., 2019).
A familial study of patients with Crohn’s disease showed an increase
in the number of pathogenic bacteria, and a decrease in beneficial

bacteria. In particular, three potentially pathogenic biofilm-forming
species from both the bacteriome (Serratia
marcescens and Escherichia coli) and mycobiome (Candida
tropicalis) interact with each other and impact the host immune
system by increasing levels of proinflammatory cytokines and
mucolytic enzymes which cause oxidative and tissue damage
(Hoarau et al., 2016).

Diabetes mellitus is another disease in which its progression is

partially in response to gut microbiome dysbiosis. While there are
other factors that play into diabetes such as culture, genetics, age,
lifestyle, etc., this can be interlinked with an individual’s
microbiome. Studies over both type 1 and type 2 diabetes have
shown that a change in the gastrointestinal microbial ecology is
associated with diabetic subjects as compared with their healthy
counterparts (Giongo et al., 2010, Larsen et al., 2010, Musso et al.,
2011, Sohail et al., 2017). Type 1 diabetes stems from destruction of
pancreatic beta cells, which results in decreased insulin production
and elevated blood glucose levels. The gut microbiome may
contribute to the disease by dysbiosis-associated immune
regulation causing destruction of the beta cells and/or gut
leakiness, endotoxemia, and chronic low-grade inflammation
associated with certain enteric microbes (Cani et al., 2007, Lee et
al., 2011, McDermott and Huffnagle, 2014, Sohail et al., 2017). Type
2 diabetes is characterized by the body’s improper regulation and
secretion of insulin and is associated by hyperglycemia.

Physiological changes in the GI tract could be induced by dysbiosis
in the gut microbiome leading to gut permeability and insulin
resistance (Everard and Cani, 2013). In general, the gut microbiota
composition is less in terms of diversity and richness for those with
type 2 diabetes, though an increase in abundance of certain groups
like Bifidobacterium could improve conditions associated with
pathogenesis (Cani et al., 2007, Sohail et al., 2017). It seems that
an alteration of the microbial gut profile has considerable effects
on host metabolism, gastrointestinal physiology, gut fermentation
capabilities, and immunity which can have many other downstream
implications (Boulange et al., 2016).
Obesity is commonly associated with type 2 diabetes as well as
other comorbidities that are linked to gut microbiome dysbiosis.
As mentioned earlier, diet strongly affects the composition and
function of the gut microbial community and subsequently impacts
the host’s metabolic capabilities. In fact, a high-fat/calorie or
improper diet that results in dysbiosis is evident earlier than the
signs of the host’s metabolic abnormalities, and so gut microbiome
dysbiosis may be the principle ingredient responsible for the
progression of obesity and type 2 diabetes (Nagpal et al., 2018).
A high-sugar diet seems to promote an abundance of Mollicutes,
a class within Firmicutes, which in turn suppresses Bacteroidetes
(Turnbaugh et al., 2008), and this higher ratio of Firmicutes/
Bacteroidetes has been proposed as a biomarker and hallmark
indicator for obesity (de Bandt et al., 2011, Zou et al., 2020). However,
it is important to consider other factors such as physical activity
and medication that could cause a variation in diversity of the gut
microbiome, as this ratio does not always definitively denote obesity
(Magne et al., 2020). Though, there are similarities between many
of the microbiome-linked contributing factors of pathogenesis
progression for diet-induced diseases. For example, a high-fat diet
induces increased gut permeability that allows exogenously
produced bacterial compounds (e.g. lipopolysaccharides) to diffuse
through the intestinal barrier, which then can interact with immune
cells and lead to inflammation (Cani et al., 2007, Nagpal and Yadav,

2017). However, diet isn’t the sole factor that can promote gut
leakiness, and it seems that obesity in general causes an altered
gastrointestinal state (Nagpal et al., 2018).

Chemotherapeutic Intervention and C. diff
Medication and antimicrobial drugs can also have drastic effects

on the gut microbiome that invoke risk of secondary infections,
allergies, and other diseases like obesity (Becattini et al., 2016).
Though many of these prescribed treatments are necessary to
combat infectious diseases, the aftermath may have more serious
consequences. Not only does antimicrobial therapy disrupt the
resident microbiome, but misuse, suboptimal dosing, and patient
noncompliance can create conditions conducive to fostering
antimicrobial resistance through selective pressure.
Clostridioides difficile (commonly called C. diff) infections are
directly associated with antibiotic-induced dysbiosis in the
gastrointestinal tract. C. difficile is part of the normal microbiota,
however, as an opportunistic pathogen it can invade or colonize
empty niches brought about by dysbiosis and cause potentially fatal
episodes of pseudomembranous colitis, which is associated with
abdominal cramping, pain, sepsis, and bouts of diarrhea (Kho and
Lal, 2018). Infection and transmission of this organism has been well
known for its prevalence in hospital settings, primarily affecting the
elderly and immunocompromised, however, community-associated
infections have recently increased in what was once considered

low-risk populations (Rouphael et al., 2008, Baker et al., 2010,
Hensgens et al., 2012, Benson et al., 2015, Johanesen et al., 2015).
It is also alarming that this organism has resistance mechanisms
to many commonly prescribed antimicrobials, including β-lactams,
aminoglycosides, lincomycin, tetracyclines, and erythromycin
(George et al., 1978), and more recently ‘hypervirulent’ strains have
developed resistance to fluoroquinolones (He et al., 2013, Johanesen
et al., 2015). C. difficile infections have an enrichment of fungi that
associate with the bacteriome and perhaps antifungal therapy could
help improve treatment success if administered in conjunction with
specific antibacterial drugs (Stewart et al., 2019). Though,
these infections can have lingering impacts on the gut microbiome,
as further antimicrobial therapy that is usually required can
perpetuate the situation. The inflammation as a result of the disease
induces the production of antimicrobial peptides by epithelial cells
and neutrophils which inhibit the growth of the natural resident
commensal microbes (Leber et al., 2015).
While many events of gut dysbiosis are directly linked to the
chemotherapeutic effects on microbes since they are prescribed
to target microbes responsible for the infection, some medications
which are meant to address other diseases, like antidepressants for
mental health, have undesired effects on the microbiome (Maier
and Typas, 2017). In the cases of multi-drug combinations (e.g. non-
steroidal anti-inflammatory drugs (NSAIDs), antidepressants,
laxatives, proton-pump inhibitors (PPIs), etc.), it is not the number
of drugs that affect gut microbiome diversity, but rather the types of
drugs (Rogers and Aronoff, 2016). Though these scenarios become
complicated as it is difficult to ascertain whether the alterations
observed on the microbiome are from the drug’s mechanism of
action, a downstream side effect, or originate from the condition
that is being treated, and it is likely a complex combination of all
factors for each disease and medication (Rogers and Aronoff, 2016,
Maier and Typas, 2017, Jackson et al., 2018).

Fecal Microbiota Transplant
Although pharmaceutical drugs are of dire importance to treat

various diseases, whether they are infectious in nature or not, other
avenues must be pursued for those that may benefit from
restoration of the gut microbiome. Probiotics and fecal microbiota
transplant (FMT) can serve as viable options for the prevention and
treatment of gut microbiome dysbiosis. Probiotics are considered
foodstuffs with microorganisms, usually bacteria (many being lactic
acid bacteria) and yeast, and their byproducts that have a beneficial
effect on human health when introduced into the body. Many
probiotics are commercially available to consumers in the forms of
products like yogurt, kefir, buttermilk, sauerkraut, pickles, premade
vitamin supplements and many others. Specifically, probiotics can
be used for the treatment and prevention of many of the
aforementioned gut microbiome dysbiosis-associated diseases,
especially those induced by antibiotics (Kim et al., 2019). The
beneficial microbes outcompete pathogens for resources or prevent
them from establishing a niche in which to grow (Ouwehand et al.,
1999).
In more extreme cases of gut microbiome dysfunction and
disease, like those from C. difficile infection (CDI) in which
antibiotics are ineffective and can potentially exacerbate the
problem, other measures must be taken. Fecal microbiota
transplant therapy takes a stool sample containing the gut
microbiome from a healthy donor and relocates it into the infected
patient’s colon. The introduced microbiota then helps move the
gut microbiome towards homeostasis by restoring the structure
of beneficial microbes and metabolites (Fujimoto et al., 2021). This
procedure is usually reserved for those patients with recurrent CDI
and has shown to be highly successful and is considered safer and
more effective than prolonged antibiotic usage (Mattila et al., 2012,
Cammarota et al., 2015), though is also being investigated for first-
line treatment of CDI (Camacho-Ortiz et al., 2017). FMT has gained
traction for its success and is being further considered as a

therapeutic option in other treatment protocols, such as those for
cancer patients undergoing cancer immunotherapy to help improve
response or manage toxicity (McQuade et al., 2020), and individuals
undergoing allogenic hematopoietic stem cell transplant for
hematological disorders that experience graft-versus-host disease
complications from it (Zhang et al., 2021). However, precautions
must be taken for FMT, as the donor’s sample could potentially
harbor other pathogenic microbes, like multi-drug resistant
Escherichia coli, that can result in pathogenesis, further
complications, and even death for the recipient (DeFilipp et al., 2019,
Martinez-Gili et al., 2020). More comprehensive research and FMT
trials must be performed in order to optimize this procedure to
better match donors with recipients and to further understand the
exact mechanisms of microbiome rehabilitation.



Conclusion
Gut microbiome intervention may be the key to future treatments

of diseases associated with dysbiosis like IBD, diabetes, obesity,
colorectal cancer, etc., and offers a viable alternative to many
traditional pharmaceutical interventions. Though, the gut
microbiome is plastic and continually changes with its host’s
environment and lifestyle, so stabilization is constant work.
Additionally, creating an ideal ‘cocktail’ of microbes that will
maintain homeostasis when implemented can be challenge. While
there are general members of the gut microbiome that exist at a
constant level and show some correlation to normal health, there
may not be a true ‘standard’ gut microbiome due to the vast
differences between people across the world. So, this type of
therapy may require a more unique and individualized approach
that depends on the disease and characteristics of both the host and
their microbiome.
• What factors influence the composition of the gut

microbiome?
• How is gut microbiome dysbiosis brought about?
• What diseases are associated with gut microbiome
dysbiosis? Are features of pathogenesis always a cause
or effect of these events? Explain.
• Explain the treatment options that are available for

gut microbiome dysbiosis.
Media Attributions
• Video 1 – Gut microbiome and individual

genetics by Latest Thinking. Licensed under
• Video 2 – Diet and gut microbiome interactions
in irritable bowel syndrome by Research
• Video 3 – Microbiome and Obesity – Martin
Blaser by National Human Genome Research
Institute. Licensed under Creative Commons: By
• Video 4 – Gut microbiome composition after
multi-donor fecal microbiota transplantation for
obesity by Research Square. Licensed under
• Video 5 – Rob Knight: Fecal Transplants? The
Disgusting is Par for the Course by World
Economic Forum. Licensed under Creative

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6. The Oral Microbiome
The Oral Microbiome
The oral microbiome as with other site-specific microbiomes on

and inside the human body is very distinct for each individual and
its makeup and function is reflective of a variety of factors. Even
within the context of the oral cavity, there are several unique niches
with their own microbial ecosystem, including saliva, tongue, teeth,
gingiva, throat, tonsils, and others. Each of these habitats exhibit
diverse and complex interactions between bacteria, archaea, fungi,
viruses, and protozoa, where dysfunction can lead to a number
diseases, both rare and common (Wade, 2013, Sampaio-Maia et al.,
2016).
Factors Affecting Composition
The composition of the oral microbiome and its respective niches

is inherently dependent on host genetics, but other factors and
habits such diet and smoking have a substantial effect on diversity.
Members of the oral cavity demonstrate more heritability than the
gut microbiome, and higher abundances of certain heritable
organisms, such as Prevotella pallens, are associated with a lack
of dental caries, while on the other hand, Streptococcus
mutans and Lactobacillus species are linked to cavities (Davenport,
2017, Xiao et al., 2018). Detection of the specific groups of microbes
has traditionally been difficult since many are fastidious and cannot
be cultivated. So other techniques like metagenomics,
metatranscriptomics, and proteomics have been implemented to
better characterize the microorganisms present and their
respective roles (Parahitiyawa et al., 2010, Grassl et al., 2016). These
The Oral Microbiome | 85

studies have found that the oral microbiota is quite different even
between healthy individuals, and so it may be the microbial
functionality that is more important in the progression of various
diseases, such as the role of biofilm formation, plaque buildup, and
sugar metabolism in the development of dental caries (Segata et al.,
2012, Takahashi et al., 2010, Duran-Pinedo and Frias-Lopez, 2015,
Sato et al., 2015, Davenport, 2017).
The oral cavity is such a dynamic location that constantly

experiences a variety of different foods, drinks, oral hygiene
products, and other environmental stimuli with each composed of
a multitude of macromolecules, compounds, and potentially other
microorganisms, and so it makes sense that its microbiome often
fluctuates in diversity (Parahitiyawa et al., 2010). Though there are
differences in composition between individuals, specific sites, and
over periods of time, species such as Streptococcus mitis
and Granulicatella adiacens are conserved and generally present
throughout the oral microbiome, while the presence of other
specific microbes are associated with a particular disease (Aas et
al., 2005). However, diseases like Periodontitis, which is the chronic
inflammation of the gums and tooth supporting structures and one
of the most common oral diseases, has many organisms associated
with the condition, so discerning which ones are primarily
responsible is a complex task. There are a variety of viruses that
cause oral-related conditions, such as the Human papilloma virus
(HPV) which is known for causing lesions and warts in the mouth,
as well as head and neck squamous cell carcinoma (Kumaraswamy
and Vidhya, 2011). An increase of the protozoa Entamoeba
gingivalis and Trichomonas tenax are observed in patients with
gingival disease, but are not the causative agents, rather just taking
advantage of the increased food sources (bacteria and food debris)
from poor hygiene (Wantland et al., 1958). There is also a variety
86 | The Oral Microbiome

of fungi present in the oral cavity with Candida species being the
most common, and many of the members in the oral mycobiome are
responsible for chronic diseases, however correlation isn’t exactly
clear (Ghannoum et al., 2010). Archaea, primarily methanogens, are
also present in the oral microbiome, and while there aren’t
technically any known pathogens in this domain, there is an
increase in abundance observed in patients with periodontitis (Lepp
et al., 2004, Mattarazo et al., 2011, Wade, 2013, Willis and Gabaldon,
2020).
Generally, the oral microbiome is linked with aspects oral health
and has primary implications in dental and periodontal diseases,
however it can contribute to vitality and diseases in other parts of
the body such as cardiovascular disease, stroke, Alzheimer’s disease,
cystic fibrosis, rheumatoid arthritis, diabetes, pneumonia, and
preterm birth (Seymour et al., 2007, Duran-Pinedo and Frias-Lopez,
2015, Kori et al., 2020, Willis and Gabaldon, 2020). Additionally, the
oral microbiome is implicated in various forms of cancer including
esophageal, pancreatic, gastric, liver, colorectal, and oral (Willis and
Gabaldon, 2020, Bakhti et al., 2021, Mohammed et al., 2021). In many
of these types of cancers the Gram-negative anaerobe,
Fusobacterium nucleatum, is a primary culprit as it can promote
cancer by activation of cell proliferation, promotion of cellular
invasion, induction of chronic inflammation and immune evasion
(Al-hebshi et al., 2017, McIlvanna et al., 2021). Another contributing
factor to cancer development is the use of tobacco products which
cause oral microbiome dysbiosis (Al-habshi et al., 2017, Gopinath et
al., 2021, Sajid et al., 2021). As with other microbiomes, deviation
from the normal composition can result in altered function and
progression of disease.

Figure 1. Oral and systemic diseases associated with the oral microbiome. A
representation of the associations found between diseases with increases or
decreases of the abundances of organisms in the oral cavity. Organisms listed
in blue have been shown to be increased in abundance in the oral cavity in
individuals presenting with the noted disease, and organisms listed in red
have been shown to be decreased. Those in purple may be either increased or
decreased depending on the conditions or progression of the disease. (Willis
and Gabaldon, 2020).

Figure 2. Examples of metagenomic studies of associations between the oral
microbiome and oral diseases. The first column indicates a disease, the second
indicates organisms that have been found at higher abundances in individuals
presenting with the disease, and the third indicates organisms at lower
abundances. (*) indicates taxa associated with oral cancer from a study in
which samples were from tumor and non-tumor sites in the same patients
and disease treatment is not specified. References: Matarazzo et al., 2011, Lepp
et al., 2004, Vartoukian et al, 2009, Griffen et al., 2012, Costalonga and
Herzberg, 2014, Liu et al., 2012, Jorth et al., 2014, Haubek, 2010, Koo and
Bowen, 2014, Gross et al., 2010, Wang and Ganly, 2014, Pushalkar et al., 2012,
Mager et al., 2005, Peters et al., 2017, Willis and Gabaldon, 2020.
Saliva
Saliva is an important component in maintaining homeostasis in

the oral cavity, as it lubricates food, initiates the digestive process,
and defends against bacteria. Disruption in secretion can lead to
changes in the oral microbiome which promotes progression of oral
and other diseases (Grassl et al., 2016). Since saliva contacts virtually
all surfaces within the oral cavity it is involved with all other duties
of the mouth, and adaptive and innate immune defense mechanisms
can be considered the most important in terms of microbiological
clinical relevance. The protein and gycoprotein content regulates
the oral microbiome by promoting the colonization of commensal

microbiota while helping eliminate pathogenic microbes (Cross and
Ruhl, 2018). These macromolecules aid in bacterial adhesion and
biofilm formation so that they aren’t dislodged by salivary flow and
other oral physiological processes (Mandel, 1987). The establishment
of beneficial microbes prevents pathogenic bacteria from gaining a
foothold, and the agglutinins found in saliva aid in removal through
binding and then swallowing (Scannapieco, 1994). The co-evolution
of the microbiota and humans has cultivated the development of
specific bacterial adhesins for colonization of the preferred
microorganisms, thus establishing a mutualism, though pathogenic
microbes are quick to adapt to changing binding motifs (Springer
and Gagneux, 2013, Cross and Ruhl, 2018).
Saliva can harbor numerous microbes, with one milliliter
containing approximately one hundred million microbial cells
(Marsh et al., 2015) and over 600 different species (Dewhirst et
al., 2020, Willis and Gabaldon, 2020). In one study, the prominent
genera found across various types of saliva samples (i.e. spit, drool,
and oral rinse) from healthy individuals were
Streptococcus (17.5%), Prevotella (15.5%),
Veillonella (15.3%), Neisseria (12.7%) and Haemophilus (10%) (Lim et
al., 2017). Though it can be difficult to differentiate a core salivary
microbiota from other specific oral niches since it coats the oral
cavity.
For those suffering from various diseases, the relative abundance
of certain microorganisms and general composition of the oral
microbiota is altered as compared to healthy controls. For example,
patients suffering from chronic obstructive pulmonary disease
(COPD) and periodontitis have varying abundances of Veillonella,
Rothia, Actinomyces, and Fusobacterium in saliva samples (Lin et al.,
2020). Periodontitis and COPD are comorbid diseases that are
commonly associated with other conditions like rheumatoid
arthritis, diabetes mellitus, and cardiovascular diseases (Scher et
al., 2014, Wang et al., 2014, Chrysanthakopoulos and
Chrysanthakopoulos, 2014, Lin et al., 2020). Dysfunction of bacterial
ecology in saliva is exacerbated by COPD and periodontitis, and

so restoration of the salivary microbiota may treat or reduce the
severity of these diseases and their comorbidities (Jeffcoat et al.,
2014, Zhou et al., 2014, Lin et al., 2020).
Alterations of the salivary microbiome are also associated with
certain human viral infections like the herpes virus, influenza, and
SARS-CoV (Blostein et al., 2021, Miller et al., 2021). Saliva generally
is beneficial to oral health, though changes in it’s makeup could be
detrimental and further aggravate disease. For instance, the saliva
microbiota and their byproducts could be responsible for increased
susceptibility of infection of gum tissues with herpes simplex virus 1,
especially in individuals with periodontitis lesions (Zuo et al., 2019).
Eukaryotic viruses can also directly interact with oral bacteria and
affect disease severity, as is the case with streptococci and influenza
which results in an increased viral load (Kamio et al., 2015). Similarly,
among patients with COVID-19 there are differences in the salivary
bacterial community based on SARS-CoV-2 viral load, though the
exact dynamics and repercussions of the interaction isn’t yet well
understood. It is possible that COVID-19-induced inflammation
could directly impact the oral microbiome and contribute to other
diseases connected with dysbiosis (Miller et al., 2021).
Dysbiosis of the fungal component in the salivary microbiome
also contributes to overall oral microbial community changes and
detrimental effects to the human host. The mycobiome is an
important constituent of the oral microbiome though its member’s
abundance is much less than that of the bacteriome. There are two
main genera in the salivary mycobiome: Candida and Malassezia,
where the former is associated with dental plaque bacteria,
carbohydrate-rich microbial communities, and acidic pH conditions
which contribute to dental caries (Hong et al., 2020). Other
interactions between the mycobiome and bacteriome in saliva has
been observed in the chronic inflammatory disease oral lichen
planus (OLP). OLP causes swelling, discoloration, and open sores of
the mucosal membranes in the oral cavity, primarily affecting the
buccal region (cheek), but the lips, gingiva, and tongue may also be
affected. Similar to plaque buildup and cavity formation, this disease

is characterized by an increased abundance of genera Candida, but
also Aspergillus, as well as a decrease in biodiversity (Li et al., 2019).
While diversity and abundance of specific groups within the
salivary microbiome are associated with various diseases, it is
important to consider other factors like diet, lifestyle habits, and
genetics that can influence the progression of any particular
disease.
Teeth, Plaque, and Cavities
Plaque formation occurs when a multispecies biofilm builds layers

on the surface of teeth over time. This structure not only contains
a variety of microbes, some of which are pathogenic, but proteins,
carbohydrates, minerals, antimicrobial peptides and other
compounds that dictate its structure and activity (Amerongen and
Veerman, 2002, Flemmig and Beikler, 2011, Zarco et al., 2011). For
normal healthy individuals, plaque biofilms are important in
maintaining oral homeostasis and good tooth conditions as they can
trap pathogens or prevent them from thriving due to competitive
inhibition. However, regular detachment of these biofilms through
oral hygiene and salivary flow are necessary to prevent pathogen
establishment and their escape from immune responses and
antimicrobial therapy (Avila et al., 2009, Filoche et al., 2009, Van
Essche et al., 2010, Flemmig and Beikler, 2011, Zarco et al., 2011).
The persistence of oral biofilms contributes directly to cavity
formation as carbohydrate-fermenting microbes within produce
acidic byproducts which lower oral pH and damage tooth enamel
(Selwitz et al., 2007, Ling et al., 2010). Cavities form once the surface
layers of the tooth wear away and lesions form in the dentin,
resulting in oral pain, tooth decay and loss (Selwitz et al., 2007,
Zarco et al., 2011). Dental caries are the most prevalent disease for
children worldwide, and dental care is the most common unmet
need among children in the United States (Loesche and Grenier,
1976, Acs et al., 1999, Low et al., 1999, Peterson et al., 2013).

The fastidious nature of several members of plaque polymicrobial
communities have made it traditionally difficult to characterize an
exact consortium responsible for dental caries, however, recent
studies using NGS technologies have detailed a few signature
genera. Streptococcus mutans and Lactobacilli spp. are the primary
culprits, but other genera such as Fusobacterium, Bifidobacterium,
and Actinomyces are found in high abundance in people with
cavities (Munson et al., 2004, Chhour et al., 2005, Corby et al.,
2005, Peterson et al., 2013). The fungal yeast, Candida albicans, is
also found regularly in children with severe early childhood caries
(S-ECC) (Xiao et al., 2018). Interestingly, other members of
Streptococcus including S. parasanguinis, S. mitis, S. oralis, and S.
sanguinis are associated with individuals exhibiting good dental
health (Corby et al., 2005, Peterson et al., 2013). Overall, during the
progression of caries there is a reduction in species diversity in
these communities (Peterson et al., 2013).
Gingivae and Periodontitis
Aside from dental caries, periodontitis, a.k.a. ‘gum disease’, is the

other most common oral disease in humans, and it also results from
alterations in oral microbial ecology. This inflammatory condition
affects the supporting structures surrounding the teeth where the
microbial communities that inhabit the subgingival area serve to
trigger its onset (Hajishengallis and Lamont, 2012, Hong et al., 2015).
Though progression of the disease comes in episodes, the continual
breakdown of periodontium tissue (gingiva, periodontal ligament,
cementum, and alveolar bone) leads to alveolar bone breakdown,
formation of pocket lesions, and tooth loss (Listgarten, 1986). This
condition is very difficult treat as specific antibiotics must be
administered, though they are often unsuccessful as pathogens can
hide in plaque, develop resistance, and quickly recolonize through
reserves in the mucous membranes of the oral cavity. Once pockets
form in the periodontium, periodontitis becomes irreversible, as the

tissues are unable to reattach to the bone and the pathogens within
cannot be completely eradicated or removed (Pihlstrom et al., 2005,
Horz and Conrads, 2007, Van Essche et al., 2011, Zarco et al., 2011).
Culture-based studies have shown that periodontitis is associated
with varying levels of abundance of three bacterial species:
Porphyromonas gingivalis, Tannerella forsythia and Treponema
denticola, which are collective referred to as the ‘red complex’
(Socransky et al., 1998, Rocas et al., 2001, Socransky and Hafajee,
2005, Teles et al., 2010). More recent genetic analysis has revealed
other bacteria associated with the disease, including those from the
classes Clostridia, Negativicutes, and Erysipelotrichia; the genera
Synergistes, Prevotella, and Fusobacterium; and the species Filifactor
alocis (Vartoukian et al, 2009, Griffen et al., 2012, Costalonga and
Herzberg, 2014, Willis and Gabaldon, 2020). Some methanogenic
archaeal species have even been implicated in the disease, and
perhaps serve a metabolic role as a ‘hydrogen sink’ for secondary
fermenters (Lepp et al., 2004, Matarazzo et al., 2011). Viruses, both
eukaryotic and bacteriophages, are also thought to play a part in
the etiology of periodontitis. While bacteriophages manage biofilm
formation through bacterial predation, the role of eukaryotic
viruses found in the oral cavity such as those in the Herpesvirus
family, is more enigmatic (Martinez et al., 2021). The
protist Trichomonas tenax is frequently found in patients with
severe periodontitis, though it is not known if its presence is a cause
or a result of the disease (Benabdelkader et al., 2019. It is generally
thought that this condition arises from events of dysbiosis causing
an increase in community diversity and richness which then alters
antagonistic/synergistic relationships, metabolic functions, and the
oral environment (Kolenbrander et al., 2006, Shi et al., 2015, Ng et
al., 2021). Those bacteria that may be absent or inhibited during
dysbiosis conditions, and are associated with good periodontal
health include those from the phylum Proteobacteria and the
Firmicutes, class Bacilli, and the genera Streptococcus,
Actinomyces, and Granulicatella (Griffen et al., 2012, Liu et al., 2012,
Willis and Gabaldon, 2020).

Periodontitis resulting from oral microbiome dysbiosis has been
linked to other diseases like chronic kidney disease, as well as
several types of cancers including oral, esophageal, gastric, lung,
pancreatic, prostate, hematologic, and breast (Fitzpatrick and Katz,
2010, Ioanndiou and Swede, 2011, Michaud et al., 2017). These
associations stemming from the oral cavity could be due to
dysbiosis-induced microbiome changes and recruitment of disease
causing pathogens, production of harmful microbial-derived
byproducts, and/or modulation of the immune system causing an
increase in proinflammatory cytokines (Abnet et al., 2005,
Kurkivuori et al., 2007, Chalabi et al., 2008, Meurman, 2010, Willis
and Gabaldon, 2020). In reality, there are likely a multitude of
factors and interactions between microbial communities and the
host that lead to the progression of these diseases, however, it is still
important to determine certain organismal signatures or functions
that could indicate a condition and perhaps aid in diagnosis or
treatment.

Tongue
The tongue is another niche for a variety of microbial communities,

and though it is a maneuverable oral centerpiece that comes into
contact with the rest of the cavity, it has its own unique makeup
of microbes. The tongue microbiome serves as an ideal model to
analyze changing microbial consortia and understand microbial
community dynamics. By employing a novel computational method

known as oligotyping, which relies on identifying certain nucleotide
content of genetic sequences, a better resolution of microbial
taxonomic distribution can be achieved (Eren et al., 2014, Welch et
al., 2014, Wilbert et al., 2020). As expected, the tongue experiences
large fluctuations in relative abundance across taxa, but this isn’t
completely explained by obvious human behavior such as oral
hygiene and food/liquid intake. The complex microbial organization
that is observed is likely a combination of many factors, like other
microbiomes, such as host immunity, physiology influenced by
circadian rhythm, epithelial cell renewal, other microbial (e.g.
bacteriophage) interactions, as well as certain host behaviors and
lifestyles (Welch et al., 2014, Wilbert et al., 2020).
The changing microbiome of the tongue can also be linked to
various diseases and conditions in both the oral cavity and
throughout the rest of the body. Viewing the tongue for diagnosis
isn’t a novel approach either, as traditional Chinese medical
practices have viewed tongue phenotypes to discern various
illnesses for thousands of years, and currently physical aspects of
the tongue are being connected with its microbiome composition to
better diagnosis certain diseases like oral, liver, gastric, colorectal,
and pancreatic head cancers (Jiang et al., 2012, Han et al., 2014,
Mukherjee et al., 2017, Cui et al., 2019 Lu et al., 2019, Mohammed
et al., 2021). It is also interesting that members of the tongue
microbiome can help regulate blood pressure and cardiovascular
health through metabolism of dietary nitrate, and oral hygiene (e.g.
tongue cleaning) can help promote the growth of these beneficial
organisms (Tribble et al., 2019). While good habits like proper oral
hygiene can cultivate a healthy oral microbiome and improve overall
health, bad habits like smoking negatively affect microbial
communities which contributes to various diseases. For example,
metagenomic analysis investigating bacterial species and their gene
content showed significant differences of certain species, strains,
and metabolic pathways within the tongue microbiome between
smokers and never smokers (Sato et al., 2020). This further
demonstrates the usefulness of tongue microbiome analysis as a

reliable technique to explore and diagnosis certain diseases and
conditions.
Conclusion
Though not as well characterized as the gut microbiome, the oral

microbiome has a significant impact on human health. Within the
oral cavity, specific niche microbiomes of saliva, teeth, gums, and
tongue are each compositionally unique and implicated in various
conditions and diseases. Moreover still, other oral niches like the
buccal mucosa, palate, pharynx, and tonsils not detailed here are
distinctive and have certain associated diseases initiated with
respective microbiome dysbiosis (Gao et al., 2014, Fukui et al., 2018,
van der Meulen et al., 2018). Areas within the oral microbiome may
eventually become routine sites for medical observation since
samples are easily acquired, especially saliva, and microbial analysis
can serve as a fast and reliable option for diagnosis and potential
treatment of oral disorders as well as other diseases.
As the oral cavity is the initial point of contact and entrance for
foreign microbial loads into the body, oral disease and its associated
microbial ecology appear to serve as a conduit for a multitude of
other diseases. In a sense the oral microbiome can be considered
a precursor to the gut and lung microbiomes, as microbes are
undoubtedly mixed with food, drink, and air before being swallowed
or inhaled and taken further into the respective system. It is
therefore important to understand and better characterize the oral
microbiome so that medical diagnosis and intervention can be
improved. However, with vast differences in microbial community
diversity and composition between humans across the word, this
will be quite the challenge to determine normalized healthy
consortia respective to people of various geographic regions, ages,
lifestlyes, etc.

Hotspot Quiz

online here:
• What aspects of the oral microbiome allow for the

formation of unique niches?
• How does dysbiosis within the oral microbiome
contribute to certain diseases? (What aspects of
changes in microbial community diversity and
richness could cause oral and other diseases?)

• Why may saliva sampling be considered a viable
option for diagnosis of oral and other diseases?
• How do oral biofilms contribute to cavity formation
and tooth decay?
• Why are microbial infections associated with
periodontitis hard to treat?
• How do certain lifestyles influence various niches
of the oral microbiome and lead to disease?
Media Attributions
• Video 1 – GM2: Periodontal Microbiome –

Murray Brilliant by National Human Genome
Research Institute. Licensed under Creative
• Figure 1 – Oral and systemic diseases associated
with the oral microbiome by Willis and Gabaldon,
2020, caption adapted by Dylan Parks. Licensed
License http://creativecommons.org/licenses/
by/4.0/
• Images of body sites and organs in Figure 1 by
Servier Medical Art. Licensed under Creative

• Figure 2 – Examples of metagenomic studies of
associations between the oral microbiome and
oral diseases by Willis and Gabaldon, 2020,
caption adapted by Dylan Parks. Licensed under
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7. The Skin Microbiome
The Skin Microbiome
The microorganisms that inhabit the largest human organ are

important in a variety of ways in regard to host health. The skin
microbiome defends against pathogens, educates the immune
system, helps wound healing, and moderates progression of
diseases, and in return receives nutrients and real estate for
colonization, creating a symbiotic establishment (Byrd et al., 2018).
As the primary external interface to the environment, the skin
serves as the initial physical barrier against invasion of potential
pathogens. The territory of the skin itself is harsh for most
microbes; its cool, acidic, dry, and considered nutrient poor, and
so only those organisms that have adapted to these conditions can
successfully colonize it. A majority of these microbes rely on
obtaining nutrients from sweat, sebum, and dead skin cells by using
proteases and lipases to break apart various compounds to liberate
usable resources (Byrd et al., 2018). Though, there are adverse
conditions and a general lack of food for microbes on the skin,
there is still quite a diverse community which is unique to certain
individuals and body locations.
Better characterization of the skin microbiome and its site-
specific diversity can allow for greater understanding of skin
diseases such as atopic dermatitis, acne, rosacea, and psoriasis that
are associated with dysbiosis (Grice and Segre, 2011). The role of
resident and transient microorganisms are important in the onset
and progression of these types of diseases, and their study may help
in diagnosis and treatment protocols.
The Skin Microbiome | 115

Composition and Stability
Within a single square centimeter on the skin there can be up to

one billion microorganisms, and this mixed community of bacteria,
viruses, protozoa, fungi, and mites can be both good and bad for
host health (Grice and Segre, 2011, Weyrich et al., 2015). Overall,
there are four main bacterial phyla that constitute the human skin
microbiome: Actinobacteria, Firmicutes, Proteobacteria, and
Bacteroidetes (in descending order of abundance) (Grice et al.,
2009). Microbial composition primarily depends on the specific
location of skin site, and particularly whether it is dry, moist, or
sebaceous (i.e. oily). Moist areas like the bend of the elbow or the
feet harbor bacteria like Staphylococcus or Corynebacterium species
and tend to have higher species diversity, while sebaceous locations
promote the growth of Propionibacterium species and are lower in
diversity (Grice et al., 2009, Oh et al., 2016, Byrd et al., 2018).
The composition of fungi on the skin also depends on location and
physiological properties. Fungal members of the skin microbiome
primarily consist of species from the genus Malassezia, with some
from Penicillium and Aspergillus, and less of a few other genera.
Specifically, the community of fungi on the feet have a high amount
of diversity and it tends to change more over time as compared
with other locations on the body. This may be due to environmental
exposure, sock and shoe usage, or the fact that specific sites like
plantar heels, toe webs and toenails are commonly infected by
116 | The Skin Microbiome

fungal pathogens, which can be difficult to treat. (Findley et al.,
2013).
Small arthropods less than half a millimeter also colonize the skin
and makeup part of the microbiota. Two species of mites from the
genus Demodex utilize lipids produced in the sebaceous regions of
the skin; the larger species D. folliculorum tends to cluster around
hair follicles while the smaller D. brevis situated near the eyelid
rim is more antisocial (Schommer and Gallo, 2013). These lipid-
eating mites normally have a symbiotic relationship with humans,
however, they could potentially serve as mechanical vectors for
transport of pathogenic bacteria, and their population buildup and/
or associated events of dysbiosis could promote inflammatory
reactions (Lacey et al., 2009, Lacey et al., 2011).
Eukaryotic viruses on the skin are primarily transient, exhibit
lower site-specific affinity, and the most different from person to
person. This is likely due to the fact that they are obligate
intracellular pathogens with special constraints. Also, maybe as
expected, various bacteriophage abundance in certain sites is
dependent on the corresponding bacterial genera (Oh et al., 2016).
Microbial communities on the skin remain stable despite the
constantly changing external environment that humans bring about.
Over both short- and long-term time intervals, sebaceous sites are
the most stable, and moist areas like the feet are the least stable.
Interestingly, dry sites with high environmental contact and
disruption, like the palms of hands, exhibit community stability over
time (Oh et al., 2016). Though, variability and stability may be
dependent on individual host habits and lifestyles. At an early age,
however, there are drastic changes in the skin microbiome until
it becomes established. At birth, the baby transitions from an
essentially sterile environment in the womb, to open air and
constant microbial exposure where initial colonization of the skin
begins. The method of delivery also affects initial community
composition on the skin. When a baby is born conventionally
through the vaginal canal, the members of the skin microbiome
reflect the vaginal microbiome, and if born via cesarean section, the

infant’s skin community more closely resembles that of the mother’s
skin (Dominguez-Bello et al., 2010). In the initial few months, the
skin community predominately consists of
Streptococci and Staphylococci bacteria, but as aging ensues the
abundance of these genera decrease and diversity and numbers of
others begins to increase and level out (Capone et al., 2011). This
has long-term effects on health, as the evolution of an infant’s skin
microbiome helps to regulate and mature both the skin and immune
system.
Hot Spot Quiz

online here:
Immune System Interaction
Exposure to potentially pathogenic microorganisms at a young age

can help educate the immune system. Immediately after childbirth,
initial skin colonization by microorganisms is allowed without the
classical inflammatory response, though shortly after this period,

these microbes promote the development of distinct components
of the immune system for future pathogen encounters (PrabhuDas
et al., 2011, Naik et al., 2012, Naik et al., 2015, Belkaid and Harrison,
2017). This has been observed in infant’s skin microbiome and an
early colonization of Staphylococcus aureus being associated with a
lower risk of developing atopic dermatitis, as the immune system
is prepared for this organism that can exacerbate and perpetuate
the disease (Kennedy et al., 2017, Blicharz et al., 2019). S. aureus can
further modulate the immune system through toxin production and
colonization, which initiates leukocyte responses and stimulates
the adaptive and innate immune systems (Niebuhr, et al., 2011,
Nakamura et al., 2013, Nakatsuji et al., 2016). Skin colonization of
Staphylococcus epidermidis elicits similar immune responses, and a
pre-association with this microbe could help defend against certain
fungal and parasitic skin infections (Naik et al., 2012, Naik et al.,
2015). Further investigation of microbe and immune system
interactions will help uncover exact molecular functions and
relationships, which could have future implications in therapy and
treatment of skin diseases (Byrd et al., 2018).
Pathogens, Dysbiosis, and Skin Diseases
The skin microbiome also contributes to human health and

combating infectious diseases in a preventative measure. In addition
to educating the immune system, the commensal microorganisms
that call the epidermis home help to prevent pathogen invasion
by physically taking up niches and secreting certain antimicrobial
compounds. For example, S. epidermidis, which is a part of the
normal microbial flora of the skin, produces antimicrobial peptides
and proteases that selectively targets and inhibits growth of
pathogens such as S. aureus (Cogen et al., 2010, Iwase et al., 2010,
Schommer and Gallo, 2013).
Though many commensal microorganisms on the skin can help to
prevent pathogen colonization, these microbes themselves may be

opportunistic pathogens and can cause an infection when certain
conditions arise. For example, S. epidermidis is a common cause of
infections when transmitted from the skin into the body, usually
through a medical procedure like catheterization (Otto, 2012).
Staphylococci are known for their ability to form biofilms on
medical devices, which make them more difficult to treat (Otto,
2009). The fungi, Candida albicans, is another normal skin resident
that can cause opportunistic infections. It is thought that disruption
of the normal skin flora, through means such as antibiotic therapy,
could induce virulence factor production by the yeast, resulting
in penetration of epidermal tissue and a subsequent case of
candidiasis (Kuhbacher et al., 2017). Other skin diseases associated
with dysbiosis, or various skin pathogens, include atopic dermatitis,
acne, rosacea, psoriasis, and chronic wound healing.
Atopic dermatitis (AD), the most common form of eczema, is a
chronic inflammatory condition of the skin that is caused by a
mutation in several genes, including the fillagrin gene responsible
for encoding a protein that helps maintain epidermal health
(Bierber, 2008). AD is characterized by itchy, dry rashes that become
vulnerable to infection when the skin barrier is breached by
constant scratching, and occurs more frequently in areas that
become lower in microbial diversity due to an altered physiology
(Weyrich et al., 2015). S. aureus colonization is directly related to
disease severity of AD, as it produces virulence factors that disrupt
the integrity of the skin barrier. There is also an increase in
abundance of S. epidermidis, and Clostridium and Serratia species,
and this increase of select species suggests that a lower amount
of microbial diversity plays a role in the progression of AD (Kong
et al., 2012, Oh et al., 2013, Williams and Gallo, 2015). Selectively
targeting pathogens such as S. aureus for treatment of AD proves
to be difficult as the normal microbiota also suffers resulting in
dysbiosis.

Psoriasis is similar to AD in that the disease results in inflamed,

scaly skin plaques that are itchy and painful. This condition is also
associated with altered microbial diversity, and there is an
association with the development of psoriasis and oral
streptococcal infections, though the connection is not exactly
known (Norlind, 1955, Owen et al., 2000). Within psoriatic lesions,
there is an increase Proteobacteria and Firmicutes, a decrease in
Actinobacteria, and specifically a decrease within the genera
Propionibacterium (Gao et al., 2008, Fahlen et al., 2012, Statnikov
et al., 2013). Though there is a decrease in general of microbial
diversity in psoriatic lesions, there hasn’t been any specific
microbial causative agent identified for the disease.
Rosacea is a common chronic dermatosis which primarily
manifests as persisting erythema (redness), telangiectasia (dilated
or broken blood vessels), bulging, swelling, and/or raised patches
in superficial facial skin (Picardo and Ottaviani, 2014). Like other
cutaneous diseases, development of rosacea is linked with skin
microbiome composition, and how those communities influence
skin immune responses. In particular, an increase in Demodex mite
abundance and density is observed in those with rosacea. They
potentially contribute to the disease state through immune system
activation, damaging epithelial tissue, and/or the exposure of
antigenic proteins of bacteria released from their digestive tract
(Forton and Seys, 1993, Georgala et al., 2001, Lacey et al., 2007,
Koller et al., 2011, Casas et al., 2012, Forton, 2012, ). The induced
reactions from microbiome shifts, genetics, and environmental
factors then likely invoke other inflammatory triggers, which

includes overgrowth of certain bacteria like S. epidermidis
(Schommer and Gallo, 2013).
Acne (acne vulgaris) is a skin condition that results from hair
follicles and sebaceous glands that are clogged with oil, bacteria,
and dead skin cells, which creates whiteheads and blackheads.
Propionibacterium acnes is a primary etiological agent in acne; it’s
secretion of lipases, proteases, and hyaluronidases damage skin
pores and induce inflammatory responses (McKelvey et al., 2012).
Although this species of microbe is part of the normal skin
microbiota, there are differences in certain strains of P. acnes that
may explain differential virulence. Some disease-associated strains
also have genes for antibiotic resistance, making treatment options
other than chemotherapy a necessity (Fitz-Gibbon et al., 2013).
Other commensal microorganisms, like S. epidermidis, could
interact with P. acnes and be implicated in acne formation also,
further demonstrating that residents can become pathogenic when
opportunistic conditions arise (Bek-Thomsen et al., 2008, Weyrich
et al., 2015, Dreno et al., 2017).
Chronic skin wounds (duration longer than three months) and
their capacity to heal are also affected by the skin microbiome,
especially in those individuals who are elderly, obese,
immunocompromised, or diabetic (Weyrich et al., 2015, Byrd et al.,
2018). Though the lesions or ulcers may not be initially caused by a
microorganism, their presence, infection, and polymicrobial biofilm
formation can be deleterious to the healing process and cause
further complications (McKelvey et al., 2012, Wolcott et al., 2013).
Analysis of skin wound microbiomes have shown a compilation of
a diverse array of genera, but that microbial diversity is lower as
compared with healthy skin (Gardiner et al., 2017, Kalan et al., 2019).
Perhaps a higher microbial diversity allows for easier elimination of
potential pathogens from the wound and promotes faster healing.
An increase in facultative anaerobes, specifically the genus
Enterobacter, are significant indicators in the persistence of chronic
wounds and their lack of healing, possibly due to their versatile
metabolism (Verbanic et al., 2020). Antibiotic therapy is an option to

eliminate certain bacterial pathogens for chronic wounds, however,
the resulting changes in the microbiome and addressing fungal and
viral constituents may necessitate multiple different treatment
approaches (Price et al., 2009). Further studies are needed in order
to see whether individual therapy and targeted therapeutic
intervention would promote faster healing in cases with chronic
wounds (Kong, 2011, Weyrich et al., 2015, Verbanic et al., 2020).
It is likely that changes in microbial community composition and
the elicited immune response work in combination with host
genetics and other environmental factors to cause various
cutaneous disorders (Weyrich et al., 2015 ). This makes treatment
of these complex conditions difficult, and targeting a few potential
pathogens is rarely successful, especially if it is not known whether
their presence is a cause or effect. So, future therapeutic efforts
may focus on a similar approach to treating gut dysbiosis with
FMT, and as so, use healthy skin microbiota transplants to repair
and stabilize the skin microbiome for patients inflicted with skin
diseases (Williams and Gallo, 2015).
Conclusion
The skin microbiome is incredibly complex, resilient, and has a

strong influence on health and disease. Characterization and
defining a normal skin microbiome could help in future diagnosis
and treatment of disease, although factors like differences in host
genetics, lifestyles, and particular skin locations must be accounted
for. As research and medical technologies advance, it may be
possible to utilize these microbial neighbors on the skin in efforts to
promote better overall health.

• What features of human skin affect the

composition and stability of its microbiome?
• How is the skin microbiome influenced in early life,
and what implications does this have in human
health?
• What diseases are associated with dysbiosis of the
skin microbiome? How do these come about and
what particular microorganisms are associated with
each?
• How do certain members of the skin microbiome
impede the healing process of chronic wounds?

Media Attributions:
• Video 1 – A mixed community of skin

microbiome representatives influences cutaneous
processes by Research Square. Licensed under
• Video 2 – Eczema, Immunity, and the Skin
Microbiome – Heidi Hong by National Human
Genome Research Institute. Licensed under
• Video 3 – Expansion of known skin microbes
could aid skin health research by Research
• Hot Spot Quiz Image – Skin Microbiome by Jane
Ades, NHGRI under Public Domain
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10.1111/j.1469-0691.2012.04001.x

8. The Respiratory
Microbiome
The Respiratory Microbiome
The respiratory microbiome (specifically the lower portions) was

once thought to be nonexistent, as many considered healthy lungs
to be a sterile environment. Like other microbiomes though, the
upper and lower respiratory system are environments rich with
bacteria, fungi, archaea, and viruses, and because of the novelty
of study many of the members in these groups have not been
identified. However, it is known that many of such organisms are not
only responsible for or exacerbate a number of pulmonary diseases,
but that the respiratory microbiome can help to mitigate infection
and influence treatment of certain illnesses (Unger and Bogaert,
2017, Watson et al., 2019).
The human respiratory system begins with the upper respiratory
system; starting with the nose and nostrils, then proceeds to the
nasal cavity, pharynx, and larynx. The lower respiratory system then
starts with the trachea, moves to the bronchi and bronchioles, and
ends with the lungs. Each of these structures and areas have their
own microbial niche which serve various roles in maintaining
health, but also have the potential to be compromised. The
respiratory system is similar to the oral and digestive system, in that
they interact intimately with external stimuli and bring in foreign
matter and microbes during regular biological processes, like
inhalation. These systems also have overlapping parts (e.g. oral
cavity and pharynx), where their respective microbiomes serve
similar roles such as resistance to disruptive environmental factors
and host defense (Dickson et al., 2014, Zaura et al., 2014, Hakansson
et al., 2018). The mucosal surfaces of the respiratory tract also have
The Respiratory Microbiome | 133

a natural healthy biofilm where the resident microbiota reside to
maintain homeostatic functions, however, as an extension of
dysbiosis, these biofilms can become altered and obtain pathogenic
microbes which contribute to respiratory diseases (Hamilos, 2019).
Composition
Immediately after birth the respiratory microbiome becomes

colonized, though the collection of microbes found here during
infancy is not much different than other locations in the body,
and generally reflect the mode of delivery (Dominguez-Bello et al.,
2010). The respiratory microbiome, as well as others, become
differentiated over time when the different species of microbes
adapt and outcompete each other based on their niche, host
genetics, and environmental factors (Bosch et al., 2016).
Colonization additionally depends on microbial immigration,
microbial elimination, and relative reproduction rates of its
members (Figure 1) (Dickson and Huffnagle, 2015). With age, the
respiratory microbiome of each particular individual reflects the
aforementioned factors as well as other lifestyle habits, and so they
can be vastly different between people. Although these respiratory
microbiomes are unique, a core set of microbes can be defined, with
most of them being aerobic or facultatively anaerobic (Stearns et al.,
2015, Hakansson et al., 2018).
Microbial composition also depends on the specific structure or
niche within the respiratory system. The nasal microbiome largely
consists of Staphylococci, Corynebacteria, and Streptococci, which is
most likely due to its similarity and proximity to the skin (Mika et al.,
2015, Shilts et al., 2016). Communities within the paranasal sinuses
are highly diverse and include lactic-acid producing bacteria such
as Lactobacilli, Enterococcus, and Pediococcus, and once in the
nasopharynx the collection of microbes becomes more complex
and favors oxygen-utilizing groups (Abreu et al., 2012, Biesbroek
et. al, 2014, Teo et al., 2015). In the lower respiratory system, the
134 | The Respiratory Microbiome

microbiome resembles a bit of a mix from both the nasal and oral
cavities, with a majority of bacteria from the genera
Streptococcus, Fusobacteria, Pseudomonas, Veillonella,
and Prevotella (Cui et al., 2014, Beck et al., 2015, Dickson and
Huffnagle, 2015, Hakansson et al., 2018).
While much focus of the respiratory microbiome has been on
bacteria (like many other microbiomes), study of the mycobiome
and virome components have revealed their importance in
pulmonary health and disease. Fungal spores are commonly inhaled,
especially in high numbers during peak seasons, and depending on
the particular organism and host immune health, they can cause
infection and respiratory complications (Pashley et al., 2012,
Denning et al., 2014, Nguyen et al., 2015). For healthy people, the
respiratory mycobiome is predominated by environmental fungi
including Aspergillus, Cladosporium, and Penicillium species
(Charlson et al., 2012, van Woerden et al., 2012, Underhill and Iliev,
2014). However, members from these genera and other more well-
known fungal pathogens, like Candida albicans, can be implicated in
respiratory illnesses due to microbial community changes (Nguyen
et al., 2015). The respiratory virome can also modulate various
pulmonary diseases through respective bacterial interactions and
host immune response. For bacteriophage composition, there are
observed differences between healthy individuals and patients
suffering from diseases associated with respective bacterial
infections (Wilner et al., 2009). There is a diverse range of
eukaryotic viruses that make up the normal human and respiratory
virome, with the majority consisting of adenoviruses, herpesviruses
and human papillomaviruses (HPVs), though these may only be
transient and quickly cleared by the immune system or via the
mucociliary escalator (Wilner et al., 2009, Popgeorgiev et al., 2013).
More research needs to be conducted over the normal respiratory
virome to determine its functionality and importance, though
usually more focus is catered to those viruses directly involved in
pathogenesis and dysbiosis.

Figure 1. Ecological determinants of the respiratory microbiome. The
constitution of the respiratory microbiome is determined by three factors:
microbial immigration, microbial elimination, and the relative reproduction
rates of its members. In health, community membership is primarily
determined by immigration and elimination; in advanced lung disease,
membership is primarily determined by regional growth conditions. Adapted
from Dickson et al., 2014.
The normal matrix of organisms in the respiratory system maintain

homeostasis through their complex interactive network with each
other and their host. Pathogens like Streptococcus
pneumoniae and Haemophilus influenzae are actually part of the
resident respiratory microbiome and interact with commensal
microbes, however, they can incite infection when they are not
kept in check due to community perturbation (de Steenhuijsen et
al., 2015). During events of dysbiosis, pathogens and opportunistic
pathogens can contribute to and exacerbate several respiratory
diseases including asthma, chronic obstructive pulmonary disease
(COPD), cystic fibrosis, pneumonia, otitis media, and other acute
infections like influenza.
Asthma is a chronic lung disease that usually develops at an early

age, and its complex etiology makes it difficult to treat. People with
asthma experience inflammation and tightening of their airways
and a production of extra mucus impairs breathing, talking, and
being active. The prevalence of asthma has increased in recent
decades, and a decrease in microbe exposure during youth could
be partially responsible (Ober and Yao, 2011, Prescott, 2013). Proper
development of the immune system is reliant on contact with
microbes, and those individuals who are regularly subjected to a
diverse variety of microorganisms at an early age have a reduced
risk of developing asthma (Ownby et al., 2002, Fujimura et al., 2010,
Fall et al., 2015). Aside from grooming the immune system, the intake
of microbes influences the composition of the mucosal microbiota
that lines the air passageways, which could directly affect asthma
development (Durack et al., 2016). Indeed epidemiologic studies
have shown that the disruption of the microbiome, such as in the
case of antibiotic use during childhood, create a predisposition for
this allergic disease (Khalkhali et al., 2014). Specifically, the increased
presence of genus Haemophilus and other Proteobacteria, as well as
the fungus Aspergillus fumigatus in the respiratory system are
signature of asthma patients (Hilty et al., 2010, Teo et al., 2015,
Urb et al., 2015). These pathogens would normally be cleared by
the immune system or stifled by the resident microbiota, however,
early childhood dysbiosis resulting in hampered immune functions
and/or a discontent microbial consortium in the respiratory system
contribute to the development of this condition.
Chronic obstructive pulmonary disease (COPD) is another chronic
inflammatory lung disease that causes obstructed airflow to the
lungs and is exacerbated by microbial dysbiosis. Emphysema, a
condition where the alveoli are damaged due to cigarette smoke and
other irritants, and chronic bronchitis, a condition characterized
by inflammation of the lining of the airways, are the most common
contributors to COPD. An alteration of the lower respiratory
microbiome causing reduced diversity or the presence of specific
organisms can further aggravate COPD causing a worsening of
symptoms (Huang et al., 2014). Viral infections, like those caused by

the rhinovirus, are commonly detected (in about half the patients)
during COPD exacerbations (Seemungal et al., 2001, Rohde et al.,
2003, Mallia et al., 2011). Also during these bouts, there is an increase
in pathogens from the bacterial genera Haemophilus, Pseudomonas,
and Moraxella and a general shift towards the phylum
Proteobacteria in the respiratory microbiome (Huang et al., 2014,
Millares et al., 2014). Additionally, patients with COPD exhibit a
decline or absence of certain normal lung organisms that could
contribute to mucosal homeostasis and prevention of pathogen
overgrowth, such as Firmicutes (Streptococcus spp.), as well as
Bacteroidetes (Prevotella spp.) (Fukata and Arditi, 2014, Sze et al.,
2015, Hakansson et al., 2018). These compositional changes are at
least partially brought about by current COPD treatments like
steroid and antibiotic usage, and so future therapy may need to take
the respiratory microbiome into further consideration for various
approaches (Wang et al., 2016).
Cystic fibrosis (CF) is a genetic disease that causes normal cellular
secretion of mucus, sweat, and digestive juices to become thicker
and viscous. In the respiratory system, the buildup of sticky mucus
can clog airways, impair lung function, and make breathing difficult.
Bacterial pathogens, like Staphylococcus aureus, Pseudomonas
aeruginosa, and Haemophilus parainfluenzae, tend to grow in the
sputum and can cause infections which worsening CF conditions
(Keogh and Stanojevic, 2018). Their biofilm-forming capabilities also
contribute to the excess mucus production, further causing
complications as infectious agents which can be resilient and
persistent (Høiby et al, 2010, Orazi and O’Toole, 2017). While changes
in the microbiome are not typically responsible for exacerbations
of CF, antibiotic administration is the primary treatment option
to eliminate these pathogens residing in the mucus, however,
prolonged use can result in decreased microbiome diversity and
dysbiosis in the respiratory system as well as other areas (Zhao et
al., 2012, Carmody et al., 2013 Price et al., 2013, Dickson et al., 2014).
Pneumonia is a microbial infection of the lungs that can cause
the alveoli to fill with pus or other liquids, and severe cases can

be life-threatening, especially to children, the elderly, and those
who are immunocompromised (Wardlaw et al., 2006, Chalmers et
al., 2011, Valley et al., 2015). The intensity of the infection is directly
correlated with the microbial load and diversity, so etiologic
determination is important for proper treatment, as the onset of
pneumonia can have bacterial, fungal, or viral origins (Dickson et
al., 2014, Iwai et al., 2014). Viral pneumonia is commonly caused
by influenza or rhinovirus, however, severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) has now also become a major
cause post 2019 (Jain et al., 2015, Zhou et al., 2020, Pettigrew et
al., 2021). Respiratory viral infections are the most common cause
acute respiratory illnesses, and often times, these viral infections
then lead to secondary bacterial infections, like pneumonia, due
to host immune reactions and induced dysbiosis in the respiratory
tract and gastrointestinal system (Hanada et al., 2018). Streptococcus
pneumoniae is the most common culprit for bacterial pneumonia,
and while it regularly resides in the upper respiratory tract, under
dysbiotic conditions this organism can proliferate and spread to the
lower respiratory tract and cause illness (File, 2003, de Steenhuijsen
Piters et al., 2014). In patients with pneumonia, there is a decrease
in many Gram-negative anaerobic bacteria that are apart of the
resident microbiota, some of which are associated with a reduced
risk of hospital-acquired pneumonia and clearance of S.
pneumoniae (Bousbia et al., 2012, de Steenhuijsen Piters et al., 2014,
Krone et al., 2014). Although, changes in the respiratory microbiome
are more apparent in bacterial pneumonia rather than viral, it is
not known whether this alteration in microbial consortia is a cause
or effect of the disease, though it is likely conditional of both
circumstances (Ramos-Sevillano et al., 2019, Pettigrew et al., 2021).


Conclusion
The respiratory microbiome is an up-and-coming avenue for

research, diagnosis, and treatment of several pulmonary illnesses.
While much of its characterization is still in it’s infancy, proper
identification of microorganisms is important for management of
respiratory diseases (Nguyen et al., 2016). As antibiotic resistance
continues to surge, novel approaches are necessary to mitigate
not only respiratory illnesses, but to maintain general health. And
similarly to other microbiome-related treatments, like fecal
microbiota transplant for the gut, eventually oral or aerosol
administered microbiota may be implemented to treat pulmonary
conditions (Huang and Boushey, 2015). Continual research detailing
both core and individual respiratory microbiomes will push the
advancement of these ‘futuristic’ medical motions.
Drag and Drop Quiz
Drag each contributing factor of the respiratory

microbiome composition into the appropriate category.

online here:
• How is the colonization of the respiratory

microbiome influenced, and what factors affect its
composition over time?
• What factors of the respiratory microbiome affect
the development of asthma?
• Which microorganisms are implicated in COPD and
periods of exacerbation? How does dysbiosis
contribute to this chronic condition?
• Why do you think a greater change is observed in
the respiratory microbiota in patients with bacterial
pneumonia versus viral?

Media Attributions
• Figure 1 – Ecological Determinants of the

Respiratory Microbiome by Dickson and
Huffnagle, 2015. Licensed under Creative
Commons: Attribution 4.0 International (CC BY
4.0) License https://creativecommons.org/
licenses/by/4.0/
• Video 1 – The Lung Microbiome: Challenging
Old Paradigms about Microbes by National Human
Genome Research Institute. Licensed under
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S0140-6736(20)30566-3

9. The Vaginal Microbiome
The Vaginal Microbiome
The vaginal microbiome is a reproductive organ-specific niche that

harbors a unique collection of microorganisms that are important in
a variety of aspects to human health. This microbial community has
significance in maintaining vaginal homeostasis, protecting against
urogenital infections, host immunity, and reproductive capacities.
In recent years, next generation sequencing (NGS) techniques have
been employed to classify the vaginal microbiome into community
state types (CSTs), or vaginotypes, based upon composition, which
can enhance epidemiological studies to make better associations
between the microbiome and host-vaginal health (France et al.,
2020, Mancabelli et al., 2021).
Composition and Role
The vaginal microbiome is composed of over 200 different bacterial

species, though it is primarily dominated by members from
Lactobacillus genus (Ma et al., 2012, Auriemma et al., 2021). The
Lactobacilli protect the vagina from potential pathogen invasion
through their fermentation of vaginal epithelial cell-produced
glycogen into lactic acid, which lowers vaginal pH, as well as their
production of various antimicrobial compounds, and resource/
space competitive inhibition (Boskey et al., 2001, Amabebe and
Anumba, 2018, Chee et al., 2020, Jang et al., 2019). Though, the
bacteriome is usually the emphasized feature of the vaginal
microbiome, it also harbors protists, fungi, archaea and viruses,
with each occupying their own niche in the normal healthy network
(Belay et al., 1990, Bradford and Ravel, 2017, Happel et al., 2020,
The Vaginal Microbiome | 153

Chacra and Fenollar, 2021). The composition isn’t always fixed
however, as factors like the menstrual cycle, hormone fluctuation,
sexual partners, hygiene, genetics, age, the environment, drug use,
and other aspects of lifestyle can affect the microbial makeup
(Aagaard et al., 2012, Fettweis et al., 2014, Hyman et al., 2014, Zapata
and Quagliarello, 2015, Martin and Marrazzo, 2016, Diop et al., 2019,
Chacra and Fenollar, 2021).
As mentioned earlier, using 16S rRNA sequencing, the vaginal
microbiome has been categorized into CSTs and allowed for deeper
analysis, where comparisons of the consortia within these
classifications can be used to make associations with host and
vaginal health. There are 5 main CSTs each predominated by a
particular species of Lactobacilli, except for group IV, which has
been further dissected into additional subgroups: CST I—L.
crispatus dominated, CST II—L. gasseri dominated, CST III—L.
iners dominated, and CST V—L. jensenii dominated (Ravel et al.,
2011, France et al., 2020, Chacra and Fenollar, 2021, Mancabelli et
al., 2021). CST IV is not dominated by any particular species, and
contains a mixture of both strict and facultative anaerobes including
Gardnerella, Atopobium, Lactobacillus, Bifidobacterium, etc., where
certain controversial subgroups (e.g. CST IV-A, CST IV-B, CST IV-C,
CST IV-D, CST IV-G, etc.) have various combinations depending on
the study (Gajer et al., 2012, Albert et al., 2015, France et al., 2020,
Mancabelli et al., 2021). Each of the five groups has correlations with
vaginal pH, microbial colonization and biodiversity, as well as host
characteristics such as pregnancy, ethnicity, and age, though it is
not always absolute (France et al., 2020, Mancabelli et al., 2021). With
reproducible organization of the vaginal microbiome, connections
can be made with medical conditions, which could benefit
treatment and diagnosis of vaginal diseases and associated affairs.
154 | The Vaginal Microbiome

As with other microbiomes associated with the human body,

disturbance of the resident vaginal microbiome can result in
complications of health. Many diseases of the urogenital tract such
as bacterial vaginosis (BV), urinary tract infections (UTIs), yeast
infections, and several sexually transmitted infections (STIs) are
caused by pathogenic microbes associated with dysbiosis (Taha et
al., 1998, Donders et al., 2000, Wiesenfeld et al., 2003; Lai et al.,
2009, De Seta et al., 2019, Mancabelli et al., 2021). Normally, the
local flora maintains homeostasis, however, changes in composition
provide opportunistic pathogens a window to proliferate and
invade.
Bacterial vaginosis, associated with CST IV, stems from a
perturbance of the vaginal flora, specifically a decrease in
Lactobacilli and an increase in other microbes like Gardnerlla
vaginalis, Atopboium vaginae, Ureaplasma urealyticum, and others
that are only usually found in low numbers (Gajer et al., 2012,
Margolis and Fredricks, 2015, Onderdonk et al., 2016, Zozaya et
al., 2016). This condition can be asymptomatic in up to half the
women with BV, and in the others can be diagnosed by observed
changes in vaginal discharge or by the Nugent scoring system which
utilizes a Gram stain (Figure 1) (Nugent et al., 1991, Amsel et al.,
1983, Schwebke, 2000). While not exactly considered a sexually

transmitted disease, bacterial vaginosis is commonly associated
with certain sexual practices, though other factors such as hygiene,
nutrition, intrauterine devices, hormonal changes, and certain
comorbidities can contribute to susceptibility (Avonts et al, 1990,
Calzolari et al., 2000, Neggers et al., 2007, Verstraelen et al., 2010,
Zabor et al, 2010, Margolis and Fredricks, 2015). Women with this
disease also have a higher risk to contract sexually transmitted
infections, and it can be linked to reproductive complications and
poor infant health (Wiesenfeld et al., 2003, Prince et al., 2015, Chacra
and Fenollar, 2021). Typically, antibiotics targeting anaerobic
bacteria are administered to treat BV, however recurrence is
common, likely due to the antimicrobial-resistant nature of biofilms
formed by the pathogens and/or regular exposure to external
reservoirs (Swidinski et al., 2008, Oduyebo et al., 2009, Marrazzo
et al., 2012, Bradshaw and Sobel, 2016). Probiotics, specifically
containing L. crispatus, may be the better option, and vaginal
microbiota transplantation from a healthy donors is a promising
treatment course of action (Hemmerling et al., 2010, Ma et al., 2019).
Figure 1. Examples of Gram-stained slides from women with (right, Nugent

score = 9) and without (left, Nugent score = 1) BV. In women with a
lactobacilli-dominated microbiome (left), long purple rods are the main
morphotype. In contrast, women with BV tend to have a lot more bacteria
overall, with a significant fraction that do not stain as Gram-positive (i.e.
pink). Morphotypes in BV tend not to be long Gram-positive rods. Although
not part of the Nugent scoring system, women with BV tend to have large
conglomerations of bacteria associated with epithelial cells as seen in the
upper right quadrant (Lewis and Gilbert, 2020).

Urinary tract infections are a common problem, especially for
females, which are disproportionately affected. This infection is
most likely to occur in the urethra or bladder (though in severe
cases the kidneys can be affected), which causes pain in the pelvic
area and during urination, the frequent urge to urinate, and the
presence of blood in urine (Lee and Neild, 2007, Sheerin, 2011,
Hooton, 2012). The vaginal microbiome, or more specifically some
normal residents, like Escherichia coli, can move to the urogenital
tract and cause issues resulting in a UTI, which often occurs via
sexual activity (Nicolle et al., 1982, Foxman, 2014, Stapleton, 2016,
Lewis and Gilbert, 2020). Other fastidious microorganisms, like
those abundant in women experiencing BV, contribute to a higher
risk of contracting an infection in the urinary tract as compared
with those not suffering from dysbiosis in the vagina (Hillebrand
et al., 2002, Sumati and Saritha, 2009). In these cases, the vaginal
opening may harbor potential uropathogens (Figure 2) and transient
exposure to the urinary tract could prompt colonization or other
reactions (e.g. immunomodulation) that results in a UTI (Lewis and
Gilbert, 2020). Antibiotics are traditionally used to treat UTIs,
however probiotics and estrogen administration could help to
restore the Lactobacillus colonization and protect against
complicated and recurrent infections (Raz and Stamm, 1993,
Eriksen, 1999, Prais et al., 2003, Stapleton et al., 2011, Tan and
Chlebicki, 2016, Lewis and Gilbert, 2020).

Figure 2. Schematic illustrating vaginal bacteria with potential to impact the
urinary tract. The vagina can serve as a reservoir for several bacterial species
known to be causes of UTI (E. coli, GBS, Staphylococcus) as well as
underappreciated potential uropathogens (G. vaginalis, Aerococcs
Ureaplasma) that can cause UTI and have been associated with urological
conditions such as urgency incontinence and “sterile” pyuria (Lewis and
Gilbert, 2020).
Yeast infection of the vaginal region caused by Candida species,

also known as vulvovaginal candidiasis (VVC), is a common
condition with severe symptoms and a high recurrence rate
(Oerlemans et al., 2020). Those affected experience vaginal itchiness
or soreness, dyspareunia (painful intercourse), abnormal vaginal
discharge, redness, swelling, and thinning of the vaginal wall (Chew
and Than, 2016, Oerlemans et al., 2020). While it is the second-most
common infection of the vagina behind BV, this disease primarily

affects premenopausal women with a low vaginal pH value under
4.5 (Kim and Park, 2017, Gupta et al., 2019). The exact cause of
VVC isn’t exactly clear, though it is thought it can come as a result
of microbiome dysbiosis induced by prolonged antibiotic usage,
which allows various Candida species to overgrow and establish an
infection (Goldacre et al., 1979, Mitchell, 2004, Peters et al., 2014, van
de Wijgert and Verwijs, 2020). Traditionally, antifungal medication
has been used to treat VVC, however administration of probiotic
vaginal microbes may be more prudent as their mechanisms of
pathogen colonization and biofilm formation inhibition are more
effective to prevent disease recurrence (Petrova et al., 2016,
Tachedjian et al., 2017, Allonsius et al., 2019, Oerlemans et al., 2020,
van de Wijgert and Verwijs, 2020).
Individuals with vaginal microbiome dysbiosis characterized by a
decrease in abundance of Lactobacilli species (i.e. BV) are at a higher
risk of contracting sexually transmitted infections, such as those
caused by Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia
trachomatis, and Mycoplasma genitalium (Martin et al., 1999,
Cherpes et al., 2003, Peipert et al., 2008, Brotman et al., 2010,
Molenaar et al., 2018, De Seta et al., 2022). Infections caused by
these organisms usually result in symptoms similar to other genital
infections such as vaginal itching, pain, unusual discharge, rash,
etc. which like other conditions (e.g. vaginitis), result in decreased
epithelial integrity and can exacerbate pathogen invasion (Miller
and Shattock, 2003, Greenbaum et al., 2019). In those individuals
with BV and non-inflamed tissue, the increased risk for STIs could
be due to the negative effects dysbiosis-related bacteria have on
the innate immune system (Murphy and Mitchell, 2016, Liebenberg
et al., 2017). Similarly, contraction of sexually transmitted viral
infections like human immunodeficiency virus (HIV), herpesviruses,
and human papillomavirus (HPV) are frequently associated with
vaginal dysbiosis, immunomodulation, and disruption of the
epithelial barrier (Sewankambo et al., 1997, Borgdorff et al., 2016,
Siqueira et al., 2019, Torcia, 2019, De Seta et al., 2022). While there
is correlation between vaginal dysbiosis and STIs, the protective

mechanisms of the resident vaginal microbiota are unknown and
still need to be uncovered (De Seta et al., 2022).

Reproduction, Pregnancy, and Infant health
The vaginal microbiome has further implications in host immunity,

fertility, pregnancy, spontaneous preterm birth, and infant health
(Anahtar et al., 2015, Fettweis et al., 2019, Gupta et al., 2020, Xu
et al., 2020). Indeed, there are a multitude of factors that affect
reproduction like age, genetics, hormone levels, fallopian tube
blockage, menstrual cycle, and vaginal pH, however only recently
has the vaginal microbiome been studied for its association with
various fertility factors (Xu et al., 2020, Fan et al., 2022).
Changes in the resident vaginal flora and infections by certain
pathogens can cause complications for reproductive health and
pregnancy. For example, infection by Group B Streptococcus (GBS)
has been associated with a decline in ovarian function, pregnancy
loss, preterm delivery, and is the leading cause of bacteremia and
meningitis in newborns (Zaleznik et al., 2000, Phares et al., 2008,
Kolter and Henneke, 2017, Tazi et al., 2019, Xu et al., 2020). The
vaginal microbiome of pregnant women is less rich and diverse than
non-pregnant individuals, likely caused by changes in sex hormone
levels (Farage et al., 2010, Aagaard et al., 2012). These alterations
can cause shifts in the vaginal microbiome that could then result in

infection and a risk of preterm or spontaneous labor (Wylie et al.,
2018, Fettweis et al., 2019, Feehily et al., 2020, Gupta et al., 2020).
Infants become introduced to the microbial world via their
mother, and predominately right after birth where vaginal versus
cesarean delivery has a great impact on composition (Chu et al.,
2017). However, exposure may happen earlier in utero, as this
environment may not be as sterile as once thought as some studies
have shown the presence of microbes in the placenta and amniotic
fluid (Aagaard et al., 2014, Collado et al., 2016, Kolter and Henneke,
2017). The establishment of a newborn’s microbiome has profound
effects on the development of immunity and metabolism as well as
the onset of diseases like atopic dermatitis and obesity in later life
(Rautava et al., 2012, Collado et al., 2016, Ta et al., 2020).
Conclusion
The vaginal microbiome is a complex community which affects

many facets of human health and disease including urogenital,
reproductive, immune, and infant. Characterization of the vaginal
flora has allowed categorization into community state types which
can help to predict and diagnose disease states. Within so, these
dynamic changes that occur under various conditions produce a
unique fingerprint for the vaginal microbiome which can be
analyzed and potentially treated in a specific manner (Ceccarani et
al., 2019, Lagenaur et al., 2020, Abou Chacra and Fenollar, 2021).
While antibiotic administration has its benefits, novel approaches
using targeted application of probiotic microbiomes (e.g. a gel
containing specific Lactobacilli species) could help in treating
diseases associated with vaginal dysbiosis by restoring those
disrupted communities, as well as alleviating certain negative
consequences of chemotherapy (Pino et al., 2019, Lagenaur et al.,
2020 Oerlemans et al., 2020). More in-depth and continued
research of the vaginal microbiome is necessary to illuminate the
interactions and connections of it members to human health.

Drag and Drop Quiz
Drag the species of bacteria that dominates each vaginal

community state type.

online here:

• Explain vaginal microbiome CSTs. Why are they

important?
• Which CST is associated with BV, and which genus
is typically absent?
• How does BV contribute to the development of
other conditions like UTIs, VVC, and STIs?
• How does the vaginal microbiome influence
reproduction?
• Where does a newborn acquire its microbiome, and
what factors affect the composition?
• What alternatives to antimicrobials are promising
for the treatment of vaginal microbiome associated
diseases?
Media Attributions
• Video 1 – VALENCIA: A nearest centroid

classification for vaginal microbial
communities by Research Square. Licensed under

• Figure 1 – Examples of Gram-stained slides from
women with (right, Nugent score = 9) and without
(left, Nugent score = 1) BV by Lewis and Gilbert,
2020. Licensed under Creative Commons
• Figure 2 – Schematic illustrating vaginal
bacteria with potential to impact the urinary tract
by Lewis and Gilbert, 2020. Licensed under
Creative Commons Attribution 4.0 License
• Video 2 – Beyond bacterial vaginosis: vaginal
lactobacilli and HIV risk by Research
• Video 3 – A temporal model of cervicovaginal
microbiota identifies targets to promote
reproductive health by Research Square. Licensed
License https://creativecommons.org/licenses/
by/3.0/
References
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10. Mental Health and
Multi-Microbiome
Interactions
Mental Health and Multi-Microbiome

Interactions
It may seem obvious that localized microbiomes are responsible

for diseases related to their respective areas (e.g., IBD and the gut
microbiome or AD and the skin microbiome), however, it is a
fascinating phenomenon that various microbiomes can affect each
other and influence health in different parts of the body. Even more
captivating is the link between certain microbiomes and mental
health conditions presuming the brain is devoid of microbes.
These links between microbiomes are referred to as axes, and
initial connections began with the most well-studied microbiome,
the gut. Since many gut-derived microbial-produced molecules and
compounds are spread through the bloodstream, associations with
various organs are formed, such as the gut-brain axis, gut-skin
axis, gut-lung axis, or a combination of multiple; gut-brain-skin
axis. There are likely overlapping connections between all microbial
components that form the human holobiome, thus teasing apart the
exact members and their functions is a dutiful task.
Gut Interaction with Other Microbiomes
As mentioned earlier, the dissemination of molecules and

compounds from the gut to the rest of the body creates an
interconnected highway affecting most, if not all, parts of the body.
180 | Mental Health and Multi-Microbiome Interactions

Certainly, juxtaposed regions such as the oral cavity and the
proximal portions of the GI tract have a relationship between their
microbiomes, which it termed the gut-oral axis (GOA). These linked
microbiomes have been shown to have immunomodulatory roles
in the development of rheumatoid arthritis (RA) and osteoarthritis
(OA) under dysbiosis, where the abundance of oral Porphyromonas
gingivalis and intestinal Prevotella copri could be responsible (Drago
et al., 2019, du Teil Espina et al., 2019). It has also been proposed that
dysbiosis of the gut-oral microbiome axis is implicated in cirrhosis
of the liver through pathogen invasion, resultant systemic
inflammation, and impaired immunity and liver function (Acharya
et al., 2017). Even gastrointestinal conditions like IBD and cancer of
the colon, liver, and pancreas are linked with both the gut and oral
microbiome dysbiosis, further demonstrating this strong interorgan
connection to human health (Park et al., 2021).
The gut and the skin microbiomes are also linked via the gut-skin
axis (GSA), and as the primary interface to the environment, they
have major roles in physiological health (Figure 1). Dysbiosis of the
gut-skin microbiome axis has influence on both GI and cutaneous
disorders such as IBD, celiac disease, atopic dermatitis, psoriasis,
acne, and other dermatologic issues, where each of these diseases
can be associated with the ‘partner disease’ (i.e. a disease in one
system linked to a disease in another system) (O’Neill et al., 2016,
Salem et al., 2018, De Pessemier et al., 2021). Though crosstalk is
bidirectional, more research seems to have focused on skin
disorders and homeostasis as a result of gut microbiome health,
where diet, immunomodulation, intestinal permeability, and
metabolite secretion are major contributing factors (De Pessemier
et al., 2021). However, there is evidence that Mallassezia restricta, a
fungal member of the skin microbiota, is associated with Crohn’s
disease and can exacerbate colitis (Limon et al., 2019, Sinha et al.,
2021).
Mental Health and Multi-Microbiome Interactions | 181

Figure 1. Inflammatory and microbial influences between the gut and skin for
a healthy state (left) and a dysbiotic state (right): The intestinal and epidermal
barriers are connected through the systemic circulation (blood and lymph)
and are visualized here together in a simplistic manner. The dysbiotic state is
characterized by an impaired gut barrier (imbalance in gut microbiome,
reduced mucus layer, reduced IgA secretion, barrier disruption, intestinal
permeation into the bloodstream, and gut inflammation) and an impaired
skin barrier (imbalance in skin microbiome, reduced human and microbial
antimicrobial peptides (AMP) production, skin rashes/thickening/lesions, and
skin inflammation). Gut and skin dysbiosis are connected through an immune
imbalance (Th2 skewing in this example), whereas crosstalk can be
bidirectional (De Pessemier et al., 2021).
The gut-lung axis (GLA) has roles in the regulation of immunity

and the development of various respiratory diseases. (Frati et al.,
2019). The respiratory system and GI tract are connected by the
mesenteric lymphatic system, where intact or fragmented microbes
and their metabolites enter systemic circulation after passing
through the intestinal barrier and can migrate to the pulmonary
system to modulate immune responses (Enaud et al., 2020). The
pathophysiology of diseases such as atopy and asthma are
complicated and can be contributed to a variety of factors, however,
there is evidence that dysbiosis of the gut microbiome contributes

to the development of asthma, particularly in youths who exhibit a
decrease in Lachnospira and increase in Clostridium spp. (Penders
et al., 2007, Watson et al.,2019). Gut dysbiosis is also involved in
chronic obstructive pulmonary disease (COPD) exacerbation, where
fiber deficiency in an individual’s diet can contribute to chronic
inflammation. Metabolism of fiber by gut microbes produces anti-
inflammatory short-chain fatty acids (SCFAs), which could reduce
inflammation both systemically and in the lungs, and so targeted
dietary intervention for these patients may be a viable treatment
addition for COPD and other respiratory diseases associated with
inflammation like COVID-19 (Li et al., 2018, Vaughan et al., 2019,
Allali et al., 2021). Moreover, SCFAs also have an important role in
the defense against secondary infections in those afflicted by viral
respiratory infections, further demonstrating the importance of gut
microbiome health in connection with the GLA (Sensio et al., 2020).
Like other axes, immunomodulation by the gut microbiome also
has influence on the gut-vagina axis (GVA), though it is less
extensively studied. One promising avenue of treating vaginal
diseases, specifically cervical cancer associated with human
papilloma virus (HPV), is the use of mucosal lactic-acid bacteria
(LAB)-based vaccines to modulate the gut microbiome. The
approach could work as a prophylactic or for direct therapy, and
be more easily administered via an oral route instead of parenteral
(Taghinezhad-S, et al., 2021). Endometriosis and infertility are also
disorders associated with sex hormone levels and inflammation,
which once again are influenced by the gut microbiome
composition and state. Specifically, there are some members in
the gut who can affect the levels of circulating estrogen through
metabolic processes and an altered state could lead to increased
risk or symptom severity of these disorders (Salliss et al., 2022).
Other female reproductive-associated diseases such as polycystic
ovarian syndrome (PCOS), also associated with gut dysbiosis, could
be ameliorated by diet modification and restoration of gut
homeostasis. Here, the use of flaxseed oil could increase diminished

levels of SCFAs observed in those with PCOS and protect against
inflammation characteristic of the disease (Wang et al., 2020).
The maternal microbiome, including gut, vagina, and breast milk,
can greatly influence the colonization and health of the infant after
birth, but also affect the fetus prior to delivery. Fetal immune
development is most likely influenced in the womb from
translocation of maternal gut microbes and/or their metabolites
across the placental barrier or ingestion of amniotic fluids (Walker
et al., 2017, Nyangahu and Jaspan, 2019). Post-reproduction, the
infant gut microbiome is continually developed through initial diet,
and more specifically vertical transmission of the contents in the
mother’s breast milk which includes its own unique microbiome
(O jo-Okunola et al., 2018, O jo-Okunola et al., 2019, Quin et al., 2020).
The human milk microbiome promotes infant gut colonization of
probiotic strains which have roles in programming the immune and
metabolic systems, as well as anti-infective, anti-allergic, and anti-
tumor properties (Heikkila and Saris, 2003, Olivares et al., 2006,
Lara-Villoslada et al., 2007, Civardi et al., 2015, Hassan et al., 2015,
Walker and Iyengar, 2015, Boix-Amoros et al., 2016, O jo-Okunola et
al., 2018).

Mental Health Axes
The thought of microorganisms controlling aspects of cognitive

function in their host, especially humans, is fascinating to say the
least. While connections between other microbiomes, organ

systems, and diseases may be less surprising, the microbial link
to psychiatric, neurodevelopmental, age-related, and
neurodegenerative disorders is very much intriguing. The human
microbiome can communicate with the brain in a variety of ways;
through the immune system, metabolism, endocrine system,
circulatory system, and the nervous system, where microbes, their
induced immune response, and their metabolites such as short-
chain fatty acids, branched chain amino acids, and peptidoglycans
are involved (Figure 2) (Liang et al., 2018, Cryan et al., 2019, Olsen
and Hicks, 2019, Hadian et al., 2020, Bear et al., 2021). Several local
microbiomes each contribute affects to the mental state, and
whether it is gut-, skin-, oral-, lung-, etc. derived can dictate roles
in various disorders. This is not just a one-way street though, as
microbes around the body can alter mental states but also be
affected by governance of the brain in a bi-directional manner (Ma
et al., 2019).

Figure 2. The proposed mechanisms of the microbiome-gut-brain axis (MGBA)
are complex and intertwined. Emerging research shows that psychological
stress interacts not only directly with the brain and mood, but also with many
of the MGBA mechanisms thought to contribute to changes in mood with
alteration of the gut microbiota. Solid lines indicate strong evidence of an
effect, and dotted lines show proposed mechanisms with limited but emerging
evidence. Abbreviations: HPA; Hypothalamic-Pituitary-Adrenal (Bear et al.,
2021).
Gut-Brain
Similar to other types of microbiome and organ interactions, the

gut’s role in mental health is central and most well studied, which
makes sense as it is the largest repository of microorganisms
associated with the human body. The gut-brain axis (GBA; and gut

plus essentially every other microbiome and brain axis, e.g., gut-
skin-brain) is implicated in a number of cognitive functions and
disorders including autism, anxiety, depression, stress, pain
sensitivity, learning capacities, memory loss, moods and emotions,
behavior (dietary, social, and reproductive), schizophrenia,
Parkinson’s disease, and Alzheimer’s disease (Desbonnet et al., 2013,
Stumpf et al., 2013, Dash et al., 2015, Gareau, 2016, Luczynski et
al., 2016, Hoban et al., 2017, Liang et al., 2018, Manderino et al.,
2017, Nishida and Ochman, 2017, Vuong et al., 2017, Cowan et al.,
2018, Cryan et al., 2019, Bear et al., 2021, Narengaowa et al., 2021).
The gut microbiome actually develops in sync with the brain and
psychology, and disturbances during different stages of growth can
result in the onset of different diseases (Figure 3) (Borre et al., 2014,
Gur et al., 2015, Sampson and Mazmanian, 2015, Dinan and Cryan,
2016, Luczynski et al., 2016, Sharon et al., 2016, Kundu et al., 2017,
Vuong et al., 2017, Carlson et al., 2018, Liang et al., 2018).
Figure 3. The gut-brain, brain, and mentality develop almost synchronously

throughout the lifespan. The gut-brain, brain, and mentality undergo similar
developmental patterns; all three are susceptible to several factors that
influence the gut microbiota. Myelination, intestinal length, and the gut
microbiota develop almost synchronously. Diet plays an important role in the
maturation of the gut-brain and brain, and mentality is regulated by the
development of the brain and gut-brain. Microbiota disruption at different
stages is likely to increase the incidence of different mental disorders (Liang et
al., 2018).

Early perturbation of the gut microbiome during the post-natal
period, and even within the womb, can especially increase
susceptibility to developing mental disorders since these are critical
stages for development of the gut-brain axis and mind (Borre et al.,
2014, Gur et al., 2015, Diaz Heijtz, 2016, Mika et al., 2016, Slykerman
et al., 2016, O’Mahony et al., 2017, Liang et al., 2018). Even up through
senescence, an abnormal gut microbiota is linked with several
mental disorders, though the good news is that they can be
remedied or improved by returning the gut to a homeostatic state.
As the prevalence of mental disorders and neurological diseases
have been steadily increasing, healing mental health by exploring
and implementing options such as fecal microbiota transplant, diet
intervention, probiotics, prebiotics, and psychobiotics are a must
(Cryan and Dinan, 2012, Dinan et al., 2013, Liang et al., 2015,
Pirbaglou et al., 2016, He et al., 2017, Kang et al., 2017, Mika et al.,
2017, Bruce-Keller et al., 2018, Liang et al., 2018, Yang et al., 2018,
Kesika et al., 2021, Margolis et al., 2021).
There are several ideas as to why there has been an increase
in mental and neurological disorders in relation to the human
microbiome. The “Gut Microbiota” hypothesis suggests this
escalation is a direct result from gut microbiome dysbiosis due to
factors of modern society such as diet, antibiotics, and stress (Liang
et al., 2018b). The “Old Friends” hypothesis proposes that the co-
evolution of early humanity with its microbiota in a less civilized and
more natural ecosystem promoted the development of a stronger
immune system, but now changes in lifestyle and environment leads
to weaker immunity and subsequent disorders (Strachan, 1989, Rook
and Lowry, 2008, Kramer et al., 2013, Rook, 2013). Lastly, the “Leaky
Gut” theory implies that damage to the mucosal barrier of the gut
increases intestinal permeability, which allows biomolecules and
microorganisms to gain access various parts of the body that they
normally couldn’t, and thereafter cause disease (Leclercq et al., 2012,
Smythies and Smythies, 2014, Kelly et al., 2015, Potgieter et al., 2015,
Slyepchenko et al., 2017, Liang et al., 2018). All of theses propositions
are clear in their indication that the gut microbiome and its part

in immune development has a definite role in the functioning of
the mind, though the exact extent and mechanisms remain to be
discovered.

Other Microbiomes and Mental Health
The majority of human microbiome interaction with regards to

human health usually includes the gut to some degree, though there
are some questions as to whether certain microbiomes create their
own axes with the brain.
The oral microbiome can potentially get direct or indirect access
to the brain through the olfactory tract, or via the circulatory
system where blood can transport microbes to the blood-brain
barrier (BBB), perivascular spaces, and circumventricular organs
(Figure 4) (Olsen and Singhrao, 2015, Ranjan et al., 2018, Olsen and
Hicks, 2019). Microbial access to the bloodstream can commonly
occur during dental procedures, and pathogen invasion of the brain
can impact neuro-immune activity and inflammation (Olsen, 2008).
These oral-brain axis-derived infections could contribute to mental
disorders such as Alzheimer’s disease (AD), dementia, Down’s
syndrome, bipolar disorder, and autism spectrum disorder (ASD;
which can lead to oral dysbiosis) (Ilievski et al., 2018, Olsen and
Hicks, 2019, Maitre et al., 2020). Oral dysbiosis can also promote
the development of ASD by affecting the metabolome and thus can
create a troublesome positive feedback loop (Mussap et al., 2016,
Wang et al., 2016).

Figure 4. Direct and indirect mechanisms of infecting the brain. In the direct
mechanism, the oral cavity infects the olfactory tract, and the olfactory nerve
transfer the bacteria to the brain. In other mechanisms, bacteria inside the
mouth infect the blood and find their way via blood, blood–brain barrier
(BBB), perivascular spaces and circumventricular organs to the brain (Olsen
and Hicks, 2019).
Concerning the skin-brain axis, dysbiosis of the skin microbiome

and chronic wounds can elicit systemic effects and induce neural
responses that eventually affect the central nervous system (Figure
5) (Hadian et al., 2020). Chronic wounds exhibit persistent
inflammation and can have various etiologies including diabetic foot

ulcers (DFU), venous ulcers, arterial ulcers, pressure ulcers, surgical
wounds, and other traumatic wounds (Green et al., 2014, Renner
and Erfurt-Berge, 2017, Bui et al., 2018, Pedras et al., 2018, Hadian
et al., 2020). In these cases, a compromised skin barrier can allow
pathogens and their derivatives to enter the bloodstream, and if
the skin is in a dysbiotic state, then abundant pathogens such as
Staphylococcus aureus and Pseudomonas aeruginosa can amplify this
effect by inducing epithelial permeability (Roy et al., 2014, Basler
et al., 2017). These microbes, metabolites, pro-inflammatory
mediators, and other constituents circulate within the blood,
potentially induce permeability in the blood-brain barrier (BBB),
and eventually reach the brain to cause disorders such as wound-
associated depression, anxiety, and other cognitive disorders (Wang
et al., 2011, Zhang et al., 2015, Hadian et al., 2020). The skin-brain axis
acts in a bi-direction manner too, with studies showing connections
between certain mental disorders like post-traumatic stress
disorder, and skin diseases associated with dysbiosis like psoriasis,
chronic urticaria (hives), and atopic dermatitis (Gupta et al., 2017,
Beri, 2018).
Figure 5. The chronic wound with its microbiota induces localized

inflammatory alterations that can result in systemic effects by increasing both
epidermal and vascular permeability and activating neurons to generate
nocioceptive signals—all ultimately culminating in central nervous system
effects. Red: Proposed mediators. Yellow: Proposed downstream events. BBB:
blood brain barrier, CNS: central nervous system, PAMP: pathogen associated
molecular pattern, TLR: toll like receptor (Hadian et al., 2020).

The lung-brain axis connects pulmonary microbes to
neurodegenerative disorders and behavior characteristics, and
though not much research has focused on this specific link, there
are indications that air pollution can be a trigger. Pollutants in
the air come from a variety of sources including engine emissions,
coal combustion, biomass burning, and secondary photochemical
products, such as ground level ozone (O3) (Mumaw et al., 2016).
These compounds can be a source of chronic neuroinflammation
and persistently affect microglial cells in the central nervous
system, which in turn can increase risk of diseases such as
Alzheimer’s disease, Parkinson’s disease, and autism, as well as elicit
a decline in cognitive function in the elderly (Power et al., 2011,
Wellenius et al., 2012, Roberts et al., 2013, Volk et al., 2013, Heneka
et al., 2014, Jung et al., 2015, Kirrane et al., 2015, Mumaw et al.,
2016). It is likely that air pollution also negatively affects the lung
microbiome, and dysbiosis could further aggravate immune
responses and cognitive disruption (Mousavi et al., 2021, Whiteside
et al., 2021).
Is there a brain microbiome?
That is, are there resident microbes residing with a living brain?
With many neurodegenerative and neuropsychiatric diseases
lacking a clear etiology, determining any and all potential
associations could help in therapeutic efforts (Link, 2021). The
healthy brain is an assumedly sterile environment, though the same
was once thought about other organs like the lungs. Interestingly,
one study found evidence for the existence of viable bacteria in
the human brain. Here, researchers were interested to see whether
microbial invasion accompanied damaged brains observed in HIV/
AIDS. After sequencing total RNA from cerebral white matter, they
found bacteria and phage sequences in both experimental and
control brain samples, which was further validated by 16S rRNA gene

target amplification and in situ staining (Branton et al., 2013, Link,
2021).
If there were resident microbes in the brain, they would certainly
be at much lower abundances as compared with other regions such
as the gut or the oral cavity. Though there has been much interest
in characterizing the presence of pathogens in unhealthy brains
for disorders like Alzheimer’s disease, which is an arduous feat in
itself, finding residents in a healthy state could prove even more
difficult (Zhan et al., 2016, Alonzo et al., 2018, Dominy et al., 2019,
Link, 2021). That is, identification approaches could easily miss novel
or fastidious microorganisms, and contamination is hard to avoid.
Furthermore, a wide-range of controlled and unbiased studies are
necessary to catalogue a possible brain microbiome, however,
completing this task in humans brings about ethical implications
and sampling limitations (Link, 2021). It is fascinating to consider the
prospect of symbiotic brain microorganisms, and how they could
influence our health or who we are.
Conclusion
Mental faculties contribute greatly to human health, and therefore

characterizing the involvement of the human microbiome is of
substantial importance. Many of these select microbiomes overlap,
and the disorders associated with each are likely entangled in a
complex array. Brain functions are extremely complex as is, and
piecing together the microbial puzzle within their reach further
complicates matters. However, there is great optimism in
considering the human microbiome as a source or target for
advanced therapy to resolve mental disorders and other diseases,
which could drastically change the way we think about medicine.

Discussion Concept Mapping
With a partner or as a class, connect two or more

microbiomes using a concept map. Explain how and/or
why the various microbiomes are linked (e.g., disease cause
vs. effect, direct vs. indirect, pathways, consortia
translocation, factors affecting composition, etc.).
• In what ways does the gut microbiome connect

with other microbiomes and regions of the body?
How does it influence the development of diseases in
these areas?
• How could early use of antibiotics influence the
development of asthma in children?
• How could gut microbiome metabolites (SCFAs)
contribute to healthy human states?
• In what ways does the human microbiome
communicate with the brain?

• What options are available to treat mental
disorders through the gut microbiome?
• Why have mental illnesses been increasing in
prevalence and how could the gut microbiome be
involved?
• How are microbiomes, other than the gut,
implicated in mental health?
• What role does skin microbiome dysbiosis and
chronic wounds have in the development of cognitive
disorders?
Media Attributions
• Figure 1 – Inflammatory and microbial

influences between the gut and skin for a healthy
state by De Pessemier et al., 2021. Licensed under
the Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/).
• Video 1 – Remodeling of the maternal gut
microbiome during pregnancy is shaped by
parity by Research Square. Licensed under
• Figure 2 – Gut-brain interactions with mental
health by Bear et al., 2021. Licensed under the
Creative Commons Attribution (CC BY) license
• Figure 3 – Development of the gut-brain over

time by Liang et al., 2018. Licensed under the
Creative Commons Attribution (CC BY) license
• Video 2 – Functional GI Disorders: The role of
the gut-brain interaction, microbiome and
nutrition by UMMCVideos. Licensed under
Creative Commons Attribution license (reuse
allowed).
• Figure 4 – Direct and indirect mechanisms of
infecting the brain by Olsen and Hicks, 2019
licensed under under the terms of the Creative
Commons Attribution License
(http://creativecommons.org/licenses/by/4.0
• Figure 5 – Chronic wounds and skin-brain axis
by Hadian et al., 2020 under Public Domain
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PART IV
ENVIRONMENTAL
MICROBIOMES
Environmental Microbiomes | 213

214 | Environmental Microbiomes
11. Environmental Nutrient
Cycling and Human Health
Environmental Nutrient Cycling and Human

Health
The importance of microorganisms is unquestionable in regard to

how nutrients circulate throughout each ecosystem. There are
direct and indirect links between Earth’s ecosystems and human
health, though like other microbial networks, they are sometimes
unfathomably complex.
Climate change may be the most obvious association between
environmental and human health, however, the solution to balance
is not one likely easily achieved. Each type of ecosystem is unique,
and reflects different changes to anthropogenic activity. Global soil
microbiomes and organic foliar litter have great impact on
worldwide biogeochemical cycling, plant health, and
bioremediation, where environmental changes can disrupt
microbial taxonomic distribution and functional profiles (Albright
et al., 2020, Naylor et al., 2020). The ocean microbiome is vast
considering it is the largest ecosystem on the planet, and plays a
tremendous part in biogeochemical cycling, ecosystem dynamics,
and response to climate change(Moran, 2015, Acinas et al., 2019,
Marz et al., 2021). Also, since much of the ocean microbiome is
uncharted, it could also serve as a major untapped reservoir for
novel and progressive biosynthetic products (Paoli et al., 2021).
Other aquatic environments such rivers, lakes, wetlands, and
freshwater systems and their interaction with sediments and plants
greatly influence carbon and other nutrient cycles in their
respective ecosystems (Amado and Roland, 2017, Avila et al., 2019,
Trevathan-Tackett et al., 2021).
Environmental Nutrient Cycling and Human Health | 215

In a cyclic manner, climate change can also greatly affect

microbiome dynamics of large ecosystems like glaciers, tundra,
permafrost, and even dry deserts, which can further exacerbate
disturbances in that region and beyond. (Hamilton et al., 2013,
Tripathi et al., 2019, Vigneron et al., 2019, Hough et al., 2020, Ray
et al., 2020). These environments harbor several dormant
microorganisms that can produce greenhouse gases like carbon
dioxide and methane, and if they become metabolically active en
masse, this could drastically increase contribution to climate
change (Feng et al., 2020). Not only can ecosystem biodiversity be
affected, but these changes can impact human society and health.
Thus, it is important to consider analytical strategies to better
understand global change so future actions can be coordinated to
mitigate any negative consequences, and evaluating microbiomes
may be part of the solution.
216 | Environmental Nutrient Cycling and Human Health

Global Change and the Soil Microbiome: A
Human-Health Perspective
Article by Ochoa-Hueso, 2017 licensed under the terms

of the Creative Commons Attribution License (CC BY).
The importance of the gut and the soil microbiomes as

determinants of human and ecosystem health, respectively,
is gaining rapid acceptation in the medical and ecological
literatures. This suggests that there is a wealth of highly
transferable knowledge about the microbial ecology of
human and non-human ecosystems that is currently being
generated in parallel, but mostly in isolation from one
another. I suggest that effectively sharing this knowledge
could greatly help at more efficiently understanding and
restoring human health and the functioning of ecosystems,
which are currently under wide-spread pressure. I illustrate
this by comparing the effects of nitrogen deposition on
ecosystem carbon sequestration with unhealthy dietary
habits and human disease. The deposition of N, a key
nutrient for plant growth, may increase carbon
sequestration (equivalent to obesity) through several
mechanisms, including a reduction in the ability of soil
microbes to process organic matter, which some argue
could help mitigate climate change. However, this usually
results in a degradation of ecosystem health and, thus,
cannot represent a real solution. Similarly, human obesity is
linked to an alteration of the composition and functioning

of microbial communities inhabiting the gut, which is often
attributed to unhealthy dietary habits, including ingesting
high amounts of simple sugars and processed foods. Finally,
I advocate for the explicit recognition of the many
commonalities between the functioning of the gut and
ecosystems and a broader multidisciplinary collaboration
among experts in ecology and human health, including the
engineering of soil microbial communities designed ad-
hoc to restore ecosystem health.
Nitrogen Deposition and Carbon Sequestration in a

Changing Climate
It has been widely proposed that atmospheric nitrogen (N)

deposition could help mitigate climate change by increasing
the rates of carbon (C) sequestration in terrestrial
ecosystems (Knorr et al., 2005; Reich et al., 2006; Yue et
al., 2016). Two commonly observed responses are typically
proposed as mechanisms: first, a greater amount of N
usually implies a higher capacity for plant growth, which
would result in a greater amount of C retained within the
system (Magnani et al., 2007; de Vries et al., 2009; Laubhann
et al., 2009). Of course, for this to be true, it is necessary
that the increase in the rates of C uptake and accumulation
exceed the C emission rates, whatever the main route by
which the latter happens, including plant and/or microbial
respiration and changes in fire dynamics due to an excess
of biomass accumulation (Dezi et al., 2010; Fenn et al., 2010).
The second main mechanism is linked to a reduction in
decomposition rates, particularly of recalcitrant organic
matter, which would, therefore, accumulate within the
system (Knorr et al., 2005; Waldrop and Zak, 2006).

Otherwise, this accumulated C may be lost to the
atmosphere in the form of CO2 after being respired by soil
microorganisms (Janssens et al., 2010). Of course, the
relative importance of these mechanisms depend on how
plant communities and soil microorganisms respond,
directly and indirectly, to the additional inputs of N which,
in any case, usually ends up resulting in a disruption of
the interaction between these two key components of the
ecosystem (Liu et al., 2014).
The Need for a New Perspective
In this article, I will adopt a human health perspective,

hardly used in the discipline of global change ecology, to
substantiate why atmospheric N deposition cannot
represent a positive (i.e., healthy) alternative to mitigate
climate change. In the medical literature, it is now widely
recognized that human beings are like ecosystems (in fact,
some consider us as living ecosystems) in which the
eukaryotic cells that form part of our bodies and the
prokaryotic cells that live in and on us are deeply
interconnected, whereas the enormous importance of our
microbiome to human health is also increasingly gaining
acceptation (Bengmark, 1998; Berendsen et al., 2012; Ha et
al., 2014; Alivisatos et al., 2015; Tilg and Adolph, 2015; Blaser,
2016; Blaser et al., 2016). The fact that many modern
diseases, including conditions of the nervous and
circulatory systems, skin and heart and allergies (including
atopic dermatitis and food allergies), are directly caused by
alterations in the microbial communities that live in our
interior and exterior is also gaining rapid acceptation (Ha et
al., 2014; Tilg and Adolph, 2015; Chang et al., 2016; Tang and

Lodge, 2016). In this sense, the word ecosystem is widely
used in the current literature of integrative medicine and
gastroenterology. However, the opposite does not
frequently happen in ecology [i.e., (cautiously) comparing
ecosystems with the human body], despite the wealth of
knowledge in the medical and human health literature that
we, as ecologists, could apply in, for example, issues related
to understanding the functioning (i.e., metabolism) of
ecosystems and plant-soil-microbe interactions subjected
to human pressure (Berendsen et al., 2012; Blaser et al., 2016;
Table 1; Figure 1). Therefore, I will finally defend the need
to approach problems in ecology from a more
multidisciplinary, fresher perspective.
Table 1. The importance of the gut and the soil microbiomes as

determinants of human and ecosystem health, respectively, is
gaining rapid acceptation in the medical and ecological literatures.

Figure 1. Comparison between a healthy and an unhealthy gut
(left-hand side panel) and a healthy and an unhealthy ecosystem
(right-hand side panel). In the case of the human individual, his/her
gut microbiota is less diverse (“red” microbes are completely absent),
less abundant and contains more pathogenic taxa (“yellow”
microbes). The individual with the healthy gut has a more functional
and more abundant microbiota that provides him/her with essential
nutrients, hormones, amino acids, etc. and stimulates his/her
immune system. A healthy gut is also less prone to become infected by
pathogens and can process toxic compounds (i.e., detoxify) more
easily. The disturbed (i.e., unhealthy) ecosystem shown here has
recently been affected by a devastating fire fuelled by the
accumulation of N-loving exotic grasses that have altered the natural
fire dynamics of the system. Facilitated by the altered fire dynamics,
the system has also become chronically dominated by weedy grasses,
therefore requiring intensive (and costly) management practices
(dead tree removal, weeding and restoration), also posing a threat to
nearby human populations and their properties. Biodiversity (in
terms of microbial, faunal and plant communities) is remarkably
higher in the healthy ecosystem, which also has a higher potential to
process organic matter inputs and stabilize them in the long-term
soil pool. In contrast, undecomposed or partially decomposed litter
accumulates on the functionally impoverished soil of the unhealthy
ecosystem. This pattern is in agreement with reported observations
of higher soil carbon sequestration under increased nitrogen
deposition scenarios due to the inhibition of soil enzymes and the
reduction of microbial biomass but poses relevant questions such as:
Is this type of N deposition-induced carbon sequestration desirable?

And, does it really represent a long-term (or even short-term)
solution?
Why Nitrogen Deposition Cannot be the Solution to

Climate Change
The reason why I think that a temporary, N deposition-

induced increase in the rate of C sequestration will not
contribute to mitigating climate change in the long term is
equivalent to the reason of those that argue that an increase
in obesity rates in human populations derived from a diet
rich in simple sugars and processed food and the
consequent alteration of their microbiome will not
successfully and permanently solve any public health
problem of today’s societies. Ingesting large amounts of
simple sugars, processed foods, sugary drinks and saturated
fats is definitely better than starving, but that does not mean
that it is a healthy practice. And the same happens with N
deposition and C sequestration. In ecosystems where N is
still a limiting nutrient, which is quite common worldwide
(LeBauer and Treseder, 2008), an increase in the availability
of N can increase ecosystem productivity to levels
comparable to human obesity (Tian et al., 2016), but that
does not mean that the ecosystem is healthier and,
therefore, that this will result in a long-term benefit
(Bobbink et al., 2010; Jones et al., 2014). In this sense, a
healthy ecosystem may be defined here as a highly
multifunctional ecosystem that can maintain an adequate
supply of services, at least as compared to a previously
defined reference state.

In medicine, the term dysbiosis refers to changes in the
composition of the microbiome that are not beneficial to
the individuals, including a loss of abundance and diversity
of beneficial microorganisms and increased number of
pathogens, and that result in the development of a condition
(Ha et al., 2014; Tilg and Adolph, 2015). This term could also
be used to describe ecosystems that are dysfunctional due
to alterations of their microbial communities. In this sense,
it has been repeatedly shown through experimental studies
and meta-analyses that increased N deposition is typically
associated with changes in soil microbial communities
(usually related to a decrease in abundance and
biodiversity; Treseder, 2004, 2008; Ramirez et al.,
2010; Zeng et al., 2015), reduced ecosystem functionality
(alterations of energy metabolism; Waldrop and Zak,
2006; Treseder, 2008; Liu et al., 2014) and short- to mid-
term increases in C sequestration, especially in
aboveground biomass, but also in the soil and roots
(comparable to obesity, as previously mentioned; Xia and
Wan, 2008; Yue et al., 2016). Given that metabolic disorders
and obesity in humans are clearly associated with a
deterioration in the health status of individuals that may
even result in cases of fatality due to chronic diseases,
sudden death or, quite commonly in the natural world, to
increased sensitivity to other environmental stresses
(Mathur and Barlow, 2015; Monteiro et al., 2015), I think that
we would do well to be cautious when we consider, perhaps
naively, the potential benefits of a N that, after all, is the
result of the atmospheric pollution derived from our
activities (Gruber and Galloway, 2008).

The “Deceptively Simple” Solution
The connections between human health, disease, and the

microbiome, especially in the case of the gut, are becoming
increasingly apparent and are attracting the public
attention, especially because of the high social cost of
unhealthy dietary habits and lifestyles and the “deceptively
simple” solution of the problem (Mathur and Barlow,
2015; Tilg and Adolph, 2015; Blaser, 2016). In the case of both
people and ecosystems, (i) ensuring a healthy supply of
nutrients derived from the breakdown and cycling of
unprocessed food/organic matter, (ii) minimizing the use of
antibiotics (particularly those associated with the livestock
industry in the case of ecosystems; Park and Choi, 2008) and
chemicals (including herbicides and pesticides in the case
of ecosystems) that destroy the microbiome, unless this is
strictly necessary, and (iii) promoting practices that favor
the system’s ability to self-regenerate, something that living
systems do wonderfully well, and that increase its resilience
against pathogens and extreme events could be part of the
solution, if not all, of the problem.
Of course, there are opportunities to aid in the recovery

of our damaged and degraded ecosystems as well as there
are possibilities to recover the lost or damaged intestinal
flora (Brudnak, 2002; Sheth et al., 2016). This can be achieved
by the use of properly designed probiotics or fecal
transplants or, in the case of ecosystems, inocula assembled
in the lab from pure cultures or soil samples obtained in the
field from healthy ecosystems (Bowker, 2007; Chiquoine et
al., 2016; Wubs et al., 2016) in conjunction with a balanced

nutrient supply (i.e., organic matter inputs, the equivalent
to prebiotics; Mathur and Barlow, 2015; Sheth et al., 2016).
In this sense, the concept of synbiotics (i.e., synchronous
administration of probiotics and prebiotics) could represent
a particularly promising benchmark borrowed from the
human health literature to successfully restore degraded
ecosystems (Tang and Lodge, 2016) and, thus, the human
probiotics industry has an opportunity to play a key role in
this development.
Concluding Remarks
Recognizing and understanding the similarities and deep

connections between the gut and the belowground world,
where roots are the equivalent to our gut and the
rhizosphere is the gut microflora (Berendsen et al., 2012)
can help us advance the understanding of ecosystems by
leaps and bounds through the search of similar microbial
indicators of disease (e.g., Bacteroidetes to Firmicutes ratio
in humans; Mathur and Barlow, 2015) and, therefore, to
implement quick and successful measures in ecosystem
management rather than relying, perhaps naively, on that
the very same thing that caused climate change (i.e.,
pollutant emissions to the atmosphere) will also be part of
the solution. From here, I advocate for the development of
a new field of research that specifically aims at recognizing
and make practical use of the profound links between the
functioning of the gut and the ecosystems that extend
beyond our bodies and that benefits from a truly
multidisciplinary collaboration among experts in the areas
of global change ecology and human health.

Author Contributions
The author confirms being the sole contributor of this

work and approved it for publication.
Conflict of Interest Statement
The author declares that the research was conducted in

the absence of any commercial or financial relationships
that could be construed as a potential conflict of interest.
Acknowledgments
I am indebted to Dr. Lilia Serrano for her tirelessly

encouragement to write this opinion article.
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Info Hotspot
Click on the boxes to learn more about the importance of

microbiomes in creating and maintaining homeostatic
balance between humans and the environment.

online here:

• Which ecosystems have the greatest impact on

global nutrient cycling?
• Which microbiome is an under-explored source for
the discovery of biosynthetic products?
• How could global warming’s effect on permafrost
and tundra further increase greenhouse gases?
• What are the pros and cons of nitrogen deposition
to counteract climate change?
• What are some suggested solutions to improve
environmental and human health concerning
microbiomes?
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• Video 1 – Elevated atmospheric CO2 increases

phosphorous mineralization and alters the
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12. The Ocean Microbiome
and Marine Life
The Ocean Microbiome and Marine Life
The ocean is teeming with life, both macro and micro, where their
adaptation and success are dependent on both a local and global
scale. Oceanic microorganisms have major roles in nutrient cycling,
ecological interactions, and influence and respond to environmental
changes (Doney et al., 2012). Temperature is the strongest factor
that determines microbial community composition, which parallels
depth stratification, and these communities can serve as indicators
for anthropogenically-induced climate changes (Sunagawa et al.,
2015). It is predicted that carbon fixation by microbial primary
producers will decrease which will have downstream effects on
ocean microbiome structure and overall trophic level interactions
(Moran, 2015).
While free-living marine microorganisms can provide relevant
information and predictive models about the ocean, those host-
associated microbes can also give environmental insight into the
largest ecosystem on the planet.
242 | The Ocean Microbiome and Marine Life

Marine Animal Microbiomes: Toward
Understanding Host–Microbiome Interactions in
a Changing Ocean
Article by Apprill, 2017 licensed under the terms of

the Creative Commons Attribution License (CC BY).
All animals on Earth form associations with

microorganisms, including protists, bacteria, archaea, fungi,
and viruses. In the ocean, animal–microbial relationships
were historically explored in single host–symbiont systems.
However, new explorations into the diversity of
microorganisms associating with diverse marine animal
hosts is moving the field into studies that address
interactions between the animal host and a more multi-
member microbiome. The potential for microbiomes to
influence the health, physiology, behavior, and ecology of
marine animals could alter current understandings of how
marine animals adapt to change, and especially the growing
climate-related and anthropogenic-induced changes
already impacting the ocean environment. This review
explores the nature of marine animal–microbiome
relationships and interactions, and possible factors that may
shift associations from symbiotic to dissociated states. I
present a brief review of current microbiome research and
opportunities, using examples of select marine animals that
span diverse phyla within the Animalia, including systems
that are more and less developed for symbiosis research,
The Ocean Microbiome and Marine Life | 243

including two represented in my own research program.
Lastly, I consider challenges and emerging solutions for
moving these and other study systems into a more detailed
understanding of host–microbiome interactions within a
changing ocean.
Introduction
Marine animals are the icons of life in the oceans. They

represent about two million species (Mora et al., 2011) and
include a wide range of body designs, from the highly
simplistic sponges lacking true tissues and organs to the
complex vertebrates containing specialized tissues and
organs, such as fish and marine mammals, with some iconic
representatives presented in Figure 1. The bodies of marine
animals span several orders of magnitude in size, from the
abundant planktonic copepod (1–2 mm) to the highly mobile
blue whale (30 m), the largest animal on Earth. Marine
animals are key members of ocean ecosystems and serve
as both prey of and predators for other animals within the
complex ocean food web. In contrast to terrestrial animals,
marine animals have developed strategies for
osmoregulation as well as highly specialized approaches for
maintaining homeostasis within diverse temperature,
oxygen and pressure gradients of the ocean (Graham,
1990; Knoll and Carroll, 1999). Marine animals also possess
sophisticated specializations and functions that promote
their success on or within their benthic or pelagic habitats,
including specializations for living or enduring depths
(outlined in Figure 1) that vary widely in factors such as light
availability, access to food and predator exposure.

Figure 1. Illustration and common names of representative ocean
animal life within their approximate depth-defined ecological
habitats. Microorganisms exist on the surfaces and within the tissues
and organs of the diverse life inhabiting the ocean, across all ocean
habitats. Animals are not drawn to scale.
Marine animals share the sea with a vast diversity of

microorganisms, including protists, bacteria, archaea, fungi,
and viruses which comprise millions of cells in each milliliter
3
of the 1.3 billion km of water comprising the oceans (Eakins

and Sharman, 2010). These microorganisms are several
micrometers or smaller in size, but collectively their roles
in oxygen production, nutrient cycling, and organic matter
degradation provide critical functions to the oceans and
Earth (Arrigo, 2005; Falkowski et al., 2008). Microorganisms
that associate with marine animals are part of the animal’s
microbiome, or collection of microorganisms that reside on
or within the animal. Some of the microorganisms
comprising the microbiomes of marine animals are thought
to originate from this surrounding supply of seawater-
associated cells (e.g., Nussbaumer et al., 2006), while other
cells appear to have strict inheritance patterns, passed on
through generations from the host (Sharp et al., 2007).
Over the past two decades, the widespread application of

genomic and more integrative microbiological approaches
have advanced our understanding of animal microbiomes
(reviewed within McFall-Ngai et al., 2013). Symbiotic
relationships between microorganisms and marine animals
have been studied for decades, but technological
advancements are providing new insights into the sheer
diversity of microbial life in association with animals in the
sea (Smith, 2001; Douglas, 2010). For example, reef-building
corals are acknowledged as the icons of animal–microbial
symbiosis in the sea, with corals hosting photosynthetic
symbionts that make critical contributions to host nutrition
(Muscatine et al., 1981). New reports of diverse protists,
bacteria, archaea, and viruses in association with corals
provide insights into the role of these cells for fulfilling
diverse functional processes within the different niches of
the coral host (reviewed within Thompson et al.,
2015; Bourne et al., 2016). In fact, for many terrestrial
animals, new reports of microbial symbioses provide

insights into the variety of genetic and biochemical
interactions and the ways that microorganisms contribute
to animal health, behavior, and ecology (e.g., Ley, 2010; Cho
and Blaser, 2012).
Understanding the microbiomes of marine animals is a

growing research area within the field of marine science.
Currently, the science is heavily focused on identifying
consistent or “core” microbial members of the microbiome
(Shade and Handelsman, 2012). After first gaining an
understanding of “who’s there” generally using diversity-
based surveys targeting the small subunit (SSU) ribosomal
RNA (rRNA) gene, these microbiomes are then often
examined as a whole or in smaller units to understand the
function of the cells, the nature of the associations and
ultimately gain insight into the role of the microbiome in
animal health, physiology, ecology, and behavior (Ezenwa
et al., 2012; McFall-Ngai et al., 2013). Additionally, the ocean
environment is changing at unprecedented rates due to
climate-related and anthropogenic-induced impacts
(Halpern et al., 2008; Doney et al., 2012), and the microbiome
is also being investigated for its possible role as a sentinel of
a changing host (Ainsworth and Gates, 2016).
How environmental changes and animal life history events

affect the microbiomes of marine animals is growing area
of research, and there is an emerging focus on better
understanding interactions between the animal,
microbiome, and ocean environment, including the
elements that may define their exchanges (e.g., Meron et
al., 2011; Lesser et al., 2016; Webster et al., 2016). Therefore,
this review considers the symbiosis and dissociated stages
of animal–microbiome associations, and discusses factors
and causes that may alter interactions between animals and

their microbiome. Next, this review discusses current
research examining animal–microbiome relationships and
interactions, by focusing on select systems that represent
diverse marine animal phyla and which span the range of
being more to less developed for microbiome research. Two
of these systems, corals and marine mammals, are
represented in my own research program. Lastly, this review
concludes with a discussion of challenges in marine
animal–microbiome research and opportunities available to
further advance knowledge of animal–microbiome
interactions in the ocean.
Conceptual Model of Factors Contributing to

Host-Microbiome Interactions
Host–microbiome dynamics are generally described as

falling into two main categories: symbiosis, in which the
organisms are involved in a normal metabolic and immune
signaling interactions, and secondly dysbiosis, in which the
relationship or interactions are heavily altered, possibly
related to a major stress or infection event. While
host–microbiome symbiosis and dysbiosis has been mostly
considered in humans and humanized models (Hamdi et
al., 2011; Nicholson et al., 2012; Scharschmidt and Fischbach,
2013), many of the same concepts are applicable to
organisms in the sea (Egan and Gardiner, 2016), and are
being explored in various systems (discussed below). The
exact factors and mechanisms tipping the scale between
symbiosis and dysbiosis will probably vary with complexity
of the host anatomy and immune functioning (e.g., simplistic
sponges and corals compared to more complex fish and

sharks) as well as with the complexity of interactions that
may occur between the members of the microbiome.
A normal animal–microbiome relationship in the ocean

could be referred to as a “symbiotic” state, although the
exact nature of the relationship may vary for each cell in
the association. For example, cells residing on the surface or
within the gut cavity of an animal are physically associated,
yet do not share as intimate of an association as those
microbes residing intracellularly with the host’s cells. This
normal symbiotic state is subject to a variety of
environmental fluctuations, which are generally defined by
the characteristics of the habitat (Figure 1). For example,
in the ocean’s upper photic zone, animals are exposed to
variations in temperature and light, and host–symbiont
interactions, especially in ectothermal animals, could alter
on cycles such as seasons that generally control the
temperature and light environment. Normal fluctuations in
animal-specific patterns could also alter host–microbiome
relationships. For example, changes in diet, possibly due to
short-term prey availability, can alter gut microbiota and
host–microbiome metabolic exchanges in other systems
(e.g., David et al., 2014), and similar diet trends may also
affect marine animals. Stress is another factor more
complex animals encounter on a daily basis (e.g., squid,
crabs, fish), which could be related to social/territorial
encounters or chasing or fleeing from prey, and the short-
term production of stress hormones such as cortisol can
influence host–microbiome relationships (e.g., Moloney et
al., 2014).
There are also normal animal life events that occur on

longer time frames or that are more drastic in scope, such
as animal development, aging, and reproduction. In non-

marine animals, these factors have been shown to cause
alterations in animal-microbial relationships (e.g., Heintz
and Mair, 2014). These changes can be drastic enough to
cause a state of “altered symbiosis” that could extend for
short or longer term. For example, the gut microbiome of
women generally becomes altered during pregnancy (Koren
et al., 2012). Events resulting in normal animal stress may
also lead to a more altered symbiotic state, for example
if social conflict was more chronic, perhaps due to the
pressures of a particular habitat. Data from humans and
humanized models suggests that the microbial community
and associated genes do fluctuate with the normal
variations and animal life events, and both may be
considered “healthy” fluctuations (Nicholson et al., 2012).
However, how these fluctuations affect exchanges between
the host and microbiome is much less understood.
If symbiosis and altered symbiosis are considered as

normal host–microbiome variations throughout an
organism’s life, dysbiosis is the breakdown in the
relationship, generally related to one or more major
stressors, and can greatly alter host health and lead to a
disease state (Holmes et al., 2011). The stressor may come
from an external source, such as a pollutant, infective agent,
or a longer-term natural environmental change—and there
are probably countless other factors that could fit this
category (Figure 2). For example, one of the most visible
signs of host–microbiome dysbiosis is with scleractinian
corals, whose relationship with unicellular algae breaks
down after long-term yet small increases in seawater
temperature, causing the coral to become “bleached”
(Brown, 1997). In humanized models, major stressors such
as malnutrition are related to less physically visible changes

in innate immunity, which are linked to microbial ecology
(Hashimoto et al., 2012). Understanding the relationship
between symbiosis, dysbiosis and host health and
functioning are general topics of research in most
host–microbiome studies, but the environmental changes
occurring in the ocean environment have made this area
of research more pressing for marine animals. Overall, the
concepts behind the model presented in Figure 2, as well as
variations of this model, are generally driving much of the
current research examining animal-microbial relationships
in the ocean.

Figure 2. Conceptual diagram of a host-microbiome relationship.
Relationships are generally thought to exist in a symbiotic state, and
are normally exposed to environmental and animal-specific factors
that may cause natural variations. Some events may change the
relationship into a functioning but altered symbiotic state, whereas
extreme stress events may cause dysbiosis or a breakdown of the
relationship and interactions.
Overview of Diverse and Emerging

Animal-Microbiome Study Systems
The microbiomes of diverse marine animals are currently

under study, from simplistic organisms including sponges
(e.g., Webster et al., 2010) and ctenophores (Daniels and
Breitbart, 2012) to more complex organisms such as sea
squirts (Blasiak et al., 2014) and sharks (Givens et al., 2015).
Below I present some of the current study systems that
represent a diverse cross-section of marine animal phyla,
and trends of research in these systems including focus
on symbiosis and dysbiosis. The organisms are generally
presented in order from increasing to decreasing knowledge
about the host-microbiome relationship.
The relationship between the Hawaiian bobtail

squid Euprymna scolopes (phylum Mollusca) and the
bioluminescent bacterium Vibrio fisheri (also recognized
as Aliivibrio fisheri) is one of the best studied symbiotic
relationships in the sea and is a choice system for general
symbiosis research (Figures 3A,B). The E. scolopes-V.
fisheri relationship has provided insight into fundamental
processes in animal-microbial symbioses, and especially
biochemical interactions and signaling between the host
and bacterium (McFall-Ngai, 2000, 2014). Much of this
research focuses on establishment of the symbiosis, with
less focus on dysbiosis. Additionally, because V. fisheri exists
in the light organ, these studies have been primarily limited
to this one isolated relationship, with the remainder of the
squid’s microbiome virtually unstudied (but see Barbieri et
al., 2001; Collins et al., 2012). The E. scolopes–V.
fisheri system offers simplicity for the study of
host–microbial interactions and numerous helpful
developments in animal husbandry, genomic tools, and
experimental design that could be applied to ask more
comprehensive questions about squid–microbiome

interactions, including the conditions leading to dysbiosis of
relationships.

Figure 3. Photographs of marine animals and their associated
microbiomes from select study systems. Photographs include: the
Hawaiian bobtail squid Euprymna scolopes (A) and a transmission
electron micrograph of Vibrio fisheri cells associating with dense
microvilli (MV) and in proximity to the epithelial nucleus (N) within
the light organ (B); the reef-building coral Stylophora pistillata (C)
and a microscopy image of Endozoicomonas cells (probed yellow
using in situ hybridization) within the tentacles of a S. pistillata host
(D); the Atlantic killifish (Fundulus heteroclitus) (E) and a scanning

electron microscopy (SEM) image of the surface and scales of the fish,
with arrows pointing to bacterial-sized cells and larger cells (which
are not noted) are presumably phytoplankton (F); a humpback whale
(Megaptera novaeangliae) breaching (G) and a scanning electron
microscopy image of a humpback’s skin surface associated bacteria,
with arrows indicating two different cell morphologies (H).
Photographs (A,B) were produced by M. McFall-Ngai and were
previously published photographs (McFall-Ngai, 2014), (C,H) were
previously published by the author Neave et al. (2016) and Apprill et
al. (2014), photograph (D) was taken by Liping Xun and photograph
(E) by Evan D’Alessandro.
Similar to E. scolope, the gutless marine oligochaete

worm Olavius algarvensis (phylum Annelida) is another
relatively well-studied marine host to microbes. One major
difference is that it has been studied within the context
of a larger consortium of microorganisms compared to E.
scolope. These 3 cm long worms reside within shallow
marine sediments of the Mediterranean Sea. The worms do
not contain a mouth or a digestive or excretory system,
but are instead nourished with the help of a suite of
extracellular bacterial endosymbionts that reside upon
coordinated use of sulfur present in the environment
(Dubilier et al., 2001). This system has benefited from some
of the most sophisticated ‘omics and visualization tools
(Woyke et al., 2006). For example, multi-labeled probing has
improved visualization of the microbiome (Schimak et al.,
2016) and transcriptomics and proteomics have been
applied to examine host–microbiome interactions, including
energy transfer between the host and microbes (Kleiner et
al., 2012) and recognition of the consortia by the worm’s
innate immune system (Wippler et al., 2016). The major
strength of this system is that it does offer the ability to

study host–microbiome interactions with a low diversity
microbial consortium, and it also offers a number of host
and microbial genomic resources (e.g., Woyke et al.,
2006; Ruehland et al., 2008). Dysbiosis has not been heavily
investigated in this system, and given the growing
knowledge of host–microbial interactions, O.
algarvensis could be an imperative animal for dysbiosis
research.
As mentioned above, corals (phylum Cnidaria) (Figure 3C)

are one of the most common examples of an animal host
whose symbiosis with microalgae can turn to dysbiosis, and
is visibly detected as bleaching. Coral microbiomes have
been examined in a variety of studies, which demonstrate
how variations in the ocean environment, most notably
temperature, light, and inorganic nutrients, affect the
abundance and performance of the microalgal symbionts, as
well as calcification and physiology of the host (Dubinsky
and Jokiel, 1994; Anthony et al., 2008). Studies have also
suggested that resident bacteria, archaea, and fungi
additionally contribute to nutrient and organic matter
cycling within the coral, with viruses also possibly playing
a role in structuring the composition of these members,
thus providing one of the first glimpses at a multi-domain
marine animal symbiosis (reviewed in Bourne et al., 2016).
The gammaproteobacterium Endozoicomonas is emerging
as a central member of the coral’s microbiome, with
flexibility in its lifestyle (Figure 3D) (Neave et al., 2016, 2017).
Ocean disturbances including elevated temperature and
ocean acidification have been shown to disrupt the coral’s
associated bacteria (Thurber et al., 2009; Meron et al., 2011),
including relationships with Endozoicomonas (Morrow et al.,
2015). However, some members of this microbiome appear

to be stable across large environmental gradients
(Hernandez-Agreda et al., 2016). In addition to nutrition,
the microbiome plays a role in coral health and stress.
Temperature and light stress to corals can result in
overproduction of reactive oxygen species (ROS), which can
be detrimental to Symbiodinium and result in bleaching, but
the associated bacteria have also recently been shown to
contribute extracellular ROS (Diaz et al., 2016; Zhang et al.,
2016), which could play a signaling role with the host or
within the microbiome. Given the recent mass bleaching
occurring on reefs (Hughes et al., 2017), corals will likely
continue to be a useful and popular system for symbiosis
and dysbiosis research. There are number of resources
available to further promote study of the coral microbiome,
including integrated databases (Franklin et al., 2012; Madin
et al., 2016), a growing number of host and microbial
genomes (Shinzato et al., 2011; Bayer et al., 2012; Neave et al.,
2017), and laboratory amendable “model” systems (Weis et
al., 2008; Baumgarten et al., 2015).
Sponges (phylum Porifera) are common members of the

ocean’s diverse benthic habitats and their abundance and
ability to filter large volumes of seawater have led to the
awareness that these organisms play critical roles in
influencing benthic and pelagic processes in the ocean (Bell,
2008). They are one of the oldest lineages of animals, and
have a relatively simple body plan that commonly associates
with bacteria, archaea, algal protists, fungi, and viruses
(reviewed within Webster and Thomas, 2016). Sponge
microbiomes are composed of specialists and generalists,
and complexity of their microbiome appears to be shaped
by host phylogeny (Thomas et al., 2016). Studies have shown
that the sponge microbiome contributes to nitrogen cycling

in the oceans, especially through the oxidation of ammonia
by archaea and bacteria (Bayer et al., 2008; Radax et al.,
2012). Most recently, microbial symbionts of tropical
sponges were shown to produce and store polyphosphate
granules (Zhang et al., 2015), perhaps enabling the host to
survive periods of phosphate depletion in oligotrophic
marine environments (Colman, 2015). The microbiomes of
some sponge species do appear to change in community
structure in response to changing environmental
conditions, including temperature (Simister et al., 2012a)
and ocean acidification (Morrow et al., 2015; Ribes et al.,
2016), as well as synergistic impacts (Lesser et al., 2016).
Understanding the effect of these altered host–microbiome
interactions on sponge growth and ecology are topics for
further research. As such, there are a number of resources
to support research on sponges including a curated
database of sponge–microbial sequences (Simister et al.,
2012b), cultivated microbial isolates and sponge cell cultures
from some species (Taylor et al., 2007) to facilitate
investigations.
Atlantic killifish, (Fundulus spp., Phylum Chordata)

(Figure 3E) are one of the most abundant estuarine fishes
in North America, and are related to other families with
more global distributions in coastal areas (Fritz et al.,
1975; Lotrich, 1975). The killifish have a broad North
American geographic distribution yet limited subpopulation
movement, and thus the Atlantic killifish have become a
useful field-residing model species for examining biological
and ecological responses to natural environment conditions
(salinity, oxygen, pH, and temperature) as well as chemical
pollutants (Burnett et al., 2007). While the killifish
microbiome (Figure 3F) has not been extensively studied,

there is work examining the influence of pollutants on the
skin and mucus of the fish, which suggests that this skin
microbial community is relatively resistant to change
(Larsen et al., 2015). Populations of the fish offer a unique
host genetic resistance to toxicity (Hahn et al., 2004), and it
is possible that this resistance is also facilitated by features
of the microbiome. The Atlantic killifish appear to be an ideal
study species for microbiome investigations and especially
the response of the host–microbiome symbiosis to changing
ocean conditions. Specifically, the killifish can be maintained
in laboratory aquaria, they are hardy and amendable to
experimental manipulation, and spawning material can be
acquired for developmental (Burnett et al., 2007).
The microbiomes of marine mammals (phylum Chordata)

(Figure 3G) have recently been investigated and offer a
comparative study system to terrestrial mammals (reviewed
within Nelson et al., 2015). Marine mammals are often
viewed as sentinel species of the ocean, because they appear
to rapidly respond to ocean conditions, disturbances, and
pathogens similarly to humans (Bossart, 2011). Several
studies have examined the skin (Figure 3H), gut and
respiratory microbiomes of diverse marine mammal species,
and describe species-specific relationships (Johnson et al.,
2009; Apprill et al., 2014; Bik et al., 2016). Connections
between the community composition of the microbiome
and animal health (Apprill et al., 2014) and diet (Nelson et
al., 2013; Sanders et al., 2015) have been made, and more
detailed studies are needed to understand these specific
connections. While there are very limited resources
available for studying host–microbiome interactions in
marine mammals, there are some animals in captivity as well
as well-studied populations that will heighten investigations

of host–microbiome symbiosis and dysbiosis in these
sentinel species.
Challenges and Emerging Solutions to Studying

Animal–Microbiome Interactions
A number of the systems highlighted above are currently

examining animal–microbiome interactions, but these are
generally most developed in systems such as O.
algarvensis that offer lower complexity microbiomes, or
within the single host–symbiont relationship between E.
scolopes and V. fisheri. As such, a major challenge to the field
is exploring host–microbiome interactions within the
context of a diverse microbiome, and especially if the
microbiome includes members such a uncharacterized
protists, fungi, and viruses, which have generally not been
described in most marine animal systems. Therefore, a
through description of the microbiome is a first necessity,
but this still presents many challenges on a variety of levels.
For example, amplifying or shotgun sequencing microbial
DNA with the presence of abundant host cells often requires
optimization or high sequencing output (e.g., Rocha et al.,
2014; Weber et al., 2017). Taxonomic databases generally
contain few microbial sequences from many of these
animals, and therefore simple tasks such as assigning
taxonomy can be challenging. Developing animal-specific
databases (Simister et al., 2012b), which include the next-
generation supplied sequences generally not available in
curated taxonomic databases, could help alleviate this
problem. There are also a number of new tools for
metagenomics-based analysis, including advancements in
binning genomes from complex samples (Kang et al.,

2015; Graham et al., 2017) as well as new visualization
methods for comparing genomes (Eren et al., 2015; Wagner
et al., 2017). A challenging issue that has received less
attention is how to gain information from unknown genes
and gene families, which can make up over half of the
environmental microbial genomes. Algorithms utilizing gene
function predictions do provide some assistance with this
problem (Mi et al., 2015), and these tools may improve as
more environmental microbial genomes are available. Lastly,
computational tools are emerging to facilitate identifying
associations between host genetic variation and
microbiome composition (Lynch et al., 2016).
Once some of these hurdles are overcome and a

comprehensive view of the microbiome is available,
researchers can then explore the nature of the
host–microbiome relationship. Visualization using a variety
of different microscopy-based techniques is a powerful tool
to recognize the physical relationship between a host and
the microbiome, as well as the organization of cells within
the microbiome. Electron microscopy provides the most
detailed information about this organization, but this is less
useful for complex microbiomes because taxonomically
distinct microbial cells with similar appearances cannot be
distinguished. Fluorescent in situ hybridization (FISH), and
especially using a multi-taxonomic, simultaneous probing
technique such as Combinatorial Labeling and Spectral
Imaging FISH (CLASI-FISH) (Valm et al., 2011) can provide
significant insight into host–microbe and microbe–microbe
interactions. FISH techniques do require optimization for
some animal systems, such as corals that possess
autofluorescent host tissues (Wada et al., 2016). Visualization
techniques can also be paired with isotope probing, to

provide opportunities to trace the transfer of specific
molecules between the host and microbiome, as well as
within the microbiome using Nano-SIMS and Nano-SIP
approaches (Musat et al., 2016). There have also been many
recent instrumental and database advances in the field of
metabolomics (Beisken et al., 2015), and this approach is
beginning to be applied to examine host–microbiome
interactions (Gomez et al., 2015; Sogin et al., 2016). An
understanding of specific microbial metabolites will help
facilitate targeted investigations of how these products
affect the host nutritional and immune systems.
Lastly, experimental manipulation is a challenge to the

study of host–microbial interactions in the ocean. Studying
the animals in their natural environment is the most ideal
approach because it ensures that the surrounding seawater
microbial community is maintained. However, natural
experiments are only as helpful as the natural variability
in the host-microbe system, and generally only afford the
opportunity to study events such as seasonality, animal
growth or other life history events. Artificial systems such
as aquaria or mesocosms offer opportunities to manipulate
environmental conditions or expose the animal to
antibiotics or other molecules that are difficult to dose in
the wild. However, not all animals are ideal for these systems
(e.g., large whales, hydrothermal vent worms), and it can be
challenging to reproduce some environmental conditions.
Advances in aquaria design that offer consistency in
environmental conditions and the ability to manipulate
complex environmental interactions, such as the Australian
Institute of Marine Science’s National Sea Simulator, provide
opportunities to conduct more realistic experiments. As the
need to understand how host–microbiome interactions will

alter with the forecasted changes in ocean temperature and
pH, facilities such as this will become critical to
animal–microbiome research in the ocean.
While studies of marine animal–microbiome interactions

are certainly plagued by a number of challenges, the future
is also very bright for this emerging field. Many of the new
bioinformatics and methodological advancements now
available to marine biologists stem from the biomedical
field, and thus marine animal microbiome research, as well
as other environmental-based fields, are profiting from the
elevation in microbiome research funding and attention.
There could also be growing interest in using marine
animals as models for examining resilience, promoted by
the fact that alterations in the ocean conditions are often
outpacing those in terrestrial environments. Given the
phylogenetic breath of animals in the ocean, coupled with
the many diverse ocean environments, there is certainly
a wealth of research opportunities available to study
host–microbiome interactions in the ocean.
The author confirms being the sole contributor of this

work and approved it for publication.
Funding
Funding was provided by the WHOI’s Andrew W. Mellon

Foundation Endowed Fund for Innovative Research.

Conflict of Interest Statement
The author declares that the research was conducted in

Acknowledgments
Many thanks to Margaret McFall-Ngai and Evan

D’Alessandro for use of images and to Laura Weber for early
comments on this review.
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Studying Ocean Microbiomes Data

online here:
Prevalence of certain marine topics appearing in

combination with the term holobiont in the title, abstract,
and/or keywords of scientific publications from 1990 to
February 2021 (N = 1269). Bars indicate the percentage of
publications that contain terms related to a certain marine
topic (cnidarians, corals, sponges, etc.). It is important to
note that the different topics may overlap in several

publications. For example, many publications treat of the
topics of coral and sponge holobionts together. Data
retrieved from the curated citation and abstract database
Scopus on the date 10/03/2021. (Stevenne et al., 2021)
• Which environmental factor is the biggest driver of

change for the community composition of free-living
oceanic microbes?
• What factors affect the normal symbiotic state
between marine host and their microbiome?
• Describe one well-studied marine animal-microbial
symbiosis and its importance.
• What are some challenges to studying marine host-
associated microbiomes?

Media Attributions
• Figure 1 – Illustration and common names of

representative ocean animal life within their
approximate depth-defined ecological habitats by
Apprill, 2017. Licensed under the terms of
the Creative Commons Attribution License (CC
BY).
• Figure 2 – Conceptual diagram of a host-
microbiome relationship by Apprill, 2017.
Licensed under the terms of the Creative
Commons Attribution License (CC BY).
• Figure 3 – Photographs of marine animals and
their associated microbiomes from select study
systems by Apprill, 2017. Licensed under the terms
of the Creative Commons Attribution License (CC
BY).
References

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13. Soil Microbiomes
Soil Microbiomes
The terrestrial landscape on Earth harbors countless microbes that

have major responsibilities in shaping their surrounding
environment. One gram of soil can contain up to 10 billion
microorganisms and consist of thousands of different species. Each
ecosystem has unique soil properties that cultivate a diverse array
of microbial communities, which are primarily composed of
bacteria, however archaea, protists, fungi, viruses, and other
microscopic organisms can be found in varying abundances too.
The composition and health of the surrounding environment and
its macro inhabitants are dependent on their soil borne microbial
partners, and a disturbance in the balance of the soil microbiome
can have far-reaching effects (Omotayo and Babalola, 2021).
Environmental and agricultural sustainability is important in a
number of regards, especially maintaining biodiversity, plant and
animal health, ecosystem homeostasis, and even human health.
Anthropogenic effects on the climate and environment have
become abundantly apparent, and it is imperative to adjust mindsets
to become more environmentally conscious, not only to preserve
aspects of nature, but to accommodate an increasing human
population. The soil is foundational to agricultural practices, and
therefore food production, and so a better understanding of various
soil microbiomes could serve to benefit future applications and
practices (Tosi et al., 2020). One possibility, is to become less reliant
on synthetic nitrogen fertilizers for soil, and utilize symbiotic
nitrogen-fixing bacteria as sources for plant nitrogen.
Soil Microbiomes | 289

Microbe to Microbiome: A Paradigm Shift in

the Application of Microorganisms for
Sustainable Agriculture
Article by Ray et al., 2020 licensed under the terms of

Light, water and healthy soil are three essential natural

resources required for agricultural productivity.
Industrialization of agriculture has resulted in
intensification of cropping practices using enormous
amounts of chemical pesticides and fertilizers that damage
these natural resources. Therefore, there is a need to
embrace agriculture practices that do not depend on
greater use of fertilizers and water to meet the growing
demand of global food requirements. Plants and soil harbor
millions of microorganisms, which collectively form a
microbial community known as the microbiome. An
effective microbiome can offer benefits to its host, including
plant growth promotion, nutrient use efficiency, and control
290 | Soil Microbiomes

of pests and phytopathogens. Therefore, there is an
immediate need to bring functional potential of plant-
associated microbiome and its innovation into crop
production. In addition to that, new scientific
methodologies that can track the nutrient flux through the
plant, its resident microbiome and surrounding soil, will
offer new opportunities for the design of more efficient
microbial consortia design. It is now increasingly
acknowledged that the diversity of a microbial inoculum
is as important as its plant growth promoting ability. Not
surprisingly, outcomes from such plant and soil microbiome
studies have resulted in a paradigm shift away from single,
specific soil microbes to a more holistic microbiome
approach for enhancing crop productivity and the
restoration of soil health. Herein, we have reviewed this
paradigm shift and discussed various aspects of benign
microbiome-based approaches for sustainable agriculture.
Introduction
The health of soil plays an essential role in the ability of

plants to produce food, fuel, and fiber for a growing world
population. To keep pace, the total area of cultivated land
worldwide has increased over 500% in the last five decades
with a 700% increase in fertilizer use and a several-fold
increase in pesticide use (Banerjee et al., 2019). In addition
to being the world’s largest agricultural producers and
exporters, the EU, Brazil, United States, and China also are
some of the world’s largest pesticide users – each using
827 million, 831 million, 1.2 billion, and 3.9 billion pounds
of pesticides, respectively, in 2016 (Donley, 2019). However,
these numbers are not sustainable from either a supply-

chain or environmental perspective. Thus, because natural
resources are limited and their overuse pollutes the
environment, the continued use of fertilizers and water to
meet the demand of future global food requirements is not
sustainable. Of relevance here is that agricultural
intensification with high resource use and low crop diversity
can negatively affect soil- and plant-associated microbiota
(the so-called “phytobiome”) with subsequent impacts on
critical ecosystem services (Matson et al., 1997).
There is growing evidence that aboveground plant

diversity supports belowground microbial biodiversity,
primarily through root exudation and rhizo-deposition (Bais
et al., 2006; Eisenhauer et al., 2017; Morella et al., 2020).
These more simple carbohydrates released into the soil
primarily feed bacteria (Gunina and Kuzyakov, 2015) and are
the most abundant near the root surface and diffuse along
a gradient as distance from the root increases (Gao et al.,
2011). The microbial composition is more abundant and
complex in the rhizosphere, the narrow zone surrounding
9
plant roots, with up to 10 cells per gram in typical
6
rhizospheric soil, comprising up to 10 taxa (Lakshmanan
et al., 2017). The more complex carbohydrates (e.g., lignin,
cellulose) are largely degraded by decomposer fungi that
break down these recalcitrant compounds into forms that
can be used by other microbes. This conversion is largely
decoupled from conventional agricultural practices,
wherein the organic matter content is often lost to the
system (Craven and Ray, 2019), and the carbon flux is at
least partially unregulated in this regard. Again, defining
nutrient fluxes with techniques like Stable Isotope Labeling
(SIP) holds great potential to define and construct resilient,
functioning and beneficial microbiomes that can contribute

to future holistic agriculture. Thus, applying an efficient and
diverse soil microbiome backed by these new technologies
can facilitate and promote sustainable agriculture and can
effectively contribute to meet the triple requirements of
economic, social and environmental sustainability (Ray and
Craven, 2016).
Historically, microorganisms that promote plant growth

and nutrient acquisition have been used largely as single
strains in agriculture to offset such fertilizer inputs as
nitrogen and phosphorous. However, studies of natural
populations suggest that groups of microbes with distinct
function niches play pivotal roles in adhering and desorbing
inorganic nutrients to physical surfaces, as well as breaking
down organic residues and incorporating them into the soil
(Lakshmanan et al., 2014; Finkel et al., 2017; Kumar and
Dubey, 2020). Conceptually, such observations support the
idea of the microbiome as a second genome or an extended
genome of the plant (Vandenkoornhuyse et al., 2015). It is
now evident that improving plant performance in a
sustainable manner is beyond the binary interaction
between a specific microbe or a consortium of beneficial
microbes and a targeted host plant. This is a much more
complex set of interactions than previously thought that
requires modeling for improving predictable outcomes. In
this review, we will highlight the current state of the art
for the incorporation of specific plant growth-promoting
microorganisms and discuss the principles and management
practices for next-generation, microbiome-based
approaches for sustainable agriculture.
Application of Beneficial Microbes in Sustainable

Agriculture: Past, Present and Future
Since the early 1800s, the United States Department of

Agriculture has recommended the use of certain
rhizobacteria to improve nitrogen fertility in leguminous
crops (Schneider, 1892). Since that time, a great deal of
research has been conducted on this relationship between
legumes and these bacteria, now termed rhizobia, that
inhabit unique structures, the nodules, that form on the
roots. Rhizobia infecting these nodules are now capable of
“biological nitrogen fixation,” whereby di-nitrogen is fixed
into forms that can be used by the plant. Symbiotically, the
bacteria trade these nitrogenous compounds to the host
plant in exchange for photosynthetically derived carbon.
Despite these limited applications, much remains to be
learned regarding both the functional and taxonomic
diversity of these symbiotic bacteria and their host plants,
the role they play in the global nitrogen cycle, and
ultimately, how they can best be harnessed for improving
plant productivity. This is particularly true for marginal
lands that are not suited for row crop production but will
need to be incorporated into global food and forage
production approaches moving forward. Further, such
degraded lands must but regenerated with the goal of
restoring soil health and productivity. Any successful
endeavor in this regard must include a characterization of
the soil microbiome, both taxonomically and functionally.
Attempts currently are underway to fix nitrogen in such
non-legumes as wheat, corn and other staple crops that
produce the bulk of human food by engineering symbiotic
relationships using synthetic biology approaches (Rogers

and Oldroyd, 2014; Ryu et al., 2020). Such approaches would
significantly impact global food supplies, and may function
adequately to reduce the arable land required to meet
productivity goals.
Plant growth-promoting microbes not only play critical

and diverse roles in growth promotion per se, but also in
improving various aspects of plant resilience against a wide
array of biotic and abiotic stresses (Arnold et al., 2003; Sun
et al., 2010; Agler et al., 2016; Azad and Kaminskyj,
2016; Singh, 2016; Oleńska et al., 2020; Rai et al., 2020). In
this context, researchers globally have worked over the last
several decades on plant growth-promoting
microorganisms, such as root-associated mycorrhizal fungi,
across a broad range of crops and encompassing a wide
range of agro-climatic conditions. For
perspective, Brundrett and Tedersoo (2018) recently
reviewed 135 years of mycorrhizal research and reported
that merely 8% of the vascular plants are non-mycorrhizal,
suggesting that plant families associating with mycorrhizae
have been very successful over the evolution of the plant
kingdom.
Traditionally, agricultural application of beneficial

microorganisms involves a few types of well-characterized
microbes, such as mycorrhizal fungi or rhizobia bacteria,
for which the mechanisms underlying the plant growth
promotion effects are well understood. Further, most of
these studies focused solely on the ability of the applied
microorganisms to facilitate such specific plant growth-
promoting traits as phosphate solubilization, nitrogen
fixation, ACC deaminase production (Sarkar et al., 2018),
siderophore production, biofilm formation, plant hormone
production, biotic, and abiotic stress tolerance or

resistance, among others (Weyens et al.,
2009; Bhattacharyya and Jha, 2012; Singh et al., 2019). While
these beneficial microorganisms can impart considerable
benefits to plant growth and fitness, they are typically
documented in simple, one-on-one studies, often
conducted in sterile soils in greenhouse conditions. As a
consequence, the effects found in such simplified conditions
often fail to translate to more complex field situations
(Chutia et al., 2007; Nicot et al., 2011; Parnell et al., 2016). Soil
in field plots have more complex microbial environments
that are presumably adapted to the local eco-environment.
In recent years, next-generation sequencing has

revolutionized our understanding of microbial community
composition and function, and together with improved
culturing methodologies has greatly facilitated the use of
biologicals in the field (Schweitzer et al., 2008; Panke-Buisse
et al., 2014; Mueller and Sachs, 2015). Specifically,
metagenomics-based approaches have uncovered vast,
previously unrecognized populations of microbes that may
have new or enhanced properties that could be used for
agriculture, bioremediation, and human health. For example,
comparative analyses of rhizosphere metagenomes from
resistant and susceptible tomato plants enabled the
identification and assembly of a flavobacterial genome that
was far more abundant in the resistant plant rhizosphere
microbiome than in that of the susceptible plants. Such
findings certainly reveal a role for native microbiota in
protecting plants from phytopathogens, and pave a way
forward for the development of probiotics to ameliorate
plant diseases akin to human health (Kwak et al., 2018). In
another study, a 16S rRNA gene amplicon sequencing
analysis of maize root microbiome led to the identification

of bacteria that promote growth under low temperature
conditions (Beirinckx et al., 2020). Additionally, principles
of consortium design that rely on cross-talk, cross-feeding
and/or substrate channeling between different
microorganisms offer new opportunities for “intelligent”
consortia design (Calvo et al., 2014; Vorholt et al.,
2017; Paredes et al., 2018). We propose that the manipulation
of the plant microbiome holds tremendous potential for
agricultural improvement (Table 1). Through recent years of
research, it is elucidated how microbes worked in nature
before, and how decades of chemical fertilizer use have
silenced their ability to improve plant fitness and soil health.
Therefore, designing a microbial consortium that carefully
weighs and evaluates the relationship between inoculants
and the resident microbiome would substantially improve
the plant growth-promoting potential and resilience of
agricultural biologicals to boost plant growth. In this review,
we will discuss the key considerations that would improve
the likelihood of microbial products to improve crop yield,
decrease disease severity and/or ameliorate abiotic stress
response. Further, it is likely that such considerations would
reduce the inconsistency between the performances of
beneficial microbes from controlled greenhouse conditions
and more natural environments.

Table 1. List of recent publications in plant and soil microbiome
focusing on plant fitness and productivity.
Microbes for Plant Growth Promotion: A

Reductionist Approach
Sustainable agriculture primarily focuses on reducing the

dependency of plants on chemical fertilizers and improving

their ability to grow on marginal soil types. For such
purposes, individual microorganisms for plant growth-
promotion have largely focused on those that facilitate
growth and development by enhancing acquisition of
nutrient resources from the environment, including fixed
nitrogen, iron and phosphate, or modulating growth by
altering plant hormone levels (Figure 1) (Hayat et al., 2010).
Another approach aimed at reducing yield losses to disease
relies on microbes that decrease or prevent the deleterious
effects of plant pathogens by several different mechanisms
(Glick, 2012), i.e., by acting as a biocontrol agent. Microbe-
based plant growth-promoting products, more popularly
marketed as biofertilizer, has been commercially available
in many countries since the 1950s (Timmusk et al., 2017).
Application of such plant growth-promoting microbes in
agricultural context and more specifically as inoculants has
been nicely reviewed by Souza et al. (2015). However, under
certain cases, the results obtained in the laboratory could
not be reproduced in the field primarily due to the presence
of many crop species and crop varieties, variable
environmental conditions between fields, (Timmusk et al.,
2017; Saad et al., 2020), occasionally due to the low quality
of the inocula, and their inability to compete with the
indigenous population. In that context, it is important to
consider the fact that there is always greater likelihood of
success by introducing mixed cultures of compatible
microorganisms, rather than single, pure cultures. This is
simply because each strain in the multi-strain consortium
can compete effectively with the indigenous rhizosphere
population and enhance plant growth with its partners. For
example, sequential inoculation of nitrogen fixing
bacterium Azotobacter vinelandii, followed by plant growth-

promoting root-endophytic fungus Serendipita
indica demonstrated better growth in rice (Dabral et al.,
2020). Dual inoculation of S.
indica and Mycolicibacterium strains boosted the beneficial
effects in tomato (del Barrio-Duque et al., 2019) and that
of arbuscular mycorrhizal fungus with plant growth-
promoting bacteria Bacillus subtilis demonstrated better
growth in wheat (Yadav et al., 2020) as compared to the
singly inoculated plants. There also are numerous other
reports that showed two strains used in a consortium
promoted plant growth in a more effective manner (Nadeem
et al., 2013; Fatnassi et al., 2015; Priyadharsini and
Muthukumar, 2016). Nevertheless, to unlock the full
potential of soil microbes for such nutrient cycling as
nitrogen or phosphorus and providing plant protection
against biotic and abiotic stress microbiomes, it is necessary
to develop strategies to comprehend the functional
capabilities of soil microbial communities. Irrespective of
the approach, persistence is the first and foremost principle
underlying the design of a successful microbial consortium
for conferring plant growth promotion. This is not
surprising, as the survival and activity of microbes in any
soil system face a monumental task of competing with the
myriad of microbes naturally adapted to that same soil.
Thus, in addition to establishment of a compatible
interaction with the host, a successful microbial inoculant
has to subsequently compete and persist in the context of
indigenous microbes as well as local abiotic conditions
(Finkel et al., 2017). It has been reported that bacterial
inoculations can persist in soil up to 7 weeks, but whether
this inoculum also can provide plant growth benefits is not
clear (Schreiter et al., 2014). While persistence or resilience

of any microbial inoculum is more dependent on biotic
components of a specific soil type, their persistence can be
improved by inoculating crops with consortia rather than
single strains (Verbruggen et al., 2012; Nemergut et al., 2013).
Thus, it can arguably be stated that the diversity of a
microbial inoculum, in addition to its plant growth-
promoting traits, is critical for enhancing productivity and
longevity (Cordero and Polz, 2014).
Figure 1. A schematic comparison between individual

microorganism-based reductionist approach and microbial
community-based holistic approach.
To improve the likelihood of success for such a

management strategy, a priori knowledge of indigenous

microbial populations competing with the introduced plant
growth-promoting agent(s) is critical. While a reductionist
approach can define the currency of individual plant-
microbe interactions, the concepts of microbial community
survival and functioning require, a more holistic,
microbiome-based approach empowered by next-
generation sequencing technology to study plant-microbe
interactions at the community level (Figure 2). Indeed, this
will enable researchers to design more robust, synthetic
microbial consortia capable of reliably enhancing
agricultural productivity.
Figure 2. Responsiveness as the% gain in plant fitness attribute in

response to symbiosis over un-colonized cohorts. This figure
illustrates a hypothetical situation wherein genotype A loses less
biomass (–20%) in response to soil nutrient limitation than does
genotype B (–40%). However, if genotype B for its inherently
associated rhizosphere microbiome responds optimally to a
mycorrhizal symbiont, then it may be that it loses the least biomass
(–10%) due to soil nutrient limitation, if the symbiont is present.%
denotes loss in biomass due to soil nutrient limitation.
Microbiomes for Plant Growth Promotion: The

Holistic Approach
Soil is a vastly heterogeneous growth medium, providing

a wide spectrum of ecological niches for microorganisms

that enable diverse strains to coexist and form complex
microbial communities. When the earliest plants extended
their roots into primordial soils, they encountered a habitat
already teeming with bacterial and fungal life (Bulgarelli et
al., 2013; Kemen, 2014). Since that early time, plants have
interacted with rhizosphere microbes, evolving strategies to
forge beneficial alliances with some while keeping others at
bay. Such early associations certainly had consequences on
plant growth and development. Therefore, a more holistic
approach is needed to understand better these microbes
and the roles they play in the overall health of plant and
soil (Figure 1). Again, recent advances in next-generation
sequencing technology and the decreasing costs associated
with that technology now allow us to evaluate how microbial
populations fluctuate in both space and time or to identify
core microbiomes that appear conserved among host
genotypes or species (Sergaki et al., 2018). Thus, although
culture-independent methods have contributed
tremendously to our understanding of plant-associated
fungal and bacterial community structures, the study of
microbiome functions remains challenging because of the
inherent noise of plant-associated microbial communities.
It is now well known that there are core sets of microbes
that, depending on the host, are recognized as keystone taxa
that consistently associate with healthy plants (Banerjee et
al., 2018). Consequently, researchers working with specific
plant-microbe interactions have increasingly acknowledged
the mitigating impact these larger microbial communities
have on individual plant-microbe outcomes for plant growth
promotion or fitness. Now, plant-associated fungal and
bacterial stains from various plant species are being
isolated, which will provide in the near future an inestimable
resource for assembling taxonomically defined microbial

communities with increasing complexity. Therefore, it is
now imperative to take advantage of this knowledge to
design consortia of microbes to maintain a sustainable
rhizosphere community, with key functional properties that
include plant protection, nutrient acquisition, and
alleviating biotic and abiotic stress responses. From that
perspective, synthetic community (SynCom) approaches can
provide functional and mechanistic insights into how plants
regulate their microbiomes (Figure 1). Not surprisingly,
recent culture-independent analyses thus have paved the
way for developing SynComs more often (Bodenhausen et
al., 2014; Armanhi et al., 2018; Carlström et al., 2019).
Mycorrhizal fungi, at least the arbuscular type, were early

symbiotic partners of most land plant species, improving
nutritional conditions through soil exploration and
pathogen resistance of host plants (Klironomos et al., 2000).
In reward for the essential physiological services, they
receive ca. 20% of net photosynthetic products from plants
(HoÈgberg et al., 2001). Other mycorrhizal systems may have
different nutritional benefits and costs, as has been
proposed for the serendipitous system (Craven and Ray,
2019). Additionally, third-party partners can modulate the
outcome of the tripartite interaction, such as the case of
mycorrhizal helper bacteria (Frey-Klett et al., 2007), fungal
endobacteria (Bonfante and Desirò, 2017; Bonfante et al.,
2019) like Candidatus Moeniiplasma
glomeromycotorum within the spores and hyphae of
Glomeromycotina (Naito et al., 2017), Rhizobium
radiobacter within Serendipita indica (Guo et al., 2017), and
N2-fixing endobacteria Pseudomonas stutzeri inside
basidiomycetes yeast endophyte Rhodotorula
mucilaginosa (Paul et al., 2020). Hence, it is imperative to

consider the composition and functioning of these
microbe–microbe interactions to understand
plant–microbiome associations in a holistic manner.
Principles and Management of Rhizosphere

Microbiomes for Sustainable Agriculture
Competence and Resilience of the Rhizosphere Microbiome:

Impact of Introduced Microbes on Native Microbiomes
In 1904, the German agronomist and plant physiologist

Lorenz Hiltner coined the term rhizosphere (Hartmann et
al., 2008) to describe the area around a plant root inhabited
by a unique population of microorganisms. Since then,
numerous studies have been undertaken to decipher the
interplay between plants and rhizosphere microorganisms,
encompassing a wide variety of plant growth-promoting
bacteria, fungi, insects, protozoans, viruses, etc. (Marschner,
2012; McNear, 2013). The majority of these studies have
traditionally followed a simple principle for maximizing
successful host infection by pre-inoculation onto the
targeted crop of choice to provide a competitive advantage
for a desired microbe. Conceptually, this increases the
relative abundance of a given beneficial microbe in the
rhizosphere, at least temporarily, to achieve the desired
benefit. Such studies typically take place in a controlled,
artificial condition, such as a defined growth medium in a
greenhouse, where competition from a native rhizosphere
community is relatively low or non-existent. As mentioned
above, this approach occasionally has failed once field

application is attempted or the benefits are dramatically
reduced in amplitude and/or endurance.
As an example, Lekberg and Helgason (2018) conducted

a literature survey of research papers published on
mycorrhizal functioning spanning a 30-year period
(1987–2017). The most striking finding of this survey was
that less than 5% of the work scientifically manipulated
mycorrhizal abundance in the field. While we are not
arguing the merit of greenhouse-based studies where the
number of variables can be controlled and accounted for,
yield gains in field conditions will continue to be modest
with such an approach. Rhizosphere competence must be
evaluated in a field situation if the true power of this
approach is to be realized.
Over the last few decades, mycorrhiza-based bio-

fertilizers containing one or several species of fungi were
developed in forestry and agriculture (Jeffries and Rhodes,
1987; Baraza et al., 2016; Igiehon and Babalola, 2017). These
inoculants are generally effective in plant growth promotion
under controlled lab and greenhouse conditions. However,
few targeted efforts have been made to measure
interactions between the introduced microbe(s) and the
native mycorrhizal community, let alone the more complex
rhizosphere microbiome (Svenningsen et al., 2018; Turrini
et al., 2018). To optimize outcomes from these interactions,
targeted research must be undertaken to understand how
such mycorrhiza-based biofertilizer integrate themselves
within the context of the native microbiome.
Integration of Rhizosphere Microbiomes in

Plant-Microbe-Nutrient Relationships
The soil microbial community often assists plants by

weathering minerals from rock surfaces and degrading
recalcitrant soil organic matter whereby soil microbes break
down soluble and insoluble organic matter and convert it
into inorganic, plant-available forms. Soil organic matter
turnover is thus considered a net positive, as it liberates
the nutrients locked up in organic matter. For this reason,
conventional farming has always relied heavily on soil tillage,
along with such other intensive agricultural practices as
usage of inorganic fertilizers, herbicides and pesticides.
However, it is already clear that such practices have negative
consequences on the functional diversity of soil
microbiomes. Long-term chemical fertilization has been
shown to dramatically decrease the soil pH, which leads
to a decrease in bacterial diversity and other changes in
microbial community structure (Sun et al., 2015). This was
well documented in the work of Kumar et al. (2017), who
showed that long-term application of high doses of
inorganic nitrogenous fertilizers severely reduces relative
abundance, diversity and structure of diazotrophs, which
play a key role in converting atmospheric N2 to plant-
available ammonium.
As mentioned above, soil bacterial communities play a

pivotal role in soil organic matter decomposition. In
particular, soil carbon and nitrogen are critical factors for
bacteria that rely on soil organic C and N decomposition
to obtain energy (Chen et al., 2014; Wild et al., 2014; Tian
et al., 2018). Further, different types of soil C selectively

manipulate soil microbial community composition, resulting
in changes in such belowground ecosystem functions as
decomposition and nutrient transfer and creating feedbacks
that may affect overall plant growth and productivity (Orwin
et al., 2006). For example, bacteria belonging to the
genera Chloroflexi, Nitrospirae,
and Planctomycetes preferentially feed on recalcitrant
organic C, whereas Proteobacteria and Bacteroidetes prefer
labile organic C present in the soil (Nie et al., 2018). For
this reason, amending the soil with such organic fertilizers
as compost or manure contributes to higher microbial
diversity and biomass compared to mineral-fertilized soils,
which in turn positively impacts soil health (Schmid et al.,
2018; Banerjee et al., 2019). Unfortunately, only a few
agroecosystem experiments exist that compare organic and
conventional management strategies over an extended
period for evaluation of impact on soil health and
restoration (Raupp et al., 2006; Khatoon et al.,
2020). Hartmann et al. (2015) took a metagenomics approach
to assess microbial diversity of soil in response to more than
20 years of continuous organic and conventional farming.
Not surprisingly, they found that organic farming increased
richness, decreased evenness, and shifted the structure of
the soil microbiota when compared with conventionally
managed soils under mineral fertilization (Hartman et al.,
2018; Li et al., 2020b). There also are reports of significant
alterations in the microbial community composition of both
summer maize and winter wheat in response to increased
nitrogen fertilization dose (Wang et al., 2018; Li et al., 2020a).
Clearly, a better understanding of the interactions between
the soil microbiome and conventional agricultural practices

is crucial for the development of sustainable management of
soil fertility and crop production.
Managing the Rhizosphere Microbiome to Induce Disease

Suppression in Soil
Disease suppressive soils were originally defined by Baker

and Cook (1974) as “soils in which the pathogen does not
establish or persist, establishes but causes little or no
damage, or establishes and causes disease for a while but
thereafter the disease is less important, although the
pathogen may persist in the soil.” Disease suppressive soils
are the best examples of microbiome-mediated protection
of plants against root infections by soil-borne pathogens.
Such disease-suppressive soils have been described for
various soil-borne pathogens, including fungi, bacteria,
oomycetes, and nematodes (Mazzola, 2007; Kwak et al.,
2018). To date, several microbial genera have been proposed
as key players in disease suppressiveness of soils, but the
complexity of the microbiome, as well as the underlying
mechanisms and microbial traits, remain elusive for most
disease suppressive soils (Toyota and Shirai, 2018).
Recently, Carrión et al. (2019) showed that upon pathogen

invasion, members of
the Chitinophagaceae and Flavobacteriaceae became
enriched within the plant endosphere. They proposed that
this bacterial population shift led to the induction of
enzymatic activities associated with fungal cell-wall
degradation, as well as secondary metabolite biosynthesis,
all aimed at accelerating and augmenting the plant defense
response(s). Although the disease suppressive abilities of

certain soils can be at least partially attributed to their
physico-chemical properties, the capacity of a soil to
suppress disease progression is more often attributed to
agri-management practices and crop rotation (Weller et al.,
2002). In classic studies by Gerlagh (1968) and Shipton et
al. (1973), the authors have shown soil to become disease
suppressive after mono-culturing wheat over time. More
recently, a comparative metatranscriptome analysis of
wheat rhizosphere microbiome grown in fields suppressive
and non-suppressive to the plant pathogen R. solani AG8
clearly revealed distinct dominant taxa in these two soil
types. Additionally, suppressive samples showed greater
expression of polyketide cyclase, terpenoid biosynthesis,
and cold shock proteins (Hayden et al., 2018). While
development of probiotics for the human gut microbiome
has already been an established field of research, the use
of probiotics that comprises naturally occurring bacterial
antagonists and competitors that suppress pathogens has
recently emerged as a promising strategy for disease
suppression in soil. A study on application of probiotic
consortia that comprised predefined Pseudomonas species
reported suppression of the bacterial plant
pathogen Ralstonia solanacearum in the tomato rhizosphere
microbiome (Hu et al., 2016). In another study, amendment
of Metarhizium, an insect-pathogenic fungus that is
commonly employed as biological control agents against
crop pests, in the rhizosphere of common bean (Phaseolus
vulgaris) significantly increased the relative abundance of
plant growth promoting such taxa
as Bradyrhizobium, Flavobacterium, Chaetomium,
and Trichoderma while suppressing the root rot disease
symptoms Fusarium solani (Barelli et al., 2020). Soil

suppressive properties are mostly derived from the
biological functions of soils. Therefore, elucidation of
microbial functions in suppressive soils by a next-
generation sequencing approach will facilitate the
development of effective, consistent and durable disease
management tools.
Impact of Agriculture Management Practices on the Soil

Microbiome
One important context for plant-microbe interactions is

soil structure, as it can vary greatly depending on land-use
history, plant species composition and successional stage
(Erktan et al., 2016). Besides playing pivotal roles in soil
organic matter decomposition, carbon cycling, nutrient
mobilization, etc., saprotrophic fungi also are involved in
creating soil structure through the secretion of extracellular
compounds and physical binding of soil via hyphal networks
(Bergmann et al., 2016). Interestingly, studies on the impact
of tillage on the soil fungal communities have shown mixed
results. Reports in no-till systems have varied from
increased ratios of fungal to bacterial biomass (Acosta-
Martínez et al., 2010) to decreased ratios (Mbuthia et al.,
2015), as well as no change at all (Mathew et al., 2012). More
recent studies have shown that soil fungal communities are
negatively impacted by tillage, as they typically would be
responsible for degrading crop residue left on the surface
with no-till (Yin et al., 2017). More specifically, soil bacterial
communities were primarily found to be structured by
tillage, whereas soil fungal communities responded mainly
to management type with additional effects by tillage
(Hartman et al., 2018). Additionally, it is acknowledged that

organically managed systems increased taxonomic and
phylogenetic richness, diversity and heterogeneity of the
soil microbiota when compared with conventional farming
systems (Lupatini et al., 2017). In a simple definition, organic
farming system consists of low-input agro-ecosystem farms
in which plant productivity and ecosystem functionality are
based on the natural availability of plant nutrients
(Lammerts van Bueren et al., 2002). A study aimed at
comparing the soil microbiome in conventional and organic
farming systems in central Europe revealed no major
differences among the main phyla of bacteria between the
two farming styles (Armalytë et al., 2019), whereas another
study that investigated the effects of 12 years of organic
farming on soil microbiomes in northern China reported
shifting of the community composition of dominant phyla
and significant alterations of functional groups associated
with ammonia oxidation, denitrification and phosphorus
recycling when compared to conventional farming systems
(Ding et al., 2019).
In addition to tillage, crop rotation also plays a pivotal role

in increasing belowground microbial diversity compared to
intensive mono-cropping practices. Although the United
States Department of Agriculture has advocated [via the
Conservation Reserve Program (CRP)] crop rotation to
improve eroded land as early as 1985 (Allen and Vandever,
2005), its benefit on soil health has only been recognized
recently. Several studies reported increases in such soil
quality parameters as organic matter content, microbial
biomass and respiration under crop rotation management
when compared with a mono-cropping system (Campbell et
al., 1991; Luce et al., 2013). A meta-analysis of 122 studies that
examined crop rotation revealed similar findings, namely

that adding one or more crops in rotation to a monoculture
substantially increased the soil microbial biomass along with
increases in total soil C and N, respectively (McDaniel et al.,
2014). In another study, soil microbial communities of corn
and switchgrass in mono-cropping systems when compared
with mixed prairie grasses demonstrated that bacterial and
fungal biomass, especially arbuscular mycorrhizal fungi,
were higher in plots with mixed prairie grasses (Jesus et
al., 2016). A 16S amplicon-based metagenomic analysis of
an almost 20-year-old field trial in Bernburg, Germany
revealed a significant effect of tillage practice and the
preceding crop on prokaryotic community structures (Babin
et al., 2019)
Cover crops are typically unharvested crops planted

between cash crops that augment C provisioning to the soil
system not only via unharvested residues, but also as root
exudates that can support many rhizosphere microbes
during the active growing season of the cover crop. Other
benefits attributed to cover cropping include improved N
fertility by incorporating legumes as a cover crop, reduced
soil compaction via deep-rooted plants, and reduced
erosion by keeping a plant and its root system in the field
year round (Fernandez et al., 2016). Of various crop rotation
management practices, those that include cover crops
sustain soil quality and productivity by enhancing soil C,
N and microbial biomass (Kim et al., 2020), making them a
cornerstone for sustainable agroecosystems. Nonetheless,
very few studies have assessed the relationship between
cover crop stands and their associated belowground
microbial communities. Early research in unfertilized
grasslands demonstrated that fungal communities respond
positively to plant-derived C inputs, suggesting that

inclusion of cover crops in a rotation may promote fungal
community development (Denef et al., 2009). More recently,
a field study tested this hypothesis by specifically examining
the impact on soil microbial communities of eight fall-sown
cover crop species grown singly and in multispecies
mixtures following a spring oats (Avena sativa L.) cropping
season and found that certain cover crops selectively
favored particular microbial functional groups. Arbuscular
mycorrhizal fungi were more abundant beneath oat and
cereal rye (Secale cereale L.) cover crops, while non-AM
fungi were positively associated with hairy vetch (Vicia
villosa L.) (Finney et al., 2017). Beyond positively affecting
soil C and increasing the diversity of such beneficial fungi
as arbuscular mycorrhiza, clover as a cover crop is often
reported to suppress the relative abundance of pathogenic
fungi (Benitez et al., 2016). Contrarily, in a 2-year field study,
cover crops reportedly increased overall phylogenetic
diversity of fungi but did not change the relative abundance
of saprophytes, symbionts or pathogens, implying that cover
cropping does not always appear to contribute to functional
changes in the fungal community (Schmidt et al., 2019).
Reassessment of Plant Responsiveness to Symbiosis
It is now increasingly evident that plants employ fine-

tuned mechanisms to shape the structure and function of
their microbiome, with different genotypes of the same
plant species growing in the same soil yet associating with
distinct microbial communities (Berendsen et al., 2012). This
is demonstrated in the findings of Bazghaleh et al. (2015),
who clearly demonstrated the importance of intraspecific
host variation in the association of chickpea cultivars with

AM and non-AM fungi. Therefore, specific traits of a plant
that modulate its microbiome should be considered as a
trait for plant breeding (Wallenstein, 2017).
Despite the obvious importance of beneficial

microorganisms for plant growth and fitness, and the
impact of plant genotype on shaping their microbiome
composition, plant germplasm is typically screened in the
absence of microbes, and the selection of best breeding
lines made solely based on the interaction between plant
genotype and performance under various abiotic factors.
We propose that an a priori examination of the interaction
between a plant genotype(s) and the symbiotic microbes
upon which it likely depends is an important factor in the
selection of plant breeding lines. It seems very likely that a
subset of rejected germplasm could outperform others, but
only when coupled with a beneficial microbe or microbiome
(Figure 2). Arguably, current breeding and selection efforts
most likely result in decoupling of the soil microbiome from
plant fitness. As a result, modern varieties may have lost
their ability to support diverse microbiomes and thus, fail to
gain the most from these interactions (Wallenstein, 2017).
It is now acknowledged that transitioning from a highly

intensive mono-cropping system to a more diversified
cropping system consisting of multiple host genotypes leads
to increased bacterial and fungal diversity (Calderon et al.,
2016). Hence, future plant breeding efforts should
incorporate plant characteristics that are related to
microbiome diversity. For example, efforts focusing on
manipulating plant root exudates likely play a critical role
in selective recruitment of the rhizosphere microbiome
(Bakker et al., 2012). In support of this notion, it has been
shown that plants can select which microbial populations

receive the lion’s share of root exudates, demonstrating a
capacity by the host to refine its microbial composition.
Hence, an unbiased screening of plant genotypes for
responsiveness in the presence of a beneficial microbe or
microbiome can set forth a new and potentially
transformative paradigm in selecting microbes for plant
growth promotion (Figure 2).
Significance of Mycorrhizas: A Critical Component of

Healthy Soil Rhizospheres
Mycorrhizae are mutualistic associations between soil

fungi and plant roots that gradually evolved to be
reciprocally beneficial to both partners (Brundrett, 2002).
The benefits are generally assumed to involve an exchange
of photosynthetically derived carbon from the host plant
in exchange for soil nutrients provided by the foraging
mycorrhiza. While likely true of arum-type arbuscular types
of mycorrhizae, there are other types that can derive carbon
from organic matter in the soil, or even “steal” it from one
host plant to supply to another (Allen and Allen, 1991). A
recent study has reported that in contrast to Arum
maculatum, in which carbon is entirely derived from photo-
assimilation, the green leaves of Paris quadrifolia contain a
striking 50% carbon of fungal origin. Such partial
mycoheterotrophy could thus potentially be widespread
among the roughly 100,000 plant species that are known
to develop a Paris-type AM, with far-reaching implications
for our understanding of C trading in plant-microbe
communities (Giesemann et al., 2019). Exactly what the
mycorrhiza gains from this interaction is still under debate,
but benefits may involve a safe haven from the open, more

competitive soil space and a second, more reliable carbon
source (Sapp, 2004).
Mycorrhizae not only shape plant communities, but they

also affect the functional diversity of their cohabitants in
the rhizospheric microbiome. The mycelium of mycorrhizal
fungi transports plant-derived carbon into the soil in the
form of sugars, amino acids and polyols to help sustain the
microbiome (Tarkka et al., 2018). More recent studies
focusing on soil microbial ecology revealed that mycorrhizal
fungi mediate many diverse interactions within the soil
“mycorrhizosphere,” including pathogens and mutualists
that fix atmospheric nitrogen, take up phosphorus, produce
vitamins, and/or protect against antagonists (Buée et al.,
2009; Tedersoo et al., 2020). The “ectomycorrhizosphere,”
which forms a very specific interface between soil and many
trees, hosts a large and diverse community of
microorganisms that likely play roles in mineral weathering
and solubilization processes (Uroz et al., 2007). This carbon-
rich mycorrhizosphere also supports large communities of
root-associated microorganisms that further accelerate
weathering of minerals by excreting organic acids, phenolic
compounds, protons, and siderophores (Drever and Vance,
1994; Illmer et al., 1995).
Similarly, the extraradical hyphae of arbuscular

mycorrhiza provide a direct pathway for the translocation
of photosynthetically derived carbon to the soil, leading to
the development of nutrient-rich niches for other soil
microorganisms, particularly bacteria. A quantitative real-
time PCR method detected significantly higher 16S rDNA
abundance in both the bulk and the rhizosphere soils of
zucchini (Cucurbita pepo L.) inoculated with Acaulospora
laevis and Glomus mosseae (Qin et al., 2014). Additionally,

arbuscular mycorrhizae have been reported to increase the
relative abundance of
Firmicutes, Streptomycetes, Comamonadaceae,
and Oxalobacteraceae inhabiting the mycorrhizosphere
(Offre et al., 2007; Nuccio et al., 2013). While there is clear
evidence that microbial communities in the rhizosphere
function cohesively with their mycorrhizal partner in
nutrient mobilization from soil minerals, nitrogen cycling
and protection of plants against root pathogens, such
bidirectional synergy is not always universal. There are
reports that indicate suppressive effects of bacterial
communities on mycorrhizal functioning and vice versa.
While one study reported (Svenningsen et al., 2018) that soil
with a higher abundance of Acidobacteria suppresses the
normal functioning of extra-radical mycelium in arbuscular
mycorrhizae, another study found that Glomus
intraradices and Glomus mosseae suppressed most of the
associated soil microbial community (Welc et al., 2010).
A Novel Type of Endophytic Symbiont:

The Serendipitaceae
A diverse group of fungi in the Basidiomycota,

the Serendipitaceae (formerly Sebacinales Group B)
(Oberwinkler et al., 2014) encompasses endophytes and
lineages that repeatedly evolved ericoid, orchid and
ectomycorrhizal types. Accordingly, in many natural
ecosystems these fungi form mycorrhizal symbioses with an
astounding variety of host plants – every mycorrhizal type,
in fact, except for arbuscular. Previous research performed
in our lab with a strain of this group, Serendipita vermifera,
demonstrated plant growth-promoting properties in a

variety of plants (Ghimire and Craven, 2011; Ray et al.,
2015; Ray and Craven, 2016; Ray et al., 2020). Unfortunately,
the agronomic utility of these fungi is hampered by the
paucity of strains available, the large majority isolated from
Australian orchids. We have begun to address this constraint
by isolating the first North American strain of Serendipita,
named Serendipita vermifera subsp. bescii NFPB0129, from
the roots of a switchgrass plant in Ardmore, Oklahoma
(Craven and Ray, 2017; Ray et al., 2018).
As mentioned above, soil organic matter has a tremendous

influence on the biological, chemical, and physical
properties of soils, making it a vital component of healthy
agricultural systems. Whether a natural soil or an
agricultural one, the release of the nutrients locked within
SOM requires decomposers, primarily insects, fungi, and
bacteria, to secrete organic acids and enzymes that can
loosen and break down the cellulose and the recalcitrant
lignin into nutritive forms that can be used by other
microbes and plants. Unlike arbuscular mycorrhizae, which
exchange inorganic, mineralized nutrients mined from the
soil for carbon derived from host photosynthesis, members
of the Serendipitaceae studied thus far have a complete
arsenal of carbohydrate-active enzymes (CAZymes),
representing approximately 4% of the entire gene set and
rivaling the more well-studied saprophytic white and brown
wood rotters, and much more than other symbiotic fungi.
Additionally, genome analysis of S. bescii and S.
vermifera suggests that Serendipitaceae fungi have the
metabolic capacity to assimilate N from organic forms of
N-containing compounds (Ray et al., 2019). We hypothesize
that this carbohydrate-degrading enzyme complement
endows these Serendipitaceae fungi with saprotrophic

abilities (Craven and Ray, 2019). Unlike free-living
decomposers that maintain a solitary lifestyle, seeking only
dead or dying plant tissues as their source of
subsistence, Serendipitaceae fungi seem to maintain a
largely symbiotic lifestyle with the roots of living host plants.
It currently is unclear whether there is expression of
CAZymes while strains of Serendipita are in symbiosis with
host plants, and if so, whether there is spatial or temporal
separation from more mutualistic traits. Still, the capacity of
some strains to form mycorrhizal relationships with orchids,
where the seeds require carbon from the fungus for
germination and often well into the plant’s lifespan, suggests
that these Serendipitaceae symbionts may be less of a
carbon cost to their host plant. Presumably, this saved
carbon could potentially be used for other symbiotic
relationships or developmental processes. In any case, these
intriguing fungi and their seemingly unlimited host range
provide a novel symbiosis that could be used in a broad
variety of cropping systems.
Conclusion
Soil-dwelling microorganisms are critical components of

soil health, itself a determinant of plant productivity and
stress tolerance. Deploying microbes to improve agriculture
productivity is an extremely attractive approach that is non-
transgenic and can be viewed collectively as the extended
plant genome. Because these same microbes can contribute
to restoring soil health and productivity, they have a bright
future in low-input, sustainable agriculture that extends
beyond more classically defined plant-microbe symbioses.

PR and KC conceived and planned the overall idea of the

review manuscript. PR, VL, JL, and KC wrote the manuscript.
All authors contributed to the article and approved the
submitted version.
Funding
This work was supported by the Center for Bioenergy

Innovation (CBI) project. The Center for Bioenergy
Innovation (CBI) was a United States Department of Energy
(DOE) Bioenergy Research Center supported by the Office
of Biological and Environmental Research (OBER) in the DOE
Office of Science.
Conflict of Interest
The authors declare that the research was conducted in

Acknowledgments
The authors thank Josh Meo for graphic design.

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Hotspot Quiz

online here:

• What roles does the soil microbiome have in the

movement towards sustainable agriculture?
• What is a rhizosphere?
• What type of soil fungi are associated with
promoting plant health as well as the soil
microbiome? How do they do so?
• What are the challenges with studying plant growth
promoting microorganisms?
• Explain the difference between a reductionist vs.
holistic approach for improving plant growth with
microorganisms.
• What types of agricultural practices can alter the
soil microbiome?
• How could the use of ‘probiotics’ suppress disease-
causing soil microorganisms?
Media Attributions
• Video 1 – Understanding Our Soil: The Nitrogen

Cycle, Fixers, and Fertilizer by Jimi Sol. Licensed

License https://creativecommons.org/licenses/
by/3.0/
• Hot Spot Quiz Figure – Plant and Soil
Microbiomes by Gopal & Gupta, 2016 licensed
under Creative Commons Attribution-Share Alike
4.0
References
1. Gopal, M., & Gupta, A. (2016). Microbiome Selection Could Spur

Next-Generation Plant Breeding Strategies. Frontiers in
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Microorganisms for Sustainable Agriculture. Frontiers in
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three to tango: the importance of microbes, host plant, and soil
management to elucidate manipulation strategies for the plant
microbiome. Canadian Journal of Microbiology, 66(7), 413–433.
https://doi.org/10.1139/cjm-2020-0085

14. Plant Microbiomes
Plant Microbiomes
Plant health is influenced by a variety of environmental factors,

and though the soil content (including the microbial community
present) is a major element, the distinct plant microbiome serves
an important role. Similar to organ systems in the human body
(e.g. skin, gut, etc.), the components of various plants (e.g. internal
tissues, leaves, roots, etc.) can have unique microbial communities
that contribute to its respective and overall vitality. A better
understanding of the corresponding roles of plant microbiomes can
be combined in a synergistic effort with other environmental
microbiomes to maintain and promote sustainable agricultural
practices, ecosystem biodiversity, and research model studies.

Plant Microbiomes | 357

Plant microbiome–an account of the factors
that shape community composition and diversity
Article by Dastogeer et al., 2020 licensed under the

terms of the Creative Commons Attribution License (CC
BY).
Abstract
Plants live in association with diverse microorganisms,

collectively called the microbiome. These microbes live
either inside (endosphere) or outside (episphere) of plant
tissues. Microbes play important roles in the ecology and
physiology of plants. Significant progress has been made in
revealing structure and dynamics of plant microbiome in the
last few years. Various factors related to host, microbes as
well as environment influence the community composition
and diversity of plant microbiome. This review aimed to
provide a general account of the factors (host, microbe and
environment) that drive the microbial community
composition in plant. First, we gave an overview of the
aboveground and belowground plant microbiomes. Next, we
discussed which host factors are involved in variation in
plants followed by importance of microbe-microbe
interactions and the elements of environment that influence
358 | Plant Microbiomes

composition and community structuring of plant
microbiomes.
1. Introduction
A diverse kind of microorganisms associated with a higher

organism (human, animal, plants etc.) is together defined
as its microbiome. All higher organisms examined to date,
including plants, insects, fish, rats, apes, and humans, harbor
microbiomes [1,2]. Research on the human microbiome has
progressed very quickly. Recently, researchers have also
paid much attention to elucidating the composition and
functions of plant and soil microbiomes. It is now believed
that plants are not separate entities, but rather they live in
association with a large variety of microbes. These microbes
live either inside (endosphere) or outside (episphere) of plant
tissues. Among these microorganisms, bacteria and fungi
are predominant. About a few thousand bacterial and fungal
taxa have been reported from plant tissues [3,4]. They play
important roles such as increased nutrient availability,
uptake by plants and increased plant stress tolerance. Thus,
plant fitness (growth and survival) is the result of physical
and physiological functions of the plant per se as well as the
associated microbiome, which together are known as a plant
holobiont [5].
The study of the association of plants with

microorganisms precedes that of the animal and human
microbiomes, notably the roles of microbes in nitrogen and
phosphorus uptake. The most notable examples are plant
root-arbuscular mycorrhizal (AM) and legume-rhizobial
symbioses, both of which greatly influence the ability of

roots to uptake various nutrients from the soil. Some of
these microbes cannot survive in the absence of the plant
host (the ‘obligate symbionts’ including viruses, some
bacteria and fungi), which provides space, oxygen, proteins,
and carbohydrates to the microorganisms. The association
of AM fungi with plants has been known since 1842, and
over 80 % of land plants are found associated with them [6].
It is thought that AM fungi helped in the domestication of
plants [7]. Traditionally, culturable microbes have been used
for plant-microbe interaction studies with the enormous
unculturable microbes remain uninvestigated and
consequently, our knowledge of the roles of these
unculturable microbes remains largely unknown.
Unraveling the types and outcomes of plant-microbe

interactions has received considerable interest among
ecologists, evolutionary biologists, plant biologists, and
agronomists [4,8,9]. Recent developments in meta-omics
and the establishment of large collections of
microorganisms have dramatically increased our knowledge
of the plant microbiome composition and diversity. The
sequencing of marker genes of entire microbial
communities, referred to as metagenomics, sheds light on
the phylogenetic diversity of the microbiomes of plants. It
also adds to the knowledge of the major biotic and abiotic
factors responsible for shaping plant microbiome
community assemblages [8].
However, our understanding on the roles of microbiomes,

with respect to their impact on plant ecology and
physiology, is still far from complete, and we are at the
beginning of knowing their functions [10]. The outcome of
this improved knowledge will have significant bearings on
a variety of experiments and applications, such as

development of biofertilizer and biopesticides for
sustainable agricultural production with less reliance on
agrochemicals, while augmenting yield and nutritional value
[11].
In this review, we will present an account of recent studies

and prospects of studying the plant microbiome. Firstly, we
will present an overview of aboveground and belowground
plant microbiomes. Next, we will discuss the factors driving
the composition and community structuring of plant
microbiomes. We will not address plant pathogenic
microbes, although inclusion of this fraction of microbiome
would make sense from a broader and more holistic
viewpoint.
2. Rhizosphere microbiome
The rhizosphere comprises the 1–10 mm zone of soil

immediately surrounding the roots that is under the
influence of the plant through its deposition of root
exudates, mucilage and dead plant cells [12]. A diverse array
of organisms specialize in living in the rhizosphere,
including bacteria, fungi, oomycetes, nematodes, algae,
protozoa, viruses, and archaea [13]. The most frequently
studied beneficial rhizosphere organisms are mycorrhizae,
rhizobium bacteria, plant growth promoting rhizobacteria
(PGPR), and biocontrol microbes. Gans, Wolinsky [14]
projected that one gram of soil could harbor more than a
million distinct bacterial genomes. İnceoğlu, Al-Soud [15]
reported 55,121 OTUs (operational taxonomic unites) from
the potato rhizosphere. Among the prokaryotes in the
rhizosphere, the most frequent bacteria are within the

Acidobacteria, Proteobacteria, Planctomycetes,
Actinobacteria, Bacteroidetes, and Firmicutes [3,16]. In some
studies, no significant differences were reported in the
microbial community composition between the bulk soil
(soil not attached to the plant root) and rhizosphere soil
[17,18]. Certain bacterial groups (e. g. Actinobacteria,
Xanthomonadaceae) are less abundant in the rhizosphere
than in nearby bulk soil [3].
Mycorrhizal fungi are abundant members of the

rhizosphere community, and have been found in over
200,000 plant species, and are estimated to associate with
over 80 % of all plants [19]. These mycorrhizae–root
associations play profound roles in land ecosystems by
regulating nutrient and carbon cycles. Mycorrhizae are
integral to plant health because they provide up to 80 %
of N and P requirements. In return, the fungi obtain
carbohydrates and lipids from host plants [20]. Recent
studies of arbuscular mycorrhizal fungi using sequencing
technologies show greater between-species and within-
species diversity than previously known [21].
3. Phyllosphere microbiome
The aerial surface of a plant (stem, leaf, flower, fruit) is

called the phyllosphere and is considered comparatively
nutrient poor when compared to the rhizosphere and
endosphere. The environment in the phyllosphere is more
dynamic than the rhizosphere and endosphere
environments. Microbial colonizers are subjected to diurnal
and seasonal fluctuations of heat, moisture, and radiation.
In addition, these environmental elements affect plant

physiology (such as photosynthesis, respiration, water
uptake etc.) and indirectly influence microbiome
composition. Rain and wind also cause temporal variation
to the phyllosphere microbiome [22]. Overall, there remains
high species richness in phyllosphere communities. Fungal
communities are highly variable in the phyllosphere of
temperate regions and are more diverse than in tropical
7
regions [23]. There can be up to 10 microbes per
2
cm present on leaf surfaces of plants, and thus the bacterial
population of the phyllosphere on a global scale is estimated
26
to be 10 cells [24]. The population size of the fungal
phyllosphere is likely to be smaller [25]. Phyllosphere
microbes from different plants appear to be somewhat
similar at high levels of taxa, but at the lower levels taxa
there remain significant differences. This indicates that
microorganisms may need finely tuned metabolic
adjustment to survive in phyllosphere environment [24].
Proteobacteria seems to be the dominant colonizers, with
Bacteroidetes and Actinobacteria also predominant in
phyllospheres [26]. Although there are similarities between
the rhizosphere and soil microbial communities, very low
similarity has been reported between phyllosphere
communities and those in open air [27].
4. Endosphere microbiome
Some microorganisms, such as endophytes, penetrate and

occupy the plant internal tissues, forming the endospheric
microbiome (Fig. 1). The AM and other endophytic fungi are
the dominant colonizers of the endosphere [28]. Bacteria,
and to some degree Archaea, are important members of
endosphere communities. Some of these endophytic

microbes interact with their host and provide obvious
benefits to plants [[29], [30], [31]]. Unlike the rhizosphere
and the rhizoplane, the endospheres harbor highly specific
microbial communities. The root endophytic community
can be very distinct from that of the adjacent soil
community. In general, diversity of the endophytic
community is lower than the diversity of the microbial
community outside the plant [18]. The identity and diversity
of the endophytic microbiome of above-and below-ground
tissues may also differ within the plant [28].
The ‘Plant Microbiome’ consists of diverse microbial communities on

the outside surface and in internal tissues of the host plant. The
rhizosphere, endosphere, and phyllosphere constitute the major
compartments of plant microbiome. The soil microbiome is an
important source of the plant microbiome.

5. Drivers of plant microbiome composition
Plant microbiome structure is influenced by complex

interactions between hosts, microbes, and associated
environmental factors such as climate, soil, cultivation
practices etc. (Fig. 2). Below, we provide an assessment of
current knowledge of these factors, providing insight to
plant-microbe interactions in a broader-sense.
A simple schematic representation of various factors that shape

microbiome community structures. The host, microbe, and
environment all factors are interconnected and together build the
microbiome community in plants.

6. Host factors that influence plant microbiome
community composition
6.1. Plant species
The identity of the host plant has a significant influence

on the identity of its microbiome. Different plant species
growing adjacent to one another can harbor distinct
microbiomes. A comparative survey of root microbiomes in
maize, sorghum, and wheat showed different community
composition among these plants [32]. Samad et al. [33]
investigated the microbiome compositions of roots and
rhizospheres using 16S rRNA gene from grapevines and
some weed species growing in the same field, and their
findings suggested that these species hosted significantly
different microbiomes in the roots and rhizosphere, with
the more pronounced difference in the root communities.
Plants that are distantly-related phylogenetically show
greater variation in associated microbiome compositions,
suggesting a role of plant phylogeny in structuring root
microbiomes [32]. Plant species also influences the identity
and diversity of endophytic communities. Manter et al. [34]
reported differences in endophytic community composition
in potato and Eucalyptus plants. The most abundant
bacterial root endophytes in potato were rare or absent
in Eucalyptus and vice-versa [34] suggesting that the host
plant selects its endophytic microbes. Analysis of
endophytic fungi of three native
Australian Nicotiana species revealed that they are host-
specific but not plant organ- or host location-specific [28].

In addition to the rhizospheric and endophytic
microbiomes, phyllosphere community composition also
depends on plant identity [24]. Kembel et al. [35] showed
that the leaf microbiome community is highly correlated
with plant evolutionary relatedness similar to the
endospheric microbiome.
The effects of host plant species in recruiting microbes

from the surrounding environment indicate that plants have
evolved traits that govern root microbiome assemblages
[36]. For example, endosphere, rhizosphere community
composition are correlated with host taxonomy [36]. Xiao
et al. [37] found that the rhizosphere and root microbiomes
are mostly influenced by soil type, and the nodule and root
endophytes are influenced by plant species. Differential
microbiome assembly in different plant species is attributed
to variation in plant resource consumption [36]. Plant traits
such as leaf permeability, wettability and topography and
physicochemical properties, cuticle chemistry, root
exudates, antibiotic production, and inherent plant
immunity to invasion by microbes may also to play a role.
6.2. Plant genotypes
Evidence suggests a difference in microbe community

composition between genotypes of a particular species
[17,26,38]. Genetics of the host is one of the factors that
shape the plant-microbiome structure. For example, OTUs
in three different potato varieties were cultivar-specific
[36]. Similarly, cultivar-dependent effects have been
reported for the bacterial communities in young potato
rhizospheres [15]. Peiffer et al. [39] demonstrated that OTU

richness and β-diversity are influenced by plant genotypes
in maize. Bulgarelli et al. [38] reported that genotype
contributed to about 6% of the variation of the microbiome
composition in the rhizosphere region. A larger influence
of host genotype on community composition has been
reported [40]. Genotype-dependent microbiome
community structuring has been reported for sweet potato,
wheat, pea, and oat [9,41]. Bacteria such as Acinetobacter,
Chryseobacterium, Pseudomonas,
Sphingobium, and Stenotrophomonas were more abundant
in low-starch cultivars than those having high-starch
contents [41]. The rhizosphere communities of different
genetic clones of wild-type and transgenic lines have been
reported to be distinct in Populus [42]. Within-species
genetic variability can influence microbiome composition in
leaf tissues [43]. Wagner et al. [44] conducted an extensive
field experiment to unravel drivers of community
composition of bacteria associated with leaves and roots
of Boechera stricta. Their findings suggested that the host
genotype influences leaf community, but the root
microbiome was variable at different collection sites.
Agler et al. [45] proposed that host genotype influences

on keystone microbes, which then pass these effects onto
the total microbiome by changing microbe–microbe
interactions and altering plant fitness [3]. The host
specificity of plant microbiome could also be attributed to
the nutrient preferences of plants [46]. However, whether
the observed host influences are heritable, how strong the
effect is, and whether these associations will be actionable
for plant breeding is yet to be established.

6.3. Plant organ
Different plant tissues host distinct microbiome

communities. Edwards et al. [47] demonstrated that each
surface and internal tissue of plants may harbor distinct
microbial communities and that the role of tissue-type was
greater than host type and the microbiome of the soil. This
may be because the adaptation strategies of various tissues
may affect the microbes in colonizing them for community
composition. For instance, surface tissues are exposed to
constant fluctuations of weather and have relatively poor
nutritional status compared to the root or internal tissues.
Therefore, microbes colonizing the leaf surface need to be
adapted in these conditions [25]. Other studies found very
little or no effect of plant organ in community composition
of fungi and bacteria [28].
Study with agaves determined that prokaryotes are

largely influenced by the plant compartment, whereas the
rhizosphere, the phyllosphere, and endosphere
communities are clearly different from each other and from
adjacent soils [48]. Many studies have reported that fungal
communities show a different pattern, where the
biogeography of host were the major influencing factor [48].
This may be explained by the dispersal limitation in fungi
[49], because fungi are eukaryotes like plants and animals,
but bacteria are different in this respect.

6.4. Plant age and developmental stage
In the cases of interactions between plants and

pathogens, ontogenic resistance (age-related resistance) is
widely reported and correlated with plant developmental
stage [50]. Symbiosis research has also indicated that plant
age and developmental stage are important factors affecting
microbial communities [51]. Analysis of the bacterial
rhizosphere community of Arabidopsis revealed that the
seedling stage selects distinct microbiomes at
developmental time points. Plants produce mixtures of
compounds and specific phytochemicals in the root
exudates. Some of these chemicals are indeed distinct at
plant developmental stages and appear to shape
microbiome community assemblages [52]. The effect of the
plant age on microbiome using DGGE fingerprint analysis
revealed that it significantly influences bacterial community
composition of all groups investigated for all three sweet
potato cultivars [41]. The effect of plant age on the
composition of bacterial microbiome in the rhizosphere has
been demonstrated in potato, maize and soybean [15,53,54].
Age-related microbiome differentiation may be associated
with root growth, physiology, root architecture, root
morphology, root exudate, and its composition [51,52].
However, further research is needed on the identity and
effect of root exudates at different plant developmental
stages to determine how plants communicate in the
rhizosphere. This knowledge might offer a basis for
augmenting agricultural crops by the application of
rhizosphere microbes.

6.5. Plant canopy type
Plant canopy type also influences microbiome community

composition. For example, bacterial communities in sugar
maple leaf samples are correlated with canopy composition
[55]. The microbial migration through rain runoff may be
an important factor for variation in microbial colonization
in different canopy types. Canopy structure influences the
composition of endophytic community but not the
rhizospheric community, indicating less effect of rain
runoff, and there may be other mechanisms such as soil
factors or some unknown factors involved in this variation
[55].
6.6. Plant immunity and signaling
Plant health status may influence microbiome

composition. Plants employ two layers of defense against
pathogens: pattern-triggered immunity (PTI), which is
triggered by conserved molecular structures such as
microbe/pathogen-associated molecular patterns
(MAMPs/PAMPs), and damage-associated molecular
patterns, which are recognized by plasma membrane-
localized pattern recognition receptors [56]. It is unknown
whether plants recognize non-pathogenic microbes in the
similar way as they recognize pathogens and modulate their
response. When plants are challenged with herbivory or
pathogens they release hormones and exogenous volatiles
that alter the composition of root exudates (for review, see
[57]), and these in turn modify the microbiome community.

Aphid infestation and pathogenic microbial infection
increaseed populations of the non-pathogenic
rhizobacterium Bacillus subtilis in the sweet pepper
rhizosphere [58]. In Arabidopsis thaliana plants infected
by Pseudomonas syringae, the expression of root malate
transporter is altered, indicating a change in secretion of
malic acid that increased the number of the beneficial
rhizobacterium Bacillus subtilis [59,60]. Cucumber roots
infected by Fusarium oxysporum f. sp.
cucumerinum exhibited augmented secretion of fumaric
and citric acid, which led to the formation of biofilms
(aggregates of living bacteria in a slimy extracellular
polymeric substances) of Bacillus amyloliquefaciens. Most
investigations, however, have focused on one-to-one
interactions (plant-microbe), although in reality, plants are
subjected to attack by numerous microbial pathogens and
insect pests. Therefore, it would be interesting to see how
multiple herbivory and/or pathogens modify the
community composition of plant microbiomes. Recent
studies have reported a change in rhizosphere microbiome
community composition as influenced by specific
compounds such as sugars, sugar alcohols, or mixtures of
various chemical compounds in root exudates [52]. Plants
use various strategies in response to pathogenic infection
and insect attack. One of them is activation of defense
responses in roots, which may influence microbiome
composition in the rhizosphere and roots [61]. When aphids
feed on foliage both SAR (Systemic Acquired Resistance) and
ISR (Induced Systemic Resistance) signaling are activated
throughout the plants, which elicits sweet pepper plants to
attract B. subtilis in the rhizosphere [58]. Again, increased
JA signaling in plant either by injury or exogenously

in Medicago truncatula caused higher colonization of
beneficial mycorrhizae [62]. Different root and phyllosphere
endophyte microbial communities have been reported when
altered SA signaling was induced [63]. Plants lacking in
jasmonate-mediated defense have shown more diverse
epiphytic colonization [64]. It is evident from these studies
that the role of plant defense systems on the microbial
composition are inconstant, and that SAR is an important
factor in regulating some bacterial community composition.
Chemical signals released by plants for example, flavonoids,
activate varied responses in plant rhizosphere microbiomes
[65]. Branching in mycorrhizal hyphae is affected by
strigolactones, which enhance and promote seed
germination by parasitic plants [66].
6.7. Plant derived compounds
A diverse array of antimicrobial compounds are produced

in plants [67]. Some of them, such as different alkaloids,
phenolics, and terpenoids, are common in plants. Some are
specific to particular groups [68], for instance, Brassicales
produce glucosinolates. It was found that
transgenic Arabidopsis producing an exogenous
glucosinolate had different microbiomes in the rhizosphere
and root tissues [69]. Voges et al. 2018 reported a significant
role of plant derived coumarins in structuring the
rhizosphere community. They suggested that iron-
mobilizing coumarins are involved in redox reactions that
can mobilize ferric iron and generate reactive oxygen
species (ROS) with detrimental effects on microbial
proliferation and thus selectively inhibit certain microbial
growth while allowing proliferation other more beneficial

partners. Triterpenoid saponins, which are known as
avenacins, are found in oat (Avena strigosa), and have
antifungal properties [70]. Oat plants lacking avenacin
production attracted different culturable fungi in roots [71]
and were more vulnerable to pathogenic infections than
wild-type oat [72]. Interestingly, however, a recent
comprehensive analysis of the rhizosphere community of
these two genotypes reported little difference between the
fungal communities. The effect of avenacins on the
Amoebozoa and Alveolata was profound but has not been
reported for bacterial communities [73]. This revealed that a
small difference in plant genotype might exert multifaceted
and unpredicted effects on the plant microbiome
composition and diversity. These plants derived compound
may affect microbiome assembly in different ways. For
example, root exudates may be specific for host plant and
can modulate rhizosphere community as well as selects
specific root microbiome and thus contributing host
specific plant microbiome. Also, antimicrobial compound
may selectively enhance microbial growth by restricting
certain microbes which is a kind of ‘balance’ in mutualism.
7. Microbial factors in shaping plant microbiome

structure
Microorganisms play important roles in shaping microbial

community structures in plants. However, our knowledge
on how microorganisms influence microbiome structuring
is limited.

7.1. Microbial manipulation of hosts
Microorganisms can affect host plants, for example host

root exudations, which in turn affect the permeability of
roots and root metabolism. Some microbes in soil where
the plant if growing can also absorb certain compounds in
root exudates and excrete other compounds. Soil microbes
can produce compounds that affect plant signaling and
metabolism, which lead to production of microbe-derived
compounds in plants. Some microbes produce antibiotics
(e.g., penicillin and polymyxin) which increase the exudation
of organic materials, altered cell permeability, and increased
leakage [74] and results in a variable microbiome assembly.
7.2. Microbe-microbe interaction
The extent to which microbe–microbe interactions can

play roles in the microbiome composition is not well
understood. The outcome of microbe-microbe interactions
could be explained as cooperation, parasitism, and
competition. In cooperation, at least one species benefits,
while others are not harmed. When both species benefit, the
term mutualism is used, whereas, when one partner benefits
while the other is not affected, the term commensalism is
used. In contrast, parasitism and competition are harmful
for at least one species [75].
We know from recent studies that microbial communities

harbor highly connected taxa called keystone taxa [76].
These taxa independently or in a group show a substantial

effect on microbiome composition and functions regardless
of their spatial and temporal dynamics. They play a unique
and vital role on microbiomes, and their absence could
cause a significant alterations in microbiome composition
and functioning [76]. They use various strategies to impact
on host microbiome. For instance, they might cause changes
in intermediate or effector groups which in turn regulate
microbiome community composition and functioning [77].
Production of a secondary metabolite (2,4-diacetyl
phloroglucinol) was reported for some strains
of Pseudomonas fluorescens that suppress Gaeumannomyces
graminis var. tritici, responsible for wheat take-all [78].
Similarly, many fungi (e.g. Trichoderma and bacteria
(e.g. Bacillus) produce various secondary metabolites that
suppress microbial growth [79,80]. The keystone taxa may
produce bacteriocins to shift microbiota structure
selectively. Again, by synergism, keystone taxa may alter
the abundance of their partners, and influence community
structure and performance. For example, certain species
of Burkholderia are symbiotic with arbuscular mycorrhizae
and may change abundance and community composition
of AM fungi, thereby influencing plant community richness,
diversity, and production [19]. Agler et al. [45] studied the
roles of microorganisms (bacteria, fungi and oomycetes) in
the community composition of phyllosphere microbiomes
of Arabidopsis thaliana using a systems biology approach.
They described an interkingdom interactions network with
a profound influence on community structure. They
identified a few taxa, termed “microbial hubs’, which are
highly interlinked and have a significant impact on
communities. They used two “hub” microbes (Albugo, an
oomycete pathogen and Dioszegia, a basidiomycete yeast)

in detail. Albugo strongly affected epiphytic and endophytic
bacterial colonization. Many symbiotic microbes (including
pathogens) produce effector proteins to suppress, activate,
or alter host defense mechanisms [81], and some can
entirely reprogram the host metabolism [82]. These host
adjustments can lead to alterations of microbiome
composition because some microorganisms and not others
can benefit from altered conditions. Actually, the niches of
some microorganisms is dependent on others. For instance,
primary colonizers can aid subsequent colonizers against
hazardous abiotic factors [83] or can enhance the
competitive ability of following colonizers by producing
secondary metabolic compounds [84]. There can be direct
microbe-microbe interactions, such as the hyper-parasitism
(parasite of parasite) of primary colonizers [85] and
opportunists that utilize host’s compromised plant defenses
to colonize them [86]. Such phenomena point out why some
colonizers can affect the development of microbes on the
host even if they may be distantly related [84] and highlight
a crucial functions of such interactions in shaping
microbiome composition and structure.
One of the major strategies by which PGPR augments

plant growth is by its influence on rhizosphere microbes.
For example, Pseudomonas sp. DSMZ 13134 alters the
composition of dominant bacteria in barley roots [87]. In
some cases, however, the effects of PGPRs on resident
microbiome may not be prominent. For example, no
substantial changes in rhizosphere community were noticed
after the application of Bacillus amyloliquefaciens FZB42
[88]. Supporting results have also been reported in soybean
with the application of B. amylolique faciens BNM122 [89].
The recent investigation on the effect of B. amyloli

quefaciens on the lettuce microbiome using 454-amplicon
sequencing revealed no or only transient and minor effects
in the rhizosphere zone [90]. Interestingly, a decrease in the
bacterial number was reported. In the field only 55 % of
the inoculated bacterial DNA could be traced after a month
[91]. The effects of Bacillus subtilis strain PTS-394 on the
rhizosphere microbiome has been examined by
metagenomic profiling. Similar to the results above for B.
amyloliquefaciens FZB42 [90,91], only a minor effect on the
composition was reported. However, up until now, the
impact of Bacillus PGPR on other plant microbiota, such as
fungi, has not been investigated, and investigation on this
could reveal a more general effect of inoculated bacteria
on resident microbiota and thus on host physiology and
ecology.
8. Environmental factors as drivers of plant

microbiome assembly
Environmental elements, such as soils, climatic

conditions, geography, farming activities, and plant
domestication, could result in the differences of plant
associated microbial community composition [92]. A change
in an environmental component results in plant phenotypic
changes (e.g., [93], which consequently also change the
assemblage of distinct microbiomes harboring plant
compartments [94]. Plants grown under controlled
conditions provide specific environments for
microorganisms. For instance, when lettuce was grown
under a glasshouse, a distinct bacterial signature was found
from those grown in open fields [95]. Whitaker, Reynolds et
al. [96] reported the community composition of endophytic

fungi local environment (i.e., site), but not by host ecotype,
pointing that environmental factors are major drivers of the
endophytic mycobiome of switchgrass.
8.1. Soil
Plants recruit root microbes mainly from the soils where

they grow. Various soil factors viz. soil types, soil pH, and
the C/N ratio, as well as available P and K are frequently
reported to be the determinants of root microbial
community composition by affecting plant growth and
immunity [8,9]. Innumerable studies using high throughput
sequencing have proved that soil type is a major factor for
root microbiome structure, which is evident from the
differential initial microbial inocula present in different soil
types [18,47]. Dombrowski et al. [97] collected the arctic-
alpine Arabis alpine samples from the native location as well
as from those grown under controlled conditions and
investigated the root microbiome by 16S rRNA amplicon
sequencing analysis. They reported that soil type and length
of time the plants remained in the soil are the most
important drivers, causing variation of up to 15 % of root
microbiota. In addition, in the same soil, the root
microbiome of perennial A. alpina was similar to A.
thaliana and Cardamine hirsuta, the annual relatives of A.
alpina. The root microbiome communities are strongly
influenced by the composition of the soil microbiome close
to roots. A strong correlation between the soil and root
bacterial communities in A. thaliana has been reported by
various authors [3,18]. Similarly, the structure of fungal
communities is influenced more predominately by soil type
than by host plant [98]. The type of soil influenced the

rhizosphere bacterial microbiota composition in lettuce
[99], oak [16], Arabidopsis [3], and maize [100].
Several studies have shown that environmental variability,

such as soil pH, C: N ratio, soil carbon, water content and
biogeography may influence the microbiota composition
[39]. Lauber et al. [101] described that the impact of soil
pH on total community composition was obvious even at
a very high taxonomic level. Analyses revealed that pH has
substantial correlation with the structure of these
microbiome phyla in all soil types studied. The effects of
soil pH on soil bacterial community composition has also
been reported in other studies using various methods [102].
Zarraonaindia et al. [103] reported that the composition of
soil and root microbiome of grapevine significantly
influenced by soil pH and C:N ratio but leaf- and grape-
associated microbiota were mostly influenced by soil
carbon. Hartman, Richardson et al. [104] reported from their
study that pH was the most important factor in predicting
the alteration of soil bacterial communities, and they
detected changes in phylum-level abundances across the
pH levels. However, contrasting results have been reported
by Fierer et al. [105], where they noted soil carbon was
more important than soil pH for Bacteroidetes,
Betaproteobacteria, and Acidobacteria. This difference of
the findings was possibly linked to sample sizes and number
of soil types investigated as well as to differences in
methodologies. The exact mechanism(s) of the role of soil
pH on microbial community composition and diversity is
unknown. Two general explanations have been given [101].
Firstly, soil pH may indirectly alter bacterial community
structure with its influences on soil characteristics.
Secondly, soil pH may directly impose a physiological limit

on soil bacteria, changing outcomes of competition or
reducing survival of taxa intolerable to condition. In
addition, we hypothesize that soil pH may alter plant
microbiome by its influence on plant growth and physiology,
which also may influence soil microbiome composition.
Other soil properties, for example soil temperature and
contaminants in the soil, also influence microbiome
composition. Heat disturbance of soil results in a shift in
rhizobacterial microbiome composition [106]. Heat
disturbances due to natural wildfires can cause a decrease
in microbial activity and significant alterations in microbial
communities [107]. Increasing petroleum hydrocarbon
contamination levels results in the alteration of willow
microbiome structure. These alterations were less extreme
in the rhizosphere and plant tissues, but they were
prominent in the bulk soil. These could be because plants
provide more controlled conditions and shield microbes
against an enhanced contamination gradient [108].
8.2. Cultivation practices
Land use and cultivation practices are the most important

causes of declines in biodiversity, leading to undesirable
consequences for the environment [109]. Changes in the
vegetation influence the diversity and structure of soil
microbiomes. Agricultural activities do not necessarily have
negative consequences in soil bacterial community diversity
and structure, but they may have positive or neutral
feedback (effect not perceived) [110]. For example, the
intensity of land use (LUI) has been reported to influence
the pattern of bacterial communities. Estendorfer et al. [111]
found that under low LUI, there remains a strong interaction

between plants and adjacent soil. In contrast, no influence
of LUI on microbial diversity has been found in the
rhizosphere, which indicates that plant species have much
more influence on the rhizosphere community than soil
properties do [112]. However, Suleiman et al. [113] reported
that plants may have robust core microbiome compositions
that are less prone to alteration due to variation of land use,
soil type or edaphic factors. It was found that microbiome
of in relatively untouched deciduous forest and long-term
mowed grassland soils were comparable, although there
were significant differences in soil properties and vegetation
[114], Continuous cultivation causes changes in soil
properties, which in turn might affect the soil microbiome
communities. As per the reports of Allison and Martiny [115],
there are three potential impacts caused by land
disturbance such as the microbial structure might be
affected, it might be changed but quickly return to the
original composition (resilient), or might remain unchanged.
8.3. Climatic variables
Climate is an important driver of plant and soil

microbiome composition. The role of climate in influencing
plant microbiome community composition has to date been
largely unheeded or found to be of little importance [116]. A
recent study, however, across various ecosystems in Britain
showed that rainfall and temperature gradients are two
major climatic factors in shaping the bacterial community
composition in plants [117]. Researchers de Vries et al. [118]
reported that precipitation is an important driver of soil
microbial community composition. The biomass of fungi
and bacteria has been reported to increase with increasing

mean annual precipitation (MAP), and the effect was more
prominent for fungi, resulting in comparatively higher
fungal abundance (increased F/B ratio) under higher
precipitation levels. The above trends could be linked to
higher soil organic matter contents in higher rainfall areas.
In particular, in uplands where harsh climatic conditions
prevail, a higher organic matter build-up is noticeable,
which leads to fungi-dominated microbiome communities
[119].
9. Conclusions and future perspective
The factors that influence microbiome assemblages and

dynamics in plant and soil are now better understood, and
research in this aspect is increasing. However, our
knowledge on the underlying mechanism(s) of microbiome
assemblages and how they influence the host plants is still
lacking. Connecting the microbiome composition and
diversity to their function is a great challenge for future
research. For instance, to what extent could we use and
manipulate the plant microbiome to boost sustainable
agricultural production and environmental protection? We
now know that that host genetic factor has a significant
influence on microbiome diversity and structure, indicating
that breeding and trait selection provide opportunities to
select for desired microbiomes [45]. To have a more
profound and broader knowledge on plant microbiome,
there is a need to integrate novel molecular approaches
(e.g., meta-omics), ecological models (e.g., food web theory,
assembly of communities, or coexistence theory), and
recent bioinformatics and statistical advances with a view

to correlating community assemblages with ecological
functions.
In the future, more emphasis should be placed on

identifying the underlying mechanisms that drive
microbiome community composition and assembly. We
need to know the contributions of (1) the microbe-microbe
interactions, (2) soil and other environmental variable and
(3) various host traits in shaping community structure.
Future research will direct toward solving some pertinent
questions. For example, how stable are the drivers of
microbiome community? To what extent do agricultural
practices affect the microbiome of plant species? How
predictable are these divers? With high throughput
technologies, such as next-generation sequencing and
metagenomics, we can begin to study endophyte
microbiomes across hosts, environmental conditions, and at
different time points and focus on the mechanisms of the
plant-endophyte association.
Declaration of Competing Interest
The authors declare that they have no known competing

financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
We thank Stephen J Wylie, PhD, Murdoch University,

Australia for kindly checking the manuscript in part.

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100161 Google Scholar




Hotspot Quiz

online here:
2
• What are the various types of plant-associated

microbiomes and how does each affect plant health?
• How are the compositions of plant microbiomes
influenced by the environment?
• What aspects of the host plant can shape its

microbiome?
• In what ways can the plant microbiome be
governed by microbe-microbe interactions?
Media Attributions
• Video 1 – Disease-induced changes in plant

microbiome assembly and functional
adaptation by Research Square licensed under
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The plant microbiome in facts (3.1) by iMooX at
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References
1. Dastogeer, K. M. G., Tumpa, F. H., Sultana, A., Akter, M. A., &

Chakraborty, A. (2020). Plant microbiome–an account of the
factors that shape community composition and diversity.
Current Plant Biology, 23, 100161. https://doi.org/10.1016/
j.cpb.2020.100161

15. Pollution and
Bioremediation
Pollution and Bioremediation
Achieving sustainable life, for humans, animals, and the

environment, requires a plan of action to mitigate
anthropogenically-induced damage and develop future practices to
maintain planetary homeostasis. One of the biggest threats to
ourselves and the environment is the buildup of human waste and
pollution (e.g. plastics, oil, synthetic products, etc.) that take a
tremendously long time to naturally breakdown. With the human
population predicted to continually climb, continuing the same
destructive practices will only result in more waste generation in a
fraction of the time that it takes for decomposition. One alternative
means to solve the pollution problem is to use specialized
microbiomes for bioremediation. That is, a specially designed
microbial consortia could clean up (i.e. breakdown and assimilate)
various toxic molecules much more quickly. However, this will take
time to understand how and which type of microbes could perform
these tasks and if there are any associated (negative) effects.
406 | Pollution and Bioremediation

Alternative Strategies for Microbial
Remediation of Pollutants via Synthetic Biology
Article by Jaiswal and Shukla, 2020 licensed under the

BY).
Continuous contamination of the environment with

xenobiotics and related recalcitrant compounds has
emerged as a serious pollution threat. Bioremediation is the
key to eliminating persistent contaminants from the
environment. Traditional bioremediation processes show
limitations, therefore it is necessary to discover new
bioremediation technologies for better results. In this
review we provide an outlook of alternative strategies for
bioremediation via synthetic biology, including exploring
the prerequisites for analysis of research data for developing
synthetic biological models of microbial bioremediation.
Moreover, cell coordination in synthetic microbial
community, cell signaling, and quorum sensing as
engineered for enhanced bioremediation strategies are
described, along with promising gene editing tools for
obtaining the host with target gene sequences responsible
for the degradation of recalcitrant compounds. The
synthetic genetic circuit and two-component regulatory
system (TCRS)-based microbial biosensors for detection and
bioremediation are also briefly explained. These
Pollution and Bioremediation | 407

developments are expected to increase the efficiency of
bioremediation strategies for best results.
Introduction
The remediation processes aided by microorganisms

present at the various contaminated scenarios constitute
bioremediation (Basu et al., 2018; Kumar et al., 2019).
Microbial remediation uses multiple metabolic pathways
responsible for enzyme production (Sharma B. et al.,
2018; Dangi et al., 2019). These enzymes mainly take part
in the degradation pathways of xenobiotics (Junghare et al.,
2019). There are different customary methods for
bioremediation, primarily based on the site of
bioremediation, in and ex situ (Tomei and Daugulis, 2013). In
situ is applied to the site to minimize soil disturbance. This
method is mostly adopted due to less expenditure from
avoiding excavation and transport of contaminated soil
(Khan et al., 2004). According to Khan et al., 2004 less
disruption in in situ bioremediation causes less dust
dispersion and hence better degradation (Joshi et al., 2016)
of contaminant. Bioaugmentation, bioventing, biosparging,
and engineered in situ bioremediation are main in
situ bioremediation methods (Azubuike et al., 2016). Ex
situ bioremediation methods are solid phase system
(composting, landfarming, and biopiling) and slurry phase
system (bioreactors) (Kumar et al., 2011). Transportation of
soil to accelerate microbial degradation are done by solid
and slurry phase systems, whereby treatments of domestic,
industrial, and organic waste are done by ex
situ bioremediation (Juwarkar et al., 2010). These traditional
bioremediation methods take time and consume much cost

expenditure, giving less result output. Traditional
bioremediation (Duarte et al., 2017) processes showed the
above limitations of extra time taking, less removal or
dissimilation of pollutants, (Bharagava et al., 2019)
disturbance to nature delicacy such as more land coverage
for a long time, and a foul smell in the environment (Dangi
et al., 2019; Kumar, 2019). Therefore, researchers are eager
to discover new bioremediation technologies for best
results. Dvoøák et al. (2017) described bioremediation via
synthetic biology for boosting bioremediation strategies.
This approach can catch the catabolic (Jacquiod et al., 2014)
and metabolic complexities for reviewing the potential of
the microbial population synthetically. The preliminary
information for developing synthetic microbial models for
bioremediation can be obtained by mining genes from the
databases (Fajardo et al., 2019). The computer logics
involvement can determine the microbial cell interactions
with recalcitrant compounds (Kim et al., 2015). These
strategies can together grasp the natural metabolic
potential of microorganisms to transform into novel
biological entities of interest (Dangi et al., 2019).
Furthermore, the regulation of metabolic pathways (Alves et
al., 2018) in a controlled manner can also be achieved for
bioremediation processes (Rochfort, 2005). This transition
via synthetic biology application (Figure 1) for remediation
purposes would improve the bioremediation processes via
the involvement of potent (Zhu et al., 2017) dissimilating
particular contaminants (Trigo et al., 2008). Synthetic
biological systems mediate cellular modulations for efficient
functioning and working of existing processes. They permit
the modification of cellular processes viz. metabolic
pathway acting for a particular chemical compound. The

advancement of synthetic biology for bioremediation of
various contaminants is attaining the focus of scientists and
researchers. For instance, a sustainable synthetic microbial
community’s establishment for bioremediation is being
investigated. Microbial interactions and quorum sensing
within communities are vastly studied for application in the
area of bioremediation with synthetic biology applications.
Achievement of the synthetic genetic circuit
of Pseudomonas putida proved to be the golden gadget for
degradation studies. Besides this, genome editing by
CRISPR-Cas, TALEN, and ZFNs adds knowledge for
reviewing the progression in bioremediation studies.
Synthetic microbial biosensors and metabolic engineering
of cellular processes for utilization and detection of
contaminant residues will remediate the environment from
persistent recalcitrant pollutants. This review is focused on
the above mentioned strategies and their elements (Figure
2) applicable for bioremediation purposes and research.

Figure 1. The strategies of synthetic biology applicable for
bioremediation.

Figure 2. The components and their construction elements of
synthetic biology for bioremediation studies.
Metabolic Reconstruction for Designing Synthetic

Models
A computational platform is utilized for the

reconstruction of cellular metabolism (Agapakis et al., 2012)
via metabolic pathway analysis (MPA) (Banerjee et al., 2016).
MPA mathematically represents the reactions of
metabolism. This method is based on stoichiometric balance
reactions so as to propose steady-state metabolic flux
during cellular growth. The stoichiometry matrix imposes
constraints of flux, making the consumption and production
of the compound at a steady state (Bordbar et al., 2014).
The maximum and minimum flux of a reaction can also be

determined by providing the topper and least bound. This
helps to define the extent of permissible flux supply
(Richelle et al., 2016; Rawls et al., 2019). The next step is
defining the objective according to the biological problem
to be studied. This objective mathematically represents the
reactions responsible for the phenotype appearance. The
mathematical reactions and phenotype are combined with
linear equations and solved by computational algorithms
such as COBRA Toolbox11 and Matlab toolbox (Orth et al.,
2010; Bordel, 2014). FBA is fundamentally simple, having
immense applications in studying gaps, physiology, and
genomes via systems biology approach (Kim et al.,
2015; Hellweger et al., 2016). These gaps are missing
metabolic reactions, making the genome partially known.
FBA uses computational algorithms that can predict missing
reactions viz. OptKnock and OptCom, which can knock out
the genes responsible for producing the desired compound
(Biggs et al., 2015). These approaches are beneficial for
constructing microbial communities for bioremediation of
particular contaminants (Khandelwal et al., 2013). However,
MPA is the most challenging method when metabolic
information is incomplete, making it difficult to obtain a real
model (Covert et al., 2001). But this method shows cellular
functions in the dynamic community, and thus is very useful
for the prediction and exchange of metabolic flux in
communities of microorganisms (Khandelwal et al., 2013).
Recently, a metabolic model has been constructed by using
two Geobacter species with parameterized electron transfer
and metabolic exchange to characterize syntrophic growth
dynamics. Such a system may have useful applications in
the field of bioremediation and degradation of particular
contaminants (Butler et al., 2010). A computational platform

is also needed for better prediction of engineered genetic
pathways for community dynamics. A graph-based tool
Metabolic Tinker was developed by McClymont and Soyer to
identify thermodynamically feasible biochemical routes for
compounds deterioration (Johns et al., 2016). This may be
applied to identify the routes for degradation of recalcitrant
compounds by microbial consortia. These computational
tools are utilized along with omics (Kim et al., 2014; El
Amrani et al., 2015) and biological data for desired output
(Berger et al., 2013) and toxicity prediction (i.e., META-
CASETOX System) (Peijnenburg and Damborský, 2012).
These are also applied for functional gene identification and
their profile analysis, PCR analysis and drug discovery, etc
(Dangi et al., 2019). Computer-aided drug discovery and
development (CADDD) is used effectively with chemical and
biological aspects, i.e., chemical structures accounting the
biological role and its activity via ligand-based drug design,
structure-based drug design, quantitative structure-
property relationships, and quantitative structure-activity
(Kapetanovic, 2008). Furthermore, Table 1 depicts similar
methodologies applicable to bioremediation studies. De
Jong (2002) analyzed the multicellular feedback control
strategy in a bacterial consortium (Bruneel et al., 2011) to
define the robustness conceivable under desired conditions.
Table 1. The methodologies applied for bioremediation research.

They utilized an ordinary differential equations (ODE)-
based model and agent-based simulation on a consortium
(Hawley et al., 2014; Atashgahi et al., 2018) of interacting
species population for increasing the efficacy of the
proposed feedback control strategy. The application of
bioinformatics (Arora and Bae, 2014) resources is a
prerequisite dimension for obtaining the data to begin the
microbial bioremediation studies of recalcitrant compounds
(Gong et al., 2012; Ofaim et al., 2019). This involves the
information related to the degradation of xenobiotics by
microbes and their pathways for dissimilation (Dao et al.,
2019; Salam and Ishaq, 2019; Thelusmond et al., 2019; Wei et
al., 2019). The data related to end products and intermediate
metabolites released throughout degradation pathways can
also be retrieved (Dvoøák et al., 2017). An extended
information source linked to degradation is MetaRouter,
allowing data (Singh and Gothalwal, 2018) for life sciences
laboratories to explore degradation possibilities of
recalcitrant compounds (Mohanta et al., 2015). The
information on oxygenic degradation of xenobiotics can be
retrieved from OxDBase, a biodegradative oxygenase
database (Chakraborty et al., 2014; Shah et al., 2018).
Oxygenase is a class of enzyme which transfers the oxygen
molecule for oxidizing the chemical compound. They play a
role in the degradation of organic compounds by aromatic
ring cleavage (Jadeja et al., 2014). OxDBase is very particular
in providing knowledge of oxygenases-catalyzed reactions,
and is a powerful tool applicable to bioremediation studies
(Singh, 2018). Bioconversion and biodegradation of
persistent and toxic xenobiotics (Desai et al., 2010; Bao et
al., 2017) compounds catalyzed by oxygenases decrease the

compound sustainability and toxicity in the environment
(Kües, 2015; Kondo, 2017). Therefore, OxDBase is very
helpful in acknowledging the degradation processes
involved in bioremediation (Shah et al., 2012). The
transcriptional characterization of genes responsible for the
biodegradation and biodissimilation of a particular
compound has great significance in proposing molecular
methodologies. This can be done by the Bionemo
(Biodegradation Network Molecular Biology) database (Libis
et al., 2016a). Bionemo contains the entries for sequences
of genes coding for biodegradation (Carbajosa and Cases,
2010). It also links the gene transcription and its regulation
(Libis et al., 2016b). The data retrieved from Bionemo can be
used for designing cloning experiments and primers (Arora
and Shi, 2010). Garg et al. (2014) used eMolecules and the
EAWAG-BBD PPS database for the prediction of pathways
involved in the biodegradation of 1-naphthyl-N-methyl
carbamate. These above findings empower the researchers
to analyze and establish what prerequisites must be fulfilled
for developing synthetic bioremediation models.
Designing the Synthetic Microbial Communities
Recent advancements in the field of synthetic biology for

environmental issues have shown a great impact. The use
of GMOs in environmental biotechnology for remediation
(Malla et al., 2018) of toxic compounds, xenobiotics, and
pesticidal compounds are being done. To design a synthetic
community, it is important to understand natural microbial
communities (Schloss and Handelsman, 2008). In a natural
community, it is difficult to find out which species are
actually taking part in bioremediation (Großkopf and Soyer,

2014). Thus, a synthetic microbial community is a promising
method for constructing an artificial microbial community
with function-specific species for bioremediation purposes.
These communities may act as a model system for the study
of functional, ecological, and structural characteristics in
a controlled manner. Großkopf and Soyer (2014), defined
synthetic community by the culturing of two microbial
species under well-defined conditions on the basis of
interaction and function (Bruggeman and Westerhoff, 2007).
These factors determine the dynamics and structure of the
community. It is based upon the identification of processes
and patterns engaged in by bacterial species. These
microbial interaction patterns are metabolism-driven and
responsible for community interaction (Wintermute and
Silver, 2010). Social-based microbial interactions (i.e.,
mutualism, cooperation, and competition, etc.) and the total
outcome of these interactions between two microbial
populations can be +/+ and −/+ or +/−, respectively (Foster
and Bell, 2012). It is said that community structure and
function majorly depends on cooperation. The effect of
cooperation on community dynamics is determined by
engineered cooperation resulting in the synthetic
community. Engineered cooperation between two microbial
strains (Singh et al., 2016) can be done by manipulation of
environmental conditions, i.e., knocking the genes out and
in Zuroff et al. (2013). Beyond this, other interaction patterns
have been analyzed with engineered microbial species in
the synthetic community. Such an application of engineered
interaction is highly recognizable in bioremediation
strategies (Sharma, 2012). Synthetic biology provides greater
potential for the sustainable existence of microorganisms
(Dellagnezze et al., 2014) acting together in a large
population. Thus, synthetic microbial communities are

proved as a key strategy for the bioremediation of
contaminants, i.e., pesticides, petroleum (Kachienga et al.,
2018), oil spill, acid drainage (Serrano and Leiva, 2017), etc.
For building the synthetic microbial communities, the
engineered interspecies and intraspecies interactions can
make cellular functions robust and enhance the capabilities
of microbial consortia in various contaminated scenarios.
Quorum sensing is a bacterial signaling mechanism, which
is a density-dependent phenomenon via cell-cell
communication and population level behavior. The signaling
is done by the release and reception of chemical compounds
by microbial candidates in a population. This leads to
multicellular behavior (Obst, 2007), offering engineerable
tasks to design function specific synthetic communities.
These synthetic models can also be exploited to obtain a
rational design that can lose the function when subjected to
competition with other species in the natural environment.
With the evolution of genomic constituents and gene
transfer, the possibility of the gradual extinction of genetic
circuits is present. Thus, strategies are required to maintain
the robustness of the synthetic community, achieved via
the synthetic models by the development of synergistic and
cooperative properties that reduce instability and loss of
function (Johns et al., 2016). A recent study by Coyte et al.
(2015) suggests that competition among species is
significant in determining the stability of communities,
acting as a limiting factor in the stability of the synthetic
community. Thus, these dynamics must be accelerated in
order to design particular function specific synthetic
communities for bioremediation purposes (Coyte et al.,
2015).

Genetic and Metabolic Engineering
Enríquez (2016) said that genome editing is an umbrella

term that refers to “scientific technological advances that
enable rational genetic engineering at a local (gene) or global
(genome) level to facilitate precise insertion, removal, or
substitution of fragments of Deoxyribonucleic acid (“DNA”)
molecules, comprising one or more nucleotides into the
cell(s) of an organism’s genome.” Transcription-activators
like effector nucleases (TALEN), clustered regularly
interspaced short palindromic repeats (CRISPR-Cas), and
zinc finger nucleases (ZFNs) are major gene editing tools
used (Table 2). The most efficient and simple technique of
gene editing has been described as CRISPR-Cas
(Kanchiswamy et al., 2016). These tools can boost the
process of bioremediation. TALEN has a DNA-binding
modular which is sequence-specific for the host genome
(Utturkar et al., 2013). TALEN binding to DNA creates a
double stranded break (DSB) in the target sequence and
leaves sticky ends for stability. Similarly, ZFNs is also a DNA-
binding domain composed of 30 amino acids. It introduces
DSB at the target site of the host genome by the Fok1
cleavage domain. A new sense of using hybrid nucleases
containing TALENs and ZFNs nucleases came to act for
solving the molecular complications. The CRISPR-Cas
system, on the other hand, has unique action properties of
high sequence specificity and multiplex gene editing. This
unique property is derived from bacteria Streptococcus
pyogenes as immunity against the virus. The CRISPR-Cas
system consists of crisper derived RNA (crRNA) and trans
acting antisense RNA (trcRNA) joined by guide RNA (gRNA).

gRNA directs the Cas9 enzyme to introduce DSB in the
target DNA sequence by recognizing it. These gene editing
tools create the knock-in and knock-out and are under
processing for implementation in bioremediation studies
(Kumar V. et al., 2018). Recent reports indicate though that
the CRISPR-Cas system is mostly adopted and performed by
researchers in model organisms i.e., Pseudomonas (Karimi et
al., 2015; Nogales et al., 2020) or Escherichia coli (Chen et al.,
2018; Marshall et al., 2018; Pontrelli et al., 2018). Nowadays,
the new insights toward CRISPR tool kits and designing
gRNA for expression of function-specific genes related to
remediation in non-model organisms (i.e., Rhodococcus
ruber TH, Comamonas testosteroni and Achromobacter sp.
HZ01) are also suggested in the field of bioremediation
(Mougiakos et al., 2016; Jusiak et al., 2016; Hong et al.,
2017; Stein et al., 2018; Tang et al., 2018; Liang et al., 2020).
For gene editing and metabolic engineering, the
contaminant-inhabited bacteria are the most suitable
candidates because they are used to survive and harbor
in stress conditions of various toxic, recalcitrant and non-
degradable xenobiotics, as discussed above. Moreover,
understanding metabolic pathways seems to be important
in studying the microbial bioremediation (Plewniak et al.,
2018), i.e., bioremediation of toxic pollutants by the
haloalkane dehalogenases production pathway and
decontamination of pyrethroid from the soil via the
biodegradation pathway of fenpropathrin studied
in Bacillus sp. DG-02 (Chen et al., 2014). Metabolic
engineering leads to modification of the existing pathway
for the betterment of the bioremediation process (Michel
et al., 2007). This approach majorly covers the study of
microbial enzymes, i.e., oxidases, esterases,

monooxygenases, oxidoreductases, phenoloxidases involved
at various degradation steps (Figure 3A; Mónica and Jaime,
2019; Mujawar et al., 2019). Enzyme-based bioremediation
has many advantages because it is an eco-friendly process.
The genetic approach increases the perspective of getting
recombinant enzymes. There are research reports of
extracellular enzymes having a role in enzymatic
bioremediation. For instance, arsenic bioremediation
(Andres and Bertin, 2016; Choe and Sheppard, 2016; Akhter
et al., 2017; Biswas et al., 2019) (bioaccumulation and
biotransformation) is achieved via arsenite oxidase coded
by aioA gene of Klebsiella pneumonia (Mujawar et al., 2019);
enzymes released by white rot fungi degrade PAHs
(polycyclic aromatic hydrocarbon) (Zhao and Poh,
2008; Košnár et al., 2019), dyes, TNT (2,4,6- Trinitrotoluene)
and PCBs (polychlorinated biphenyls) (Gupte et al.,
2016; Kutateladze et al., 2018; Sadańoski et al., 2018).
Esterase D enzyme acts on β-endosulfan (organochlorine
pesticide), producing simpler compounds (Mehr et al.,
2017; Chandra et al., 2019). LiPs encoding hemoproteins
from Phanerochaete chrysosporium degrade PAHs. However,
incomplete or partial degradation of contaminants lead to
simpler non-toxic degradable compounds which can be
consumed by microbes (Kumavath and Satyanarayana, 2014)
as intermediates in biological pathways or substrate,
i.e., LiP (lignin peroxidase) dissimilate benzopyrene to three
compounds of quinine, namely 1,6- quinone, 6,12- quinine
and 3,6- quinine (Gupta and Pathak, 2020).
Furthermore, MnP (Manganese peroxidase) oxidizes organic
compounds in the presence of Mn(II) (Xu et al., 2018; Singh
et al., 2019). Laccase, MFO (mixed function oxidases),
glutathione S transferase, cytochrome P450 also acts in

biodegradation of recalcitrant compounds (Singh,
2019; Boudh et al., 2019). Catechol 1,2-dioxygenase
(intracellular enzyme) from Pseudomonas NP-6 dissimilate
catechol to muconate compounds (Guzik et al., 2011). Also,
enzyme immobilization (Cavalca et al., 2013; Sharma B. et al.,
2018) increases the half-life, stability, and enzyme activity
at a notable level. The enzymatic bioremediation is an
elementary, expeditious, and environmental friendly
method for microbial removal and degradation of persistent
xenobiotics compounds (Sharma B. et al., 2018). Isolation
and characterization of microorganisms with enzymatic
capabilities have been done with the limitation of less
productivity (Rayu et al., 2012). Organophosphates (OP) and
organochlorines (OC), major constituents of insecticides,
accumulate in the agricultural soil (Panelli et al., 2017) and
reach the water bodies via agricultural run-off. Effective
bioremediation of γ-hexachlorocyclohexane (OC) and
methyl parathion (OP) has been reported by genetically
engineered microorganisms (Gong et al., 2016). Moreover,
bioremediation of organophosphates and pyrethroids has
been experimented with using genetically modified P.
putida KT2440 (Zuo et al., 2015). With the advent of
metabolic engineering, the catabolism and degradation of
various persistent compounds has been reported. The
degradation pathways of methyl parathion and γ-
hexachlorocyclohexane in Sphingobium
japonicum and Pseudomonas sp. WBC-3 witnessed the
bioremediation strategy (Liu et al., 2005; Miyazaki et al.,
2006). Furthermore, 1, 2, 3-trichloropropane, a persistent
constituent of fumigant, is dissimilated into the
environment (Techtmann and Hazen, 2016) via heterologous
catabolism by the assembly of three enzymes from two

different microorganisms in E. coli (Dvorak et al., 2014). A
metabolic pathway (Bertin et al., 2011) degrading
organophosphorus and paraoxon is engineered by inserting
the organophosphorus hydrolase gene (opd) and pnp operon
encoding enzymes that convert p-nitrophenol into β-
ketoadipate in P. putida (de la Pena Mattozzi et al., 2006). A
study showed pobA and chcpca gene clusters of Rhodococcus
opacus R7 take part in the bioremediation of naphthenic
acid; more specifically, expression aliA1 gene codes for fatty
acid CoA ligase for degrading long chains of linear as well as
alicyclic naphthenic acid (Zampolli et al., 2020). To minimize
the accumulation, the above-mentioned strategy is attained
using microbes for partial or complete mineralization of
persistent compounds (Miyazaki et al., 2006).
Table 2. Comparative features of CRISPR, TALE, and ZFNs.

Figure 3. Schematic presentation (A) intracellular and extracellular
enzymes production; (B) TCRS based biosensor.
Synthetic Genetic Circuit and Microbial Biosensor
The synthetic genetic circuit requires chassis for

implantation. The P. putida is a HVB (Host Vector Biosafety)
strain recognized as safe by the Recombinant DNA Advisory
Committee. It is also referred to as GRAS (Generally
Recognized as Safe) to release in the environment. It is ideal
for the next generation of synthetic biology chassis panel
because it can withstand high intolerant changing
conditions including temperature, pH, solvents, toxins,
osmotic, and oxidative stress. Also, P. putida has versatile
metabolism and low nutrient requirements (Pabo and
Nekludova, 2000). These qualities make this organism the
best bacterial model for environmental bioremediation
applications (Tanveer et al., 2018). Recently, the P.

putida synthetic genetic circuit has been established for the
designing of promoter genes and expression of the gene
responsible for the degradation of persistent compounds
(Adams, 2016). An extension of synthetic biology is the
integration of genome with reporter system, and synthetic
promoters of P. putida may be developed for synthetic
bioremediation pathways. Elmore et al. use serine integrases
for synthetic genetic circuit development. Microbial cells
have the advantage of a cellular system, which controls cell
growth and response to external factors like temperature,
light, pH, and oxygen (Tropel and Van Der Meer, 2004). The
external environment of microbes inhabiting the
contaminated site will respond to concentrations of various
persistent compounds present (Ray et al., 2018; Antonacci
and Scognamiglio, 2019). Whole cell biosensors for checking
the presence, detection and biodegradation potential of
xenobiotics compounds (pharmaceutical residues,
pesticides, paraffin, PAHs and PCBs, etc.) present (Adhikari,
2019) in environmental samples are attaining attention
(Wynn et al., 2017; Heng et al., 2018; Patel et al., 2019). The
reporter proteins acting microbe makes a color signal at the
detection of particular contaminants via transducer (Zhang
and Liu, 2016). A biosensor aiming for detection and
bioremediation purposes must have enhanced contact
between microbe and contaminant (Dhar et al., 2019). This
helps the bacterium to adjust their cellular pathways in
response to external environmental conditions and encodes
the genes for utilizing the recalcitrant compounds as
substrate (Bilal and Iqbal, 2019; Skinder et al., 2020).
Synthetic biology strategies are feasible for removing a
particular toxic compound because the genetic circuits can
be developed against the exogenous environmental toxin

(Checa et al., 2012; Tay et al., 2017). The synthetic genetic
circuits are assembled via a two-component regulatory
system (TCRS) in bacteria (Futagami et al., 2014; Uluşeker et
al., 2017). This system acts upon environmental change, and
thereby, cells respond to these changes. A prokaryotic TCRS
has histidine kinase (HK) and response regulator (RR). The
HK is a sensor domain with an extracellular loop present
as an integral membrane protein. HK also has a transmitter
domain in the last cytoplasmic transmembrane, which is a
highly conserved domain. Histidine phosphotransfer (DHp)
and catalytic ATP-binding domain (CA) acts for molecular
recognition of RR and ATP hydrolysis. The transmitter
domain transmits the signal from periplasm to cytoplasm via
PAS (Periodic circadian proteins, Aryl hydrocarbon nuclear
translocator proteins, and single minded proteins), HAMP
(HKs, Adenyltatecyclases, Methyltransferases, and
Phosphodiesterases) and GAF (cGMP-specific
phosphodiesterases, adenylyl cyclases, and formate
hydrogenases) (Casino et al., 2010). Thus, HK senses the
external environmental change and adds phosphate to
conserved histidine. The HK also regulates RR by
phosphorylating the aspartate residues. This promotes the
promoter (Figure 3B) binding to activate the gene
expression or vice-versa (Ravikumar et al., 2017). Therefore,
TCRS-based synthetic biology application for biosensor
development for cell-mediated detection and
bioremediation can prove to be a new advancement.
Ecological Safety and Risk Assessment
The scientists and researchers are performing the

experimental setup to study the bioremediation (Yergeau et

al., 2012) potential against various pollutants like oil spill,
plastics, synthetic dyes, organic hydrocarbons (Yadav et al.,
2015), pesticides (Jaiswal et al., 2019a), heavy metals
(Hemmat-Jou et al., 2018; Lebrazi and Fikri-Benbrahim,
2018), and other xenobiotics, etc (Rucká et al.,
2017; Paniagua-Michel and Fathepure, 2018; Wu et al., 2020).
Considering that bioremediation is performed in an open
environment rather than in a closed fermentation tank, the
ecological safety of bioremediation performing bacteria
must be considered. Economic safety is justified by the
metabolic aptness (Gillan et al., 2015) of microorganisms
as compared to other traditional physical and chemical
bioremediation methods. Besides, regulation for using
genetically and metabolically modified bacteria is released
to evaluate the possible risks (Khudur et al., 2019). The risk
assessment is mainly done by regulatory agencies, i.e.,
Organization for Economic Cooperation and Development
(OECD) at the application level for environmental safety
(Russo et al., 2019; Alam and Murad, 2020; Pastor-Jáuregui
et al., 2020). The possible risks are gene contamination in
the native member of microbial consortium, leading to
mislaying of the natural trait (Mills et al., 2019; Pineda et
al., 2019; Rycroft et al., 2019). The competitiveness between
natural and genetically modified species can give rise to
selection pressure on non-target microflora (Kumar N. M. et
al., 2018; Mohapatra et al., 2019). Moreover, environment and
ecosystem risk assessments infer unpredictable and adverse
effects, as discussed above (Cervelli et al., 2016; van Dorst et
al., 2020). Particularly, the ecological risk assessment behind
addition of GEMs (Genetically Engineered Microorganisms)
(Benjamin et al., 2019; Ahankoub et al., 2020) to the native
environment rather than a laboratory (Fernandez et al.,
2019) is done mainly because of unnecessary delivery of

antibiotic resistance marker along with recombinant
genome of interest (Davison, 2002; Nora et al., 2019), and
unintentional uptake or transfer of induced genes to other
microorganisms (French et al., 2019; Janssen and Stucki,
2020). This phenomenon is definitely disturbing the
microbial native genome entity (Gangar et al., 2019; Petsas
and Vagi, 2019). Another aspect to consider is the change
in microbial metabolism (Okino-Delgado et al., 2019), which
may release uncertain toxic compounds into the
environment, indirectly affecting (negatively) microbial
candidates in this context (Myhr and Traavik, 2002). Under
the TSCA (Toxic Substances Control Act) (Gardner and
Gunsch, 2020), the Office of Pollution Prevention and Toxics
(OPPT) programs (Pietro-Souza et al., 2020) of the United
States Environmental Protection Agency (Leong and Chang,
2020; Saxena et al., 2020) monitors the environmental and
health risks and releases premanufacture legal notice for
the accreditation of field research outlines and prototypes
(McPartland and McKernan, 2017; Khan et al., 2020). A
magnificent example is given by University of Tennessee. In
1995, they gave application and suggested the risk evaluation
of microbial bioremediation agents (mainly Pseudomonas
fluorescens HK44) on the environment and health (Sayler et
al., 1999; Khan et al., 2016; Sharma J. K. et al., 2018; El Zanfaly,
2019). Remarkably, the literature survey points toward a
biowar weapon for humanity (Gómez-Tatay and Hernández-
Andreu, 2019; Wang and Zhang, 2019), stating that gene
editing tools left in bad hands could mislead ethical and
moral duties (Khan, 2019; Thakur et al., 2019; Sharma et al.,
2020).

Conclusion and Future Perspectives
The microbial bioremediation process for removal and

detoxification of contaminants from the environment has
now emerged as the best option. Synthetic biology is
addressing the decontamination and remediation strategies
for xenobiotics and related compounds from the
environment. It has been observed that the requisite for
understanding existing metabolic pathways is a must for
developing synthetic models of bioremediation. Moreover,
genomics reconstruction methods (Luo et al., 2014; Marco
and Abram, 2019) and technologies made a solid platform
for bioremediation studies. Satisfactory progress has been
witnessed in the field of bioremediation among various
contaminants with the role of specific genes and enzymes
applicable via synthetic biology methodologies. Therefore,
it is concluded that microbial synthetic biology remediation
strategies not only increase the efficiency of microbial
bioremediation processes for a particular contaminant, but
also provide the best methodologies for researchers.
SJ contributed to this article under the guidance of PS. SJ

acknowledges the support and guidance of PS in pursuing a
doctorate under his guidance.

Funding
The author SJ acknowledges Maharshi Dayanand

University, Rohtak, India, for providing University Research
Scholarship (URS). PS acknowledges the infrastructural
support from Department of Science and Technology, New
Delhi, Government of India, through FIST grant (Grant No.
1196 SR/FST/LS-I/2017/4).
The authors declare that the research was conducted in

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True or False?

online here:
• Explain bioremediation and the roles that

microorganisms can have in this process.
• What is synthetic biology and how can it be applied
for bioremediation?
• What are some important considerations and
challenges for designing a synthetic microbiome?

• Compare the major techniques for genetic and
metabolic engineering.
• How could microbial biosensors contribute to
bioremediation efforts?
• What aspects of ecological safety and risk
assessment must be addressed for application of
synthetic microbiomes for bioremediation in open
environments? Why?
Media Attributions
• Video 1 – [microbiome] Microbiome and

Planetary Health (6.1) by iMooX at licenced under
Creative Commons Attribution-ShareAlike 4.0
International.
• Video 2 – [microbiome] Microbiome and SDGs
(6.2) by iMooX at licenced under Creative
Commons Attribution-ShareAlike 4.0
International.
References
1. Jaiswal, S., & Shukla, P. (2020). Alternative Strategies for

Microbial Remediation of Pollutants via Synthetic Biology.

article/10.3389/fmicb.2020.00808

PART V
OTHER MICROBIOME
APPLICATIONS
Other Microbiome Applications | 477

478 | Other Microbiome Applications
16. Forensic Microbiomes
Forensic Microbiomes
Characterizing human and environmental microbiomes is not only

important to maintain host and ecological health, but can have
other far-reaching applications such as forensic science.
Determination of unexplained situations and phenomenon can be
quite challenging, and with advances in microbiomics, there is a
possibility to generate sufficient data and evidence to provide viable
answers and solutions.
Forensic Applications of Microbiomics: A

Review
Article by Robinson et al., 2021 licensed under the

BY).
The rise of microbiomics and metagenomics has been

driven by advances in genomic sequencing technology,
improved microbial sampling methods, and fast-evolving
approaches in bioinformatics. Humans are a host to diverse
microbial communities in and on their bodies, which
Forensic Microbiomes | 479

continuously interact with and alter the surrounding
environments. Since information relating to these
interactions can be extracted by analyzing human and
environmental microbial profiles, they have the potential to
be relevant to forensics. In this review, we analyzed over
100 papers describing forensic microbiome applications
with emphasis on geolocation, personal identification, trace
evidence, manner and cause of death, and inference of the
postmortem interval (PMI). We found that although the
field is in its infancy, utilizing microbiome and metagenome
signatures has the potential to enhance the forensic toolkit.
However, many of the studies suffer from limited sample
sizes and model accuracies, and unrealistic environmental
settings, leaving the full potential of microbiomics to
forensics unexplored. It is unlikely that the information that
can currently be elucidated from microbiomics can be used
by law enforcement. Nonetheless, the research to
overcome these challenges is ongoing, and it is foreseeable
that microbiome-based evidence could contribute to
forensic investigations in the future.
Introduction
For over 100 years, microbiology has played a relatively

diminutive role in forensic science (MacCallum and
Hastings, 1899). In the early 1990s, the sequencing of
amplified viral DNA was used to support a case alleging the
transmission of Human Immunodeficiency Virus from a
dentist to several patients in Florida, United States (Smith
and Waterman, 1992). The emergence of PCR-mediated
genotyping of bacteria was considered to be a valuable
forthcoming tool in forensics—e.g., van Belkum
480 | Forensic Microbiomes

(1994) suggested that forensic science would soon be a
major area for the application of PCR-mediated genotyping
due to the rapidity of technological advances at the time
(van Belkum, 1994). In the mid-1990s, fungal and pollen
spore analyses were also developed, allowing investigators
to differentiate between soil types, which in turn allowed
linking substrate items to particular sites (Bruce and
Dettmann, 1996; Bryant and Mildenhall, 1998). However, it
was not until the early 2000s and the rise of bioterrorism
that microbial forensics—the “scientific discipline dedicated
to analyzing evidence from a bioterrorism act, biocrime, or
inadvertent microorganism/toxin release for attribution
purposes”—emerged in response to the new threat
(Budowle et al., 2003; Carter et al., 2017).
Many forensic applications have been limited to

individual taxa analyses, and microbial forensics has,
historically, been constrained by a lack of available and
cost-effective sequencing technologies (Berglund et al.,
2011; Kuiper, 2016). This approach has changed dramatically
in the last decade as advances in genomic sequencing
technology, and new methods for processing complex
community datasets (and often low biomass samples) have
led to the advent of a new field of microbiomics. The
science and study of the microbiome (Statnikov et al.,
2013; Capasso et al., 2019) combined with metagenomics (all
genomes from a sample) have enhanced the development of
the microbial forensic toolkit (Clarke et al., 2017; Hampton-
Marcell et al., 2017).
As of March 2019, the conviction rate for homicides in

England and Wales (United Kingdom) was only 79% (Office
for National Statistics, 2019), slightly higher than in the US
(∼70%) (Bureau for Justice Statistics., 2019). Across the

globe, there is also a high prevalence of wrongful
convictions and often insufficient evidence to convict a
perpetrator of a crime (Sangero and Halpert,
2007; Ingemann-Hansen et al., 2008; LaPorte, 2017; Walsh
et al., 2017). According to the Innocence Project, a national
litigation and public policy organization dedicated to
exonerating wrongfully convicted individuals, to date, 375
people in the United States have been exonerated by DNA
testing, including 21 who served time on death row
(Innocent Project, 2020). There is thereby a strong interest
from the public, lawmakers, and the law enforcement
system to augment and expand the forensic toolkit,
including molecular methods. Microorganisms are
abundant in and on the human body (microbial cells can
outnumber or equal the total number of human somatic
cells) (Noel et al., 2014; Sender et al., 2016; Vázquez-Baeza et
al., 2018), in surrounding environments, and on objects
associated with a crime (Desmond et al., 2018; Oliveira and
Amorim, 2018). A growing body of evidence suggests that
forensically relevant microbial profiles could be used as
evidence or, at the very least, complement traditional
investigative methods (Metcalf et al., 2017; Schmedes et al.,
2017; Richardson et al., 2019; Phan et al., 2020). This use of
microbial profiles as evidence is done using computational
tools that are being developed alongside new approaches in
bioinformatics, processing tools, and refined protocols.
However, since the field is still in its infancy (Goudarzi et
al., 2016; Komaroff, 2018) and historically underfunded
(Morgan and Levin, 2019), there is much uncertainty as to
the true potential of microbiomic tools in forensics.
In this review, we provide an overview of past, current,

and future potential applications of microbiomics in

forensics. Specifically, we will discuss the six most
comprehensively researched themes (Figure 1): including
geolocation (e.g., substrate analysis and different spatial
dimensions and the power of machine learning), personal
identification, biological sex determination, trace evidence,
manner and cause of death (e.g., death by drowning), PMI,
and other applications (e.g., localization through animal
microbiomes).
Figure 1. A summary of possible sources of forensically relevant

microbiota identified by the literature review.

Major Themes in the Forensic Microbiology
Literature
Geolocation
In the past few years, intensive work has been carried out
to characterize environmental microbiome, particularly in
urban environments and transit systems. These studies
have demonstrated that unique community profiles may
exist in certain areas of a city (Afshinnekoo et al.,
2015; Rosenfeld et al., 2016), as well as “molecular echoes” of
environmental events, and even a forensic capacity for
geospatial microbiomic data (MetaSUB International
Consortium, 2016; Danko et al., 2019a). In the following, we
focus on two leading aspects of geolocalization.
Substrate Analysis
The potential of analyzing microbial profiles from the soil

is increasingly being recognized in forensic microbiological
research. Both the rhizosphere and bulk soil microbiomes
exhibit a high level of heterogeneity between different
sites. As such, with methodological refinement, soil
microbiome samples could provide valuable biogeographic
data to localize the origin of the soil sample. Another
potential application is the acquisition of information to
help determine the provenance of an item(s) associated
with a crime.
Habtom et al. (2019) demonstrated distance-decay

relationships between microbial samples from the soil at a
local scale (25–1,000 m) (n = 5 sites, n = 2–4 soil types, and
five replications). The results showed that the greater the
distance between the samples, the more they differed,
suggesting that both soil type and geographic location are
important factors in determining microbial community
composition. Indeed, patch discrimination using the soil
microbiome has previously been demonstrated (Macdonald
et al., 2011), and Jesmok et al. (2016) correctly classified
95.4% of soil bacterial profiles to their location of origin
using various methods including abundance charts, non-
metric multidimensional scaling, analysis of similarity,
and k-nearest neighbor. However, this was a feasibility
study with a modest sample size (n = 19). Further studies
with larger sample sizes and replications are needed to
explore the full potential of this approach.
Evaluating the microbial communities in an assemblage

of soil samples (e.g., soils from a crime scene or alibi site
and other intermediary sites) could be useful in forensics.
Samples originating from a mixture of different soil
substrates have been correctly differentiated by using a
combination of Ribosomal Intergenic Spacer Analysis and
16S rRNA gene sequencing (Demanèche et al., 2017). Recent
evidence suggests that 18S rRNA gene sequencing can also
provide greater discriminatory power over traditional Mid-
Infrared spectroscopy at fine scales for eukaryotic species
(Young et al., 2015). Furthermore, Sanachai et al.
(2016) demonstrated that the site origin of soil, obtained
from the sole of a shoe, could be elucidated by comparing
the similarity of soil bacterial 16S rDNA profiles acquired by
the denaturing gradient gel electrophoresis technique.
Despite the potential in this field, further limitations

need to be identified and addressed. For
example, Pasternak et al. (2019) identified several
potentially limiting factors to consider when interpreting
the results of microbiomic analyses. For instance, soil
samples are incredibly complex and highly heterogeneous
even at short spatial scales, which presents a major issue to
using these in a forensic context, and microbiomes exhibit
a high level of physical, chemical, and biological diversity in
both space and time. Pasternak et al. (2013) showed that
actinobacterial fingerprints significantly differed between
two seasons (summer and winter) at the same
sites—implying that temporally-associated issues could
arise. Keet et al. (2019) have also pointed out that soil
microbiome composition can change as a result of abiotic
soil conditions and plant community patterns—which poses
a considerable challenge to the accuracy of results.
Metagenomic analysis of gravesoil and the soil around

human and non-human animal cadavers has been
undertaken for forensic purposes. Carter et al.
(2015) investigated microbial community succession in soils
associated with swine cadavers across two seasons
(summer and winter). They demonstrated that postmortem
microbial communities changed in specific and
reproducible ways, but decomposition effects on soil
microbial communities differed significantly between
seasons. The authors suggest that the ecological succession
of microbial communities will be useful for forensic
investigations, but future research should aim to gain a
greater understanding of seasonality on decomposition.
The sample size for this study was not explicitly stated, but
according to the ordination plots, it appears to be modest

(n ≤ 10 per treatment). Therefore, results should be
interpreted with caution.
Adserias-Garriga et al. (2017) investigated daily

thanatomicrobiome changes in the soil as an approach to
estimate PMI. They collected soil samples from around
human cadavers (n = 1 male and n = 2 females) and
demonstrated successional changes on a daily basis. Rapid
growth of Firmicutes was observed from the bloat stage to
advance decay (<5% relative abundance at day 1 to 75%
relative abundance day 12), and the authors proposed
a Firmicutes growth curve to estimate PMI. However, the
authors state that the growth curve results may only apply
under Tennessee summer conditions and that confirmatory
research is needed using a larger number of cadavers and
under different environmental conditions. The results do,
however, corroborate those of Finley et al. (2016), who
evaluated microbial communities associated with gravesoil
human cadavers (n = 18). The researchers allowed the
cadavers to decompose over a range of decomposition time
periods (3–303 days) and showed increases in the relative
abundance of Firmicutes in surface bodies over the
decomposition period (from ∼10% at 0–3 months to ∼40%
at 7–9 months).
Singh et al. (2018) investigated the spatial (0, 1, and 5 m)

dynamics of human cadaver decomposition on soil bacterial
community structure. They collected soil samples from
each spatial buffer (n = 14 for the 0 m, n = 17 for both 1 and 5
m) and observed evidence of a predictable response to
cadaver decomposition that varied over space. Bacterial
community composition (beta diversity) at 0 m was
significantly different from the 1 and 5 m communities,
whereas there were no significant differences between the

1 and 5 m communities. The researchers also found that
bacterial alpha diversity was significantly lower in the 0 m
samples, suggesting that the additional nutrient input from
the cadavers may reduce bacterial alpha diversity. This
study provides additional spatial-compositional insights to
complement the growing body of knowledge in this area of
forensic microbiome applications.
Soil microbiome analysis has the potential to be used in

forensics; however, additional research is required to
validate the sensitivity and reproducibility of results (Young
et al., 2017). Overall, to gain a greater understanding of both
spatial and temporal dynamics associated with the
microbiome and to develop techniques to mitigate similar
pitfalls, further microbiome surveys are essential.
Different Spatial Dimensions and the Power of

Machine Learning
The growing interest in sampling and predicting

environmental microbiome profiles at different spatial
scales and orientations (e.g., between households, cities,
states, and altitudes) to provide information on the location
and provenance of people and objects resulted in the
development of a multitude of approaches. Chase et al.
(2016) identified the three cities where nine offices were
located with 85% accuracy based on analyzing office
microbiome samples using sampling plates, and although
this study suffers from a small sample size (n = 3 per office),
it demonstrates potential with further refinement. Lax et al.
(2014) analyzed samples from household occupants (n =
1625 from 18 participants in 10 houses) and their built

environments. The authors matched feet microbiome
samples to the house with 82.9% accuracy—a relatively low
degree of accuracy from an evidentiary perspective but
demonstrating the potential of such methods for fine-scale
biolocalization. Walker and Datta (2019) analyzed whole-
genome sequenced microbiota sampled from 12 cities in
seven different countries as part of the 2018 CAMDA
MetaSUB Forensic Challenge. The CAMDA dataset (n = 30)
included three mystery samples. The authors applied
machine learning techniques to identify the geographical
provenance of the microbiome samples. Up to 90% of the
samples were correctly classified, demonstrating the
potential of machine learning applications to biogeography,
although further evidence is necessary to employ these
applications in an evidentiary context. In a related
study, Ryan (2019) applied a random forest classifier built on
a dataset of 311 city microbiome samples. Their method
correctly classified 83.3% of the mystery
samples. Grantham et al. (2019) presented a different
algorithm for predicting the geolocation of fungal samples
from dust (n = 1300) in the United States using deep neural
network classifiers. Applied to a global dataset of samples
from 28 countries, the authors state that their algorithms
make “good point predictions” with >50% of the
geolocation errors under 100 km for US-wide analysis and
nearly 90% classification accuracy of a sample’s country of
origin for the global analysis. This particular field,
combining microbiomics and machine learning, is in its
infancy, and future studies would benefit from larger
sample sizes and improved classification accuracy before
such approaches can be used with confidence in a forensic
context.

Another important spatial factor to consider is that
microbiome compositions do not only differ in horizontal
space. Skin microbiomes have also been shown to differ
between humans living in high and low altitudes. For
example, Zeng et al. (2017) collected skin microbiome
samples from humans (n = 99) and pigs (n = 82) in Tibet.
They found enrichments of several bacterial taxa
(e.g., Arthrobacter sp., Paenibacillus sp.,
and Carnobacterium sp.) in samples collected from higher
altitudes. Alpha diversity was also significantly lower in skin
samples collected from individuals living at higher altitudes.
This suggests a potential future route to determine
geolocation based on altitudinal parameters via the analysis
of skin microbiome samples in the future—although here
too, methodological refinement will be essential.
Furthermore, understanding how skin microbiomes may
fluctuate throughout the life course will also be an essential
factor to consider.
Overall, all the models prioritize classification over

prediction abilities. To enable real-time prediction of
geographical coordinates from sampling data, increasing
the sample sizes geographically and temporally, and
developing more rigorous methods is essential.
Personal Identification
A growing body of evidence suggests that human

individuals may be uniquely identified based on stable
autochthonous (i.e., native to a given environment)
microbial profiles. This could have a substantial impact on
forensic science—for example, in situations where the

investigator cannot retrieve sufficient amounts of human
DNA (i.e., from human somatic and germ cells). Yet it is
unknown whether the variation in microbial communities
between people is sufficient to identify individuals within
large populations uniquely or stable enough to place them
over time.
To answer some of these questions, Franzosa et al.

(2015) tested different body site-specific microbial profiles
and attempted to match them with 25–105 microbiome
profiles during the person’s first and second visits to the
sampling site. The authors reported that these profiles
were useful in distinguishing individuals at the initial
sampling time point and that 30% of the individuals were
still uniquely identified several months later. In this study,
gut microbiome samples were used to pinpoint 80% of
individuals (n = 120) up to a year later. These results are
encouraging—particularly in shorter timescales—however,
they still suffer from relatively high variability. As such,
greater improvements, e.g., in methods and sampling effort,
will be needed before such approaches can be useful in a
forensic setting.
High resolution melting analysis that targeted the 16S

rRNA gene from oral swab samples have also been used to
demonstrate its potential in distinguishing between
individuals (Wang et al., 2019), albeit with a very small
sample size in this study (n = 5). Schmedes et al.
(2018) demonstrated accurate identification of individuals
(n = 12) based on skin swab samples from different body
sites (n = 14). They achieved 97% accuracy by sampling
shirts and 96% accuracy using palm samples based on
1-nearest neighbor classification on nucleotide diversity of
the bacterial genome. In another recent study, the

researchers utilized a similar approach to identify
individuals (n = 51). They analyzed microbiome samples
collected from three different body sites—the manubrium
(i.e., the upper-most segment of the sternum), the palmar
surface of the hand, and the ball of the foot (Woerner et al.,
2019). The researchers achieved 100% classification
accuracy when conditioned on a maximum nearest
neighbor distance for diversity, suggesting strong potential
should these results be replicable in studies with much
larger sample sizes.
Watanabe et al. (2018) suggested that minor taxa are one

of the key factors for distinguishing between individuals.
Their study analyzed microbiome samples (n = 66) from
individuals (n = 11) over 2 years and achieved 85% accuracy
in distinguishing individuals. They also used the same
analytical methods to classify publicly available skin
microbiome samples from individuals (n = 89) with a 78%
identification accuracy. However, this level of accuracy is
unlikely to be sufficient for forensic applications. The
authors suggested that although personal identification is
possible, the estimation of the accuracy decreases for
larger cohorts due to increments of similar microbiome
patterns. Overall, the use of microbiomics as a forensic tool
to determine personal identification shows potential and
technological viability and might be useful in situations
where the investigator is unable to retrieve sufficient
amounts of human DNA. Nonetheless, the findings fall short
of the burden of proof. Improvements in the model’s
sensitivity and specificity are required, and a methodology
to address potential contamination issues. Furthermore, a
better understanding of the microbial dynamics across time

and space is essential for the findings to have a forensics
value.
Biological Sex Determination
Recent evidence supports another contribution of

microbiomics toward personal identification –the
discrimination of biological sex, which could be useful
where sufficient quantities of human DNA are unable to be
retrieved. For example, airborne bacteria communities have
previously been characterized in indoor environments
(Chan et al., 2009). Luongo et al. (2017) investigated
airborne bacterial and fungal diversity (i.e., constituents of
the “aerobiome”) from different University dormitory
rooms (n = 91). They used machine learning techniques and
were able to predict the biological sex of room occupants
with 79% accuracy based on relative abundances of the
microbiota. Curiously, rooms occupied by males exhibited
higher relative abundances of the microbiota. The authors
suggested that it could be because males may shed more
biological particles or use fewer cosmetic barriers such as
skin lotions.
Biological sex-related differences in the human

thanatomicrobiome—the microbial communities colonizing
organs following death (thanatos, Greek for death) (Zhou
and Bian, 2018)—have also been demonstrated by Bell et al.
(2018). The authors compared amplicon signatures (using
the 16S rRNA gene V1-2 and V4 regions) in the corpse heart
tissue of 10 individuals and discovered key differences
between males (n = 6) and females (n = 4). For
example, Streptococcus sp. was found exclusively in male

heart tissues, whereas females had a significantly higher
prevalence of Pseudomonas sp. With refinement, such an
approach could help to determine the biological sex of a
corpse and the provenance of body parts.
In a study by Tridico et al. (2014), the authors “readily

distinguished” male (n = 3) and female (n = 4) subjects based
on the analysis of their pubic hair microbiomes. They
identified Lactobacillus spp. that were unique to female
participants. They also suggested that pubic hair is
relatively insulated from the environment and colonized
with niche-specific microbiota, which could be useful in
forensic investigations. Unfortunately, the modest sample
size of this study limits the conclusions that can be drawn
from it. Nonetheless, the findings were supported by
another small study by Williams and Gibson (2017), who
identified individuals (n = 9) and their biological sex from
pubic hair microbiota with an error ratio of 0.027 ± 0.058
and 0.017 ± 0.052, respectively. However, the sample sizes
for all these studies are modest, and as such, further
validation studies with larger sample sizes are needed
before reliable conclusions can be drawn.
Interestingly, Phan et al. (2020) analyzed skin microbiome

samples from both genders (n = 45) and found that the
absence of the bacterial genus Alloiococcus could be useful
in predicting female biological sex. The study showed a
correlation between certain bacterial species and personal
characteristics (e.g., biological sex). They specifically
explored the presence/absence of microbiota from
fingermarks left behind on surfaces and achieved a
relatively modest 67% sex prediction accuracy using leave-
one-out cross-validation analysis. Improvements in sample
sizes and machine learning accuracy are necessary to

explore the potential of this approach further. Additional
research into whether certain bacteria (and other
microorganisms) are distinct to females or simply related to
external factors (such as cosmetic products on hands)
would also be necessary.
Trace Evidence
There is an increasing interest in studying forensically

relevant microbial profiles left behind on objects and
surfaces. For instance, several studies showed that there is
often a high level of bacterial presence on personal objects
such as mobile phones (Koroglu et al., 2015; Kõljalg et al.,
2017; Koscova et al., 2018; Kurli et al., 2018). Furthermore,
human-associated items such as shoes and mobile phones
have been shown to support distinct microbiomes (Lax et
al., 2015; Coil et al., 2019).
Meadow et al. (2014) investigated the potential utility of

mobile phones as “personal microbiome sensors.” They
selected 17 individuals and collected three samples (the cell
phone’s touch surface, their index finger, and their thumb).
They demonstrated that bacterial communities sampled
from mobile phones were more similar to their owners than
other people. They found that about 22% of the taxa on
participants’ fingers were also found on their phones,
whereas only 17% were shared with other people’s phones.
An individual’s index finger shared approximately 5% more
OTUs with their mobile phone than with everyone else’s
mobile phone in the study. Furthermore, 82% of the OTUs
were shared between a person’s index finger and their

phone. Although promising, here again, the sample size and
accuracy of results need to be increased in future studies.
Kodama et al. (2019) found that postmortem skin

microbiomes could be associated with personal objects
with a high degree of accuracy. Several of the objects in the
study were associated with 100% accuracy (i.e., medical
devices, eyeglasses, bottles, and steering wheels), whereas
objects like computer devices, remote controls, and cell
phones were associated with over 67% accuracy, suggesting
that with refinement, skin microbiome samples could be
reliably linked to objects at the scene. Furthermore, studies
have found that the postmortem skin microbiomes were
stable and similar to antemortem skin microbiomes for up
to 60 h postmortem (Kodama et al., 2019).
Salzmann et al. (2019) investigated the microbial profiles

of different bodily fluids (n = 22). They identified source-
specific microbial signatures from various bodily fluids. For
example, the phyla Proteobacteria was associated with skin
and semen sources, whereas Firmicutes showed a higher
prevalence in saliva and vaginal secretions. Dobay et al.
(2019) suggest that even when body fluid is exposed to
indoor conditions for 30 days, samples continue to harbor
body-site-specific microbial signatures. Hanssen et al.
(2017) also demonstrated promising results, albeit with a
small sample size (n = 6), for the microbially-mediated
classification of body fluids. They performed pattern
recognition by fitting a linear discriminant analysis model
using Principal Component scores and were able to classify
saliva samples in 94% of the cases.
Neckovic et al. (2020) recently investigated the potential

transfer of skin microbiomes between individuals and
substrates (i.e., allochthonous microbiota). They found that

skin microbiota has been reliably transferred through
direct contact, that is, between individuals shaking hands.
Microbiota also transmits through indirect contact, as
demonstrated by individuals rubbing a substrate and then
swapping substrates with another person. The authors
suggested that such analysis could be useful to corroborate
sexual assault cases or other contact-related crimes. They
also suggested that further research should consider the
relative surface area of contact, pressure, friction, and the
duration of the contact.
Manner and Cause of Death
The ‘manner of death’ is a determination made by an

expert following an investigation (e.g., a coroner, the police,
or a medical examiner). Five manners of death are generally
considered: natural, accidental, suicide, homicide, and
undetermined (Advenier et al., 2016). Lutz et al.
(2019) recently collected microbiome samples from 265
corpses from Finland, Italy, and the United States. The
inspected cadavers differed in the manners of death:
accidental death (n = 88), natural death (n = 106), homicide
(n = 23), and suicide (n = 45). Their results suggested
that Lactobacillus, Enterobacteriaceae, Sediminibacterium,
and Rhizobiales were associated with different manners of
death. With further research, these associations could be
developed into predictive markers that help to determine
the manner of death. However, as noted by the
authors, Sediminibacterium and Rhizobiales bacteria may
also represent environmental contamination, which needs
to be controlled, and further validation through controlled

experiments is needed to improve the reliability of their
approach to determine the manner of death.
The potential of this microbiomics approach to

determining the manner of death was corroborated in a
recent study by Zhang et al. (2019) who, by obtaining
samples during routine death investigations at the Wayne
County Medical Examiner’s Office (Detroit, Michigan,
United States), found different biomarkers associated with
the manner of death. In this study, Xanthomonadaceae was
more prevalent in cases related to hospital deaths,
whereas Actinomyces sp. tended to be more prevalent in
suicide cases. Increasing the numbers of samples generally
increased the accuracy of the models. The authors
cautioned that the prediction accuracy depends on the
machine learning methods used and the number of
anatomical sites analyzed. The authors suggest this study
provides baseline information, and it could be possible to
use machine learning to develop reference databases that
allow microbially-mediated manner of death predictions in
the future.
Kaszubinski et al. (2020) modeled beta-dispersion to test

for manner and cause of death association using a
microbiome data set of n = 188 postmortem cases (five body
sites per case). The researchers demonstrated that beta-
dispersion and demographic data could distinguish among
manner and cause of death. In particular, they found that
cardiovascular disease and drug-related deaths were
correctly classified in 79% of cases. They found that binary
logistic regression models were most effective at improving
model success. This was an improvement over multinomial
logistic regression models, which confirmed the manner
and cause of death assessment only 62% of the time. The

results of this study show promise for using postmortem
microbiomes to indicate the manner of death. However, as
the researchers’ highlight, sample sizes need to be greater.
Moreover, the development of large databases will likely be
required to train models with high success rates prior to
being used in practical forensic contexts.
In terms of cause of death (i.e., the disease or injury that

produces physiological disruption in the body leading to
death), researchers such as Christoffersen (2015) have
investigated the importance of microbiological testing.
Studying autopsy results (n = 42), the author reported that
the cause of death could be determined in 42% of the cases
via microbiological analysis. The study highlighted factors
indicative of a microbiologically related cause of death,
such as a raised CRP measurements. Raised CRPs have also
been implicated in SIDS as a cause of death (Rambaud et al.,
1999; Szydlowski et al., 2013) and even for astronauts
returning from space (Garrett-Bakelman et al., 2019).
Deadly bacterial infections, such as infection or sepsis, may
also occur following neonatal circumcision (Elhaik,
2016, 2019).
A specific forensic microbiome application for

determining the cause of death is the diagnosis of ‘death by
drowning,’ which is one of the leading causes of unnatural
deaths worldwide (Domínguez et al., 2018; Cenderadewi et
al., 2019). Analyzing the presence of diatoms (single-celled
algae) has been the ‘gold standard’ for well over a decade;
however, its reliability has been questioned (Kakizaki et al.,
2009; Huys et al., 2012). Several studies have provided
support for death by drowning diagnoses by designing real-
time PCR assays with primers to detect bacterial species
associated with aquatic environments, such

as Aeromonas spp. (Aoyagi et al., 2009; Uchiyama et al.,
2012; Rutty et al., 2015; Voloshynovych et al., 2019). These
studies provided support for this cause of death diagnosis
based on relatively high detection rates of microbiota, for
example, 84% (n = 32), 75% (n = 20), and 84% (n =
43)—although to strengthen the cause of death diagnoses,
the accuracy levels, and sample sizes could again be much
improved. It has also been suggested that bioluminescent
bacteria may be biomarkers for death by drowning in
seawater. For example, Kakizaki et al. (2009) developed a
simple assay targeting the 16S rRNA gene to identify
bioluminescent colonies such as Vibrio fischeri and Vibrio
harveyi. More recently, Lee et al. (2017) analyzed
microbiome composition and pulmonary surfactant protein
(SP-A) expression to develop a marker for diagnosis of
death by drowning. They analyzed microbiota and
histological appearance of both drowned and postmortem
groups of experimental rats, comparing freshwater vs.
marine water treatments. The authors found that 5513 and
5480 OTUs were unique to marine and freshwater,
respectively. They also found that expression levels of SP-A
were higher in the lungs of drowning victims compared to
postmortem submersion. These findings could have
important forensic value (e.g., determining both the type of
environment and the timing of death) and demonstrate
good potential for future applications. Marella et al.
(2019) point out that other studies have focused on the
presence of fecal bacteria, coliforms, and streptococcal
bacteria to help determine the cause of death by drowning.
These bacteria are sampled from the femoral artery and
vein and the right and left ventricles. Fecal bacteria are
considered to be always present in subjects who drowned

compared with those with other cause of death diagnoses
(Lucci et al., 2008; Marella et al., 2019). For example, Lucci
and Cirnelli (2007) found fecal streptococcal presence in
100% of the freshwater drowning cases they studied (n =
22) and coliforms present in 90.91%. In this study, the
control subjects (n = 30) uniformly showed an absence of
fecal bacteria. In a later study, Lucci et al. (2008) assessed if
the presence of these bacteria in the drowning medium
could be detected in victims submerged after death. The
researchers collected samples from drowned victims (n = 5
freshwater and n = 5 in seawater) and victims who were
submerged after death (n = 3). Coliforms and streptococci
were detected in all drowned victims but not in those
submerged after death. These findings suggest that fecal
coliforms and streptococci could be used as markers of
drowning. However, the minuscule sample sizes must be
interpreted with caution and increased considerably in
future studies.
Postmortem Interval
The Thanatomicrobiome
Determining the PMI (the time elapsed since a person has

died) is often an essential part of a criminal investigation.
To improve PMI prediction accuracy, researchers have
begun examining the thanatomicrobiome (Javan et al.,
2016; Burcham et al., 2019). Postmortem, these
communities overwhelm the immune system allowing for
subsequent colonization (Javan et al., 2019). Preliminary
studies suggest that these microbial communities may

undergo important successional changes in organs that
could aid in determining the PMI (Adserias-Garriga et al.,
2017).
Early studies on model animals suggest that this is

feasible. For over a 48-day period of
decomposition, Metcalf et al. (2013) aimed to uncover a
“microbial clock” to provide an estimate of PMI by
sequencing the 16S rRNA gene for bacterial and archaeal
communities and the 18S rRNA gene for microeukaryotes.
Their model provided reliable PMI estimates (±3 days) (n =
223). However, the study was conducted in controlled
conditions using experimental mouse models—thereby
necessitating a degree of caution when extrapolating the
data to ‘real-life’ situations. Another study investigated the
decomposition of pig cadavers. Their model predicted the
PMI within 2–3 h of the time of death with 94.4% accuracy
(Pechal et al., 2014), demonstrating promise with further
methodological refinement. Pechal et al. (2018) carried out a
large-scale study of body microbiome samples (n = 188) that
found postmortem microbiomes were stable, reflecting
antemortem microbiomes 24–48 h after death. The
researchers also found that specific bacterial taxa were
important in predicting health status. For
example, Haemophilus and Fusobacterium were twice as
abundant in healthy individuals, whereas Rothia was 0.09
times more abundant in heart disease cases. With further
development, this could be used to indicate the state of
human health during clinical investigations into a range of
deaths, from chronic and natural to sudden and violent
(Pechal et al., 2018). It is important to note that, although
appropriate at the time, the bioinformatics approach used
to process OTUs and to make functional predictions (e.g.,

QIIME 1.8 and PICRUSt 1) is now considered to be outdated.
Furthermore, Amplicon Sequence Variants (ASV) may
provide a richer taxonomic picture (Callahan et al., 2017).
Studying human subjects, Johnson et al. (2016) sampled

the skin microbiome of decomposing human cadavers and
developed an algorithm to estimate PMI. The authors
achieved low error rates for skins samples and a PMI
estimation accuracy of ±2 days (n = 144 from 21 cadavers), a
substantial improvement compared to prior efforts (e.g., via
entomological analysis). Belk et al. (2018) used 16S rRNA
amplicon sequencing and found that creating models with
the class or phylum taxonomic levels provided the most
accurate predictions of PMI. This finding corroborated the
study by Johnson et al. (2016) and illustrated its potential
usefulness for forensics.
Another study using 454 pyrosequencing to determine

abundances and diversity of the postmortem microbiome
in several key organs such as the brain, heart, liver, and
spleen found varying PMIs ranging from 29.5 to 240 h (Can
et al., 2014). This study revealed that the most abundant
taxa in postmortem microbial communities were the
anaerobic, spore-forming Firmicute
bacteria, Clostridium sp. Javan et al. (2017) confirmed
that Clostridium sp. dominated at long PMIs, adding
evidence to support the use of microbiomics in PMI
determination in the future.
Localization Through Animal Microbiomes
Several studies have shown that animals from different

taxonomic groups and environments possess unique

microbial profiles. For example, Tibetan chickens Gallus
gallus, Chinese Rhesus macaques Macaca mulatta, and
plateau sheep Ovis spp. have unique gut microbiomes
(Zhou et al., 2016; Huang et al., 2017; Zhao et al., 2018)
shaped by genetic, geographical, and altitudinal factors. It
has been demonstrated that the skin microbiomes of
Estrildid finches, amphibians, bats, cetaceans, and
dogs Canis lupus familiaris are unique (McKenzie et al.,
2012; Avena et al., 2016; Erwin et al., 2017; Torres et al.,
2017; Engel et al., 2018; Russo et al., 2018).
Interestingly, Song et al. (2013) found that humans share
microbial communities with their dogs.
With further investigations and methodological

refinement, such capabilities point to the potential
feasibility of linking a person with a site based on shared
microorganisms with animals. Although further studies are
needed, there is potential for forensic pathways to
associate trace microbial profiles obtained from other
species (unique to the given species) to a given
environment and/or occupation (e.g., animal industries) or
to pet ownership. For example, non-human animal-specific
microbiota could potentially be detected on the body or
clothing of a suspect or victim, which may be useful in the
absence of sufficient animal DNA (i.e., from somatic and
germ cells) evidence. This profile could then conceivably be
traced to the point of contact with an animal or animal-
based environments such as equine stables, pet shops, or
zoos, thus complementing other traditional forensic
evidence. However, this approach is mostly theoretical at
the moment, and future research will be needed to test its
feasibility.

Discussion
As of today, microbiome-based forensics are almost

absent from criminal investigations and courts. To explain
why this is so, we may divide the different possible
applications of microbial forensics into two groups: first,
reconstruction issues such as the cause of death and PMI,
which ask “what happened?” and help elucidate the
circumstances of the crime; and second, comparison issues
such as geolocation and personal identification, which ask
“how similar are these two DNA profiles?” and may
(dis)connect a suspect from an object or a place (e.g.,
murder weapon or crime scene). Reconstruction
applications for forensic use are easier to develop since
competing propositions are usually well-defined and
limited in number; if sufficient research is invested in
ascertaining the microbial characteristics associated with
each combination of possible environmental, spatial and
temporal conditions, then reconstruction becomes
straight-forward. For example, if a cadaver is found buried
at a depth of 1 m in a desert in summertime, and the
temporal succession of the gut microbial community for
these environmental conditions has previously been
established, then PMI can be inferred with a high degree of
accuracy and certainty. Thus, reconstruction microbiomics
can readily pass the Daubert standard set by the US
supreme court (Daubert v. Merrell Dow Pharmaceuticals
Inc, 1993) to be recognized as admissible evidence bearing
sufficient scientific foundation, including general
acceptance in the scientific community, known and
acceptable rate of error, and so on.

Comparison microbiomic tools, on the other hand, may
provide greater benefit to the criminal investigation but are
harder to develop to a level that would satisfy
the Daubert standard. The most beneficial way to employ
such tools would be in a “one-to-many” configuration,
similar to forensic human DNA analysis: a DNA profile from
trace evidence is compared to all the profiles (from known
persons and locations) in a database, and if it exists in the
database, its frequency in the relevant population (e.g., of
soils) is calculated to enumerate the probability of
encountering this profile by chance (i.e., originating from a
location or person unrelated to the crime). At this time,
however, there are hardly any relevant forensic databases
of microbiomes that can be compared to trace evidence. In
their absence, the only way to proceed in a forensic context
is in a “one-to-one” configuration. For each criminal case,
questioned samples (e.g., from a suspect’s shoe) are
compared to context samples from the crime scene, alibi
area, and other relevant sites. This approach provides less
benefit to the investigation because it can only give
conclusions of exclusion, that two samples do not share the
same origin, or relative conclusions, such as “sample A is
more similar to sample B than it is to sample C.”
Inclusionary conclusions such as “sample A is very similar
to sample B; the probability of a different sample, from
another place, person or time, being this similar to sample
B at random is 1 in X Million” is impossible without either an
extensive database (which is costly to build and maintain)
or thorough theoretical knowledge (which we do not have
yet) regarding the factors that shape microbiomes.
However, even for “one-to-one” analyses, we can provide a
statistical evaluation of the evidence (Habtom et al., 2019)

based on the Likelihood Ratio framework as recommended
by the European Network of Forensic Science Institutes
(Willis et al., 2015).
There are three more major hurdles to forensic tools of

comparative microbiomics. The first one involves samples
of mixed origins, containing substrates or DNA from
different locations or persons. DNA analysis of mixtures is
difficult even with simple human DNA profiles, and
certainly more so with microbiomic DNA profiles which are
far more complex. It is often impossible to tell with
certainty which, or even how many, disparate microbiomes
are present in the mixture, let alone accurately infer the
DNA profile of each one. The famed 2016 report of the US
President’s Council of Advisors on Science and Technology
[President’s Council of Advisors on Science and Technology
(US), 2016] found that the prevalent subjective analysis of
complex human DNA mixtures by forensic experts is not a
reliable methodology. Consequently, several computer
programs were developed to interpret complex human DNA
mixtures in an objective manner, and these are slowly being
validated and accepted for routine forensic use. Software
for objective analysis of microbiomic DNA mixtures may be
built on this basis, but these are still years in the future. The
second major hurdle is temporal variation. Contrary to
human DNA, which can remain unchanged for years,
microbial communities (both on the body and deposited as
trace evidence) can fluctuate over time, often in correlation
with changes in environmental parameters like moisture
and pH (e.g., Pasternak et al., 2013). In cases where two
samples for comparison are obtained at different times
when markedly different environmental conditions prevail,
mitigating the temporal changes in community structure is

needed before analysis can ensue. So far, only a few studies
have addressed this topic experimentally, mainly by
applying various carbon sources to force the different
microbial communities to “converge” (Pasternak et al.,
2019), but so far with limited success. The third, and
perhaps most challenging hurdle, is the problem of DNA
transfer. In the past decade, human DNA evidence gained
widespread credibility and acceptance in the courts so that
the identification of a DNA profile from trace evidence as
originating from a specific person is rarely disputed
nowadays. Instead, it is becoming more and more common
for the defense to challenge the method of deposition of
the DNA, suggesting that it reached the crime scene by a
legitimate activity (before or after the crime occurred) or
by DNA transfer (e.g., when the innocent suspect shook the
hand of the real perpetrator). This hurdle can sometimes be
overcome by using the likelihood-ratio approach with
activity-level propositions (Mayuoni-Kirshenbaum et al.,
2020); however, similar to the former hurdles, this one is
also very much still an open question, and it will take more
time, effort, and research before microbiomics is ready to
be employed and accepted within the legal system.
Conclusion
Over the last decade, advances in genomic sequencing

and bioinformatics have given rise to microbiomics, which
fructified in a growing compendium of tools seeking to
explore the panoply of microorganisms present in our
bodies and environment. The evidence examined in this
review indicates that microbiomics could be a forensically
relevant and promising discipline with a multitude of

applications—from determining substrate provenance and
acquiring trace evidence to identifying individuals and
estimating PMI. These advances may allow various
microbiomic data, like those obtained from
thanatomicrobiome analysis, to be used by forensic
scientists to address questions related to criminal
investigations, or at least be used alongside other forensic
methods.
Throughout their life-course, humans and their

microbiomes undergo complex interactions and co-
adaptation processes involving nutrient intake and
resulting in the production of decomposition products such
as metabolites. Following a person’s death, these
interactions change dramatically, and the microbiome
composition and dynamics fluctuate accordingly.
Understanding these colonizations and fluctuations
represent major conceptual, methodological, and
computational challenges—as do antemortem microbial
dynamics. Related microbiome-based research in a
forensics context and greater exploration of fungal and viral
communities may also lead to an important enhancement
in the forensic toolkit in the future.
Many challenges remain to overcome, such as

contamination issues, modest study sample sizes, model
over-specification and misspecification, prediction
accuracies of machine learning techniques, understanding
complex spatial and temporal variations in environmental
microbiome dynamics, as well as risks and ethical concerns
(Shamarina et al., 2017). Notably, even human DNA-based
evidence, which is far better understood, is not error-
proof, as indicated by the Phantom of Heilbronn case
(Daniel and van Oorschot, 2011). Moreover, the vast majority

of published work used 16S or targeted sequencing
approaches, which have known limitation for taxonomic
resolution and could likely benefit from metagenomics
methods (Ogilvie and Jones, 2015; McIntyre et al., 2017)
and/or methods that utilize longer reads (Danko et al.,
2019b; Foox et al., 2020). Also, more field-based testing and
deployment of these sequencing methods could benefit
from rigorous, titrated standards for ensuring accuracy
(McIntyre et al., 2019). Given this, many of the applications
reported in the literature should be considered proof of
concepts rather than full-fledged forensic applications.
Nonetheless, as Ogilvie and Jones (2015, p. 1) have
summarized: “it is clear that we remain in a period of
discovery and revelation, as new methods and technologies
begin to provide [a] deeper understanding of the inherent
ecological characteristics of this [microbial] ecosystem.”
EE initiated the study. JR carried out the review. EE, JR,

ZP, and CM wrote the manuscript. All authors approved the
manuscript.
Funding
Payment for open access publication was made by Lund

University. JR was undertaking a Ph.D. through the White
Rose Doctoral Training Partnership (WRDTP), funded by the
Economic and Social Research Council (ESRC). Grant code:
ES/J500215/1.

EE consults the DNA Diagnostics Center. CM is a co-

founder of Biotia, Inc.
The remaining authors declare that the research was

conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict
of interest.
Acknowledgments
We would like to thank the Starr Cancer Consortium

(I13-0052), the Vallee Foundation, the WorldQuant
Foundation, The Pershing Square Sohn Cancer Research
Alliance, and the National Institutes of Health (R01AI151059),
the NSF (1840275), and the Alfred P. Sloan Foundation
(G-2015-13964).
Abbreviations
PCR, polymerase chain reaction; MetaSUB,

metagenomics and metadesign of subways and urban
biomes; PRISMA, preferred reporting items for systematic
reviews and meta-analyses; CAMDA, critical assessment of
massive data analysis; PMI, postmortem interval; OUT,
operational taxonomic unit; CRP, C-reactive protein; SIDS,
sudden infant death syndrome.

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Quick Quiz

online here:

• What types of microbiomes can be analyzed in

regards to geolocation for forensic science?
• How could microbiome analytical techniques be
improved for personal identification?
• How are various microbiomes different between
biological sexes?
• What are some common objects that can have
microbiomes applicable for trace evidence?
• What is the thanatomicrobiome and how can it be
used to determine postmortem interval?
• What are the major difficulties with comparative
microbiomes concerning forensic applications?
Media Attributions
• Figure 1 – A summary of possible sources of

forensically relevant microbiota identified by the
literature review by Robinson et al., 2021 licensed
under the terms of the Creative Commons
Attribution License (CC BY).

References
1. Robinson, J. M., Pasternak, Z., Mason, C. E., & Elhaik, E. (2021).

Forensic Applications of Microbiomics: A Review. Frontiers in
fmicb.2020.608101

PART VI
JOURNAL CLUB
Journal Club | 541

542 | Journal Club
17. Journal Club Articles
The following ‘Journal Club’ articles can be used as supplementary
information for various microbiome topics and included in group
discussions:
1. Microbiome definition re-visited: old concepts and new

challenges by Berg et al., 2020 under a Creative Commons
2. Role of the gastrointestinal tract microbiome in the
pathophysiology of diabetes mellitus by Sohail et al., 2017
under a Creative Commons Attribution 4.0 International
License
3. The Firmicutes/Bacteroidetes ratio: a relevant marker of gut
dysbiosis in obese patients? by Magne et al., 2020 under a
Creative Commons Attribution 4.0 International License
4. The trans-kingdom battle between donor and recipient gut
microbiome influences fecal microbiota transplantation
outcome by Kazemian et al., 2020 under a Creative Commons
5. Saliva microbiome changes in patients with periodontitis with
and without chronic obstructive pulmonary disease by Lin et
al., 2020 under a Creative Commons Attribution 4.0
International License
6. The human oral microbiome in health and disease: from
sequences to ecosystems by Willis and Gabaldon, 2020 under a
7. The lung microbiome: new principles for respiratory
bacteriology in health and disease by Dickson and Huffnagle,
2015 under a Creative Commons Attribution 4.0 International
License
8. Viral and bacteriome characterization of children with
pneumonia and asthma in Mexico City during winter seasons
2014 and 2015 by Romero-Espinoza et al., 2018 under Creative
Journal Club Articles | 543

Commons Attribution 4.0 International License
9. New insights into the intrinsic and extrinsic factors that shape
the human skin microbiome by Dimitriu et al., 2019 under
10. Probiotics and vaginal microecology: fact or fancy? by Buggio
et al., 2019 under Creative Commons Attribution 4.0
11. Human breast milk bacteriome in health and disease by O jo-
Okunola et al., 2018 under Creative Commons Attribution 4.0
12. Synergies of systems biology and and synthetic biology in
human microbiome studies by Ezzamouri et al., 2021 under
13. Microbiomes associated with foods from plant and animal
sources by Jarvis et al., 2018 under Creative Commons
544 | Journal Club Articles

PART VII
CASE STUDIES
Case Studies | 545

546 | Case Studies
18. Case Study #1 - Human
Health
Case study #1 – The Farmer’s Flu
Disclaimer: This is a fictitious scenario created for the purposes of

microbiome, health, disease, and environmental education. Any
names, characters, places and incidents either are products of the
author’s imagination or are used fictitiously. Any resemblance to
actual events or locales or persons, living or dead, is entirely
coincidental.
Part I – Background and Problem
During one week at the end of January of 2019, a high number of flu-
like cases have appeared in the small city of Bogart, Iowa which has
a population of approximately 50,000 people. 157 individuals were
hospitalized over a two-week period, and large proportion included
children under the age of 5 years old. Many common symptoms
were initially observed in the majority of the patients, however,
other less common symptoms manifested in some ill patients after
about a week and earlier in those who were immunocompromised
and with co-morbidities.
-Common symptoms included:
• fever
• headache
• muscle pain or body aches
• shortness of breath
Case Study #1 - Human Health | 547

• vomiting
• diarrhea
• cough
• congestion or runny nose
• fatigue
-Less common symptoms included:
• stiff neck
• lethargy
• chest pain
• swelling of the throat
• severe joint pain
• green, yellow, or bloody mucus production
• facial redness and swelling

Article 1: Respiratory Viral Infection-Induced Microbiome

Alterations and Secondary Bacterial Pneumonia
Article 2: Allergic inflammation alters the lung microbiome and
hinders synergistic co-infection with H1N1 influenza virus and
Streptococcus pneumoniae in C57BL/6 mice
Initial epidemiological data showed that diseased individuals all
were at or were in contact with someone who visited the farmer’s
548 | Case Study #1 - Human Health

market the previous weekend. The market was established more
than 20 years ago, and each weekend merchants open their stalls
selling everything from pottery, jewelry, and linens to produce,
homemade jams, and even livestock. The market is considered to
be the staple of town commerce and entertainment, giving Bogart
its cozy home-town feel, and even many consumers and merchants
come in from the smaller surrounding towns to benefit from the
commerce.

Figure 1. Antigenic shift of the Flu Virus. The genetic change that enables a
flu strain to jump from one animal species to another, including humans,
known as “antigenic shift.” This process can happen in 3 ways: (A) strain is

passed to an intermediate host from both human and animal where genetic
recombination occurs, creating new strain that can spread to other animals.
(B) without undergoing genetic change, the flu jumps directly from animal to
human. (C) Without undergoing genetic change, the flu jumps to an
intermediate host, then to humans.
Questions:
1. What specific pathogens could be responsible for the observed

symptoms and why would there be differences in the observed
effects in different individuals?
2. Which microbiomes may be implicated in this disease and
why?
3. Do you think this situation is a major public concern? Why or
why not?
Attributions:
• Video 1 – Raising temperatures: the immunology of influenza

by British Society for Immunology under a Creative Commons
Attribution License (reuse allowed)
• Article 1: Respiratory Viral Infection-Induced Microbiome
Alterations and Secondary Bacterial Pneumonia by Hanada et
al., 2018 under CC BY 4.0 license
• Article 2 – Allergic inflammation alters the lung microbiome
and hinders synergistic co-infection with H1N1 influenza virus
and Streptococcus pneumoniae in C57BL/6 mice by
LeMessurier et al., 2019 under CC BY 4.0 license
• Figure 1 – Antigenic Shift of the Flu Virus by NIAID licensed
under CC BY 2.0 license


coincidental.

Part II – Approach, Implementation, Reasoning
Medical personnel took nasopharyngeal (NP) swabs of sick patients

for testing. For children and older adults and those who were averse
to NP swabs, nasal and throat swabs or aspirate specimens were
collected (Flu specimen collection – CDC). Physicians prescribed
various antiviral medication; either two doses per day of oral
oseltamivir, four oral doses per day of umifenovir, or inhaled
zanamivir for 5 days, and for patients in the hospital, one dose
of intravenous peramivir or oral baloxavir for one day. They also
prescribed prophylactic antimicrobials, including quinolones
(moxifloxacin), cephalosporins (ceftriaxone and cefepime), or
macrolides (azithromycin), or a glycopeptide (vancomycin). Other
over the counter drugs were suggested to treat symptoms like fever
and headache. Public health officials advised the community to get
the flu vaccine as well.

Over the next few weeks, the number of cases increased and
symptoms began to worsen for many. The initial assumption is a
seasonal flu outbreak. Interestingly, more cases began to pop up in
surrounding rural towns, and many patients were transported to
the larger hospitals in the city to be put on ventilators. As mortality
rates also began to rise, the CDC declared this situation a flu
epidemic.
Article 1 – The respiratory microbiome and susceptibility to
influenza virus infection
Article 2 – Secondary Bacterial Infections in Patients With Viral
Pneumonia
Article 3 – Patterns in the longitudinal oropharyngeal microbiome
evolution related to ventilator-associated pneumonia
Questions:
1. What types of laboratory tests and analytical techniques do

you think were being conducted? What problems could have
arisen with the analysis of the patient’s samples?
2. Why do you think both antibacterial and antiviral medication
was prescribed? How would you explain the difference
between treatments of a viral and a bacterial infection to a

patient?
3. What reasons or factors could cause a high mortality rate in
those infected with the influenza virus?
Attributions:
• Video 1 – Influenza Virus – Viral entry and fusion inhibitor by

iCAP Université Claude Bernard Lyon 1 under a Creative
Commons Attribution License (reuse allowed)
• Article 1 – The respiratory microbiome and susceptibility to
influenza virus infection by Lee et al., 2019 under CC BY 4.0
license
• Article 2 – Secondary Bacterial Infections in Patients With Viral
Pneumonia by Manohar et al., 2020 under CC BY 4.0 license
• Article 3 – Patterns in the longitudinal oropharyngeal
microbiome evolution related to ventilator-associated
pneumonia by Sommerstein et al., 2019 under CC BY 4.0
license


coincidental.

Part III – Discussion
After several weeks of patient study and a rising number of mortality

and cases, infections were confirmed to be from a mutated variant
of influenza A (H1N1; i.e. swine flu). Medical officials believe the high
mortality rate and other severe cases may be due to a secondary
bacterial infection caused by a pathogen that is resistant to
antimicrobials.
Further epidemiological analysis shows that the initial original
cases were specifically in individuals who visited the livestock
section of the farmer’s market for an extended period of time.
Article 1 – The distribution of microbiomes and resistomes across

farm environments in conventional and organic dairy herds in
Pennsylvania
Article 2 – Antimicrobial use and production system shape the
fecal, environmental, and slurry resistomes of pig farms
Article 3 – Influence of Pig Farming on the Human Nasal
Microbiota: Key Role of Airborne Microbial Communities

***Apologies, the video and audio are a bit glitchy.***

Last line of defense antibiotics, polymixin (colistin) and
carbapenems, were prescribed for patients not responding to the
initial antibiotics. These patients were also put under quarantine in
ICU wards as their symptoms began to worsen, which additionally
included abdominal pain, bloody stool, and severe diarrhea that
persisted in patients even after being discharged from the hospital.
Article 4 – Incidence, outcome, and risk factors for recurrence of

nosocomial Clostridioides difficile infection in adults: A prospective
cohort study

Questions:
1. How do you think antibiotic resistance of the secondary

bacterial pathogen came about?
2. How could chemotherapy affect various microbiomes and
potentially contribute to pathogenesis and other disease
symptoms?
3. How would you inform the public about this situation, and
what measures would you suggest to prevent transmission or
recurrence?

Attributions:
• Article 1 – The distribution of microbiomes and resistomes

across farm environments in conventional and organic dairy
herds in Pennsylvania by Pitta et al., 2020 under CC BY 4.0
• Article 2 – Antimicrobial use and production system shape the
fecal, environmental, and slurry resistomes of pig farms by
Mencía-Ares et al., 2020 under CC BY 4.0
• Article 3 – Influence of Pig Farming on the Human Nasal
Microbiota: Key Role of Airborne Microbial Communities by
Kraemer et al., 2018 under CC BY 4.0
• Video 1 – Fact Check: Agriculture and Antibiotic Resistance by
Livestock & Poultry Environ. Learning Community under a
Creative Commons Attribution License (reuse allowed)
• Article 4 – Incidence, outcome, and risk factors for recurrence
of nosocomial Clostridioides difficile infection in adults: A
prospective cohort study by Karaoui et al., 2020 under CC BY-
NC-ND 4.0
• Video 2 – Antibiotics and the Digestive Microbiota | Jean Carlet
by World Economic Forum under a Creative Commons


coincidental.

Part IV – Resolution
Research scientists, medical professionals, and epidemiologists

finally pieced the ‘Farmer’s Flu’ epidemic puzzle together using next
generation sequencing technology and keeping detailed records of
observational data.
Two farmers from a nearby rural town contracted the novel swine
flu variant. They then brought the pigs to market in Bogart, where
the flu spread. However, they not only spread the flu variant, but
also a highly contagious antibiotic-resistant strain of Haemophilus
influenza type B (Hib). One of the farmers was sick the week prior
and therefore immunocompromised which allowed the
development of this secondary infection. It is likely that this strain
of H. influenza acquired antibiotic resistance via horizontal gene
transfer from Haemophilus parasuis, which is commonly found in
pigs.
As this respiratory disease was treated with multiple ineffective
prophylactic antimicrobial drugs, patients’ resident microbiomes
became depleted and allowed for another infection by opportunistic
pathogens. In the case of those who were hospitalized, many
developed another secondary infection by Clostridioides difficile,
resulting in gastrointestinal distress.
After a few months, with the use of both old and new treatment
options, outreach to the public with information about the diseases,
and proper community compliance, the epidemic came to an end.
Bogart resumed as a quiet cozy town, and still holds its locally famed
farmer’s market.

Reading 1: H. influenza – CDC – Epidemiology and Vaccine-
Preventable Diseases

Article 1 – Basic Characterization of Natural Transformation in a

Highly Transformable Haemophilus parasuis Strain SC1401
Article 2 – The Gut-Lung Axis in Health and Respiratory Diseases:
A Place for Inter-Organ and Inter-Kingdom Crosstalks
Article 3 – Translating Lung Microbiome Profiles into the Next-
Generation Diagnostic Gold Standard for Pneumonia: a Clinical
Investigator’s Perspective
Questions:
1. How is the gut microbiome linked with the oral and lung
microbiomes? Explain how both of the secondary infections by
H. influenza and C. difficile in this case could be related by
their respective microbiomes.
2. What other diseases, conditions, or treatments have similar
multi-microbiome effects?
3. What are some examples of novel microbiome diagnostic and
treatments that could work to restore the affected
microbiomes?

Attributions:
• Article 1 – Basic Characterization of Natural Transformation in

a Highly Transformable Haemophilus parasuis Strain SC1401 by
Dai et al., 2019 under CC BY 4.0
• Video 1 – Microbiota and Vaccines – Eric Brown by National
Human Genome Research Institute under a Creative Commons
Attribution Liscence (reuse allowed)
• Article 2 – The Gut-Lung Axis in Health and Respiratory
Diseases: A Place for Inter-Organ and Inter-Kingdom
Crosstalks by Enaud et al., 2020 under CC BY 4.0
• Article 3 – Translating Lung Microbiome Profiles into the Next-
Investigator’s Perspective by Kitsios, 2018 under CC BY 4.0

19. Case Study #2 -
Environment
Case study #2 – The Land is Sick

coincidental.
Coffee production in South America has drastically decreased in the

2021-23 growing seasons. Specifically, the major farms in Columbia
and Brazil which collectively contribute over half of each countries
coffee (both specialty and commercial) production, have had severe
crop failures. As a result, the price of a cup of coffee has noticeably
increased, putting financial strain on suppliers and businesses.
Figure 1.
Coffee
exports by
country
2019-20.
Source:
International
Coffee
Organizatio
n
Case Study #2 - Environment | 561

Figure 2. A
world map of
countries by
coffee
production,
2019.
Interestingly, the few years prior to this decline, coffee production

was at an all-time high, with more coffee in the market than
demanded. This dropped the price of coffee, and in many cases
original farmers were not fairly compensated.
The International Coffee Organization (ICO) has tasked a team of
researchers to identify potential problems to South American coffee
plant failure and provide quick and efficient resolution.
Article 1 – The Bacterial Microbiome of Meloidogyne-Based
Disease Complex in Coffee and Tomato
Article 2 – A review of three major fungal diseases of Coffea
Arabica L. in the rainforests of Ethiopia and progress in breeding for
resistance in Kenya
Article 3 – Structure and Dynamics of the Gut Bacterial
Community Across the Developmental Stages of the Coffee Berry
Borer, Hypothenemus hampei
Video 1 – The biggest threats to the coffee industry
Video 2 – #1 Specialty coffee and the price crisis | Producer
Crossover 2019
562 | Case Study #2 - Environment

Questions:
1. As a scientific researcher, what are some factors you would

take into consideration when addressing this problem?
2. What type of environmental microbiomes could be implicated
in coffee plant crop failure and why?
3. Should other coffee farms in countries besides those in South
America be worried about similar failure in their crops? Why or
why not?
Attributions:
• Figure 1 – Coffee exports by country 2019-20 by Dylan Parks.

Data source: International Coffee Organization
• Figure 2 – A world map of countries by coffee production, 2019
by Cbahrs licensed under CC BY-SA 4.0
• Article 1 – The Bacterial Microbiome of Meloidogyne-Based
Disease Complex in Coffee and Tomato by Lamelas et al., 2020
licensed under the terms of the Creative Commons Attribution
License (CC BY).
• Article 2 – A review of three major fungal diseases of Coffea
Arabica L. in the rainforests of Ethiopia and progress in
breeding for resistance in Kenya by Hindorf and Omondi, 2011
licensed under CC BY-NC-ND 3.0
• Article 3 – Structure and Dynamics of the Gut Bacterial
Community Across the Developmental Stages of the Coffee
Berry Borer, Hypothenemus hampei by Mejía-Alvarado et al.,
2021 licensed under the terms of the Creative Commons
• Video 1 – The biggest threats to the coffee industry by Startup
to Storefront licensed under a Creative Commons Attribution
License (reuse allowed)
• Video 2 – #1 Specialty coffee and the price crisis | Producer

Crossover 2019 by Coffee Circle licensed under a Creative

coincidental.

Upon initial inspection, the unhealthy coffee plants exhibited

yellowing of leaves, appearance of red-brown lesions, abnormal
shape, damaged coffee berries, and little new growth or production.
Researchers decided to take samples of healthy and unhealthy
leaves and berries, as well as soil and root samples.
Article 1 – Prokaryotic diversity in the rhizosphere of organic,

intensive, and transitional coffee farms in Brazil
Article 2 – A metagenomics approach in the evaluation of the soil
microbiome in coffee plantations under organic and conventional
production in tropical agroecosystems
Video 1 – #2 Producer Crossover 2019
Video 2 – How countries farm and make coffee differently

Over the next year, other plantations in Columbia and Central
American begin to experience similar declines in coffee plant health.
Farmers were questioned about any changes in practices, and while
there have been some adjustments do to the fluctuating coffee
prices, traditional farming techniques have remained the same for
the most part in smaller plantations. Larger plantations with
intensive farming made more changes due to trader suggestions
on improving yields including application of different fertilizers and
hiring less experienced workers.
Questions:
1. What type of analytical techniques do you think were or should

be performed on the samples taken?
2. How could the observed symptoms of the unhealthy plants be
connected to an associated microbiome?
3. How could human intervention exacerbate or ameliorate this
situation?
Attributions:
• Article 1 – Prokaryotic diversity in the rhizosphere of organic,

intensive, and transitional coffee farms in Brazil by Caldwell et
al., 2015 licensed under a Creative Commons Attribution-
ShareAlike 4.0 International (CC BY-SA 4.0) license.
• Article 2 – A metagenomics approach in the evaluation of the
soil microbiome in coffee plantations under organic and
conventional production in tropical agroecosystems by
Rodriguez et al., 2020 licensed under a Creative Commons
Attribution-NonCommercial 4.0 International License.

• Video 1 – #2 Producer Crossover 2019 by Coffee Circle
licensed under a Creative Commons Attribution License (reuse
allowed)
• Video 2 – How countries farm and make coffee differently by
Startup to Storefront licensed under a Creative Commons

coincidental.

Analysis of the negatively affected plantations’ soil showed a decline

in plant growth promoting bacteria and mycorrhizae and an
increase in bacterial and fungal pathogens like Pseudomonas
syringae and Cercospora coffeicola.
Over the next few years (2022-24) coffee plants still struggle to
grow in Central and South America. Other parts of the world, such
as Vietnam and other Asian countries, are seeing a decline in crop
health also, further contributing to global coffee market troubles.
Many of these farms switched from shade-grown to intense sun-

grown coffee to try to meet needs for demand. Larger corporations
and roasters have purchased many of the smaller family farms to
try to recoup losses and implement ‘new and improved’ growing
strategies for increased production. This included switching to
primarily sun-grown coffee, and application of large amounts of
synthetic nitrogen fertilizer.
Figure 1. Shade grown coffee in Guatemala

Figure 2. Sun-grown coffee plantation in Brazil
Video 1- #3 The future of the price crisis

Video 2 – How coffee destroys the environment
Article 1 – Root endophytes of coffee (Coffea Arabica): Variation
across climatic gradients and relationships with functional traits
Article 2 – Brazilian Coffee Production and the Future
Microbiome and Mycotoxin Profile Considering the Climate Change
Scenario
Article 3 – Effects of environmental factors on microbiota of fruits
and soil of Coffea arabica in Brazil
Questions:
1. What factors could cause these changes in the soil

microbiomes?
2. Why do you think coffee plantations across the globe are
beginning to fail as well?
3. How does natural biodiversity benefit ecosystem health on
both a micro and macro scale?
Attributions:
• Figure 1 – Shade grown coffee in Guatemala by John Blake

under Public Domain
• Figure 2. Sun-grown coffee plantation in Brazil by Knase
caption adapted by Dylan Parks licensed under the Creative
Commons Attribution 3.0 Germany license.
• Video 1 – #3 The future of the price crisis by Coffee Circle

allowed)
• Video 2 – How coffee destroys the environment by Startup to
Storefront licensed under a Creative Commons Attribution
• Article 1 – Root endophytes of coffee (Coffea Arabica): Variation
across climatic gradients and relationships with functional
traits by Fulthorpe et al., 2020 licensed under Creative
• Article 2 – Brazilian Coffee Production and the Future
Microbiome and Mycotoxin Profile Considering the Climate
Change Scenario by dos Santos et al., 2021 licensed under the
terms and conditions of the Creative Commons Attribution
(CC BY) license.
• Article 3 – Effects of environmental factors on microbiota of
fruits and soil of Coffea arabica in Brazil by Veloso et al., 2020
licensed under the terms and conditions of the Creative
Commons Attribution (CC BY) license.

coincidental.


In 2030, most varieties of coffee are extinct, and only small amounts
of coffee are produced on shade grown farms further from the
equator. It appears that coffee plantations were severely impacted
by increasing global temperatures, which altered ecosystem
dynamics and promoted pathogen and pest invasion. Fungi,
bacteria, and arthropods devastated already struggling coffee farms
and with a major switch to sun-grown coffee, soil microbiomes
were depleted and disease spread rapidly through monoculture
crops. This switchover was prompted by an energy drink
corporation, KAPOW!, which wanted to corner the caffeine market
and boost production, though their expertise in growing coffee was
lacking and the excessive use of synthetic fertilizers on already sun
baked land which requires much more watering further doomed
plantations. At least they now produce a cheap “coffee” flavored
energy drink. It tastes terrible.
Article 1 – Coffee Microbiota and Its Potential Use in Sustainable
Crop Management. A Review
Article 2 – Soil fungal communities differ between shaded and
sun-intensive coffee plantations in El Salvador
Article 3 – One health relationships between human, animal, and
environmental microbiomes: A mini-review
Video 1 – Teaching coffee farmers about the birds and the bees
Questions:
1. In what ways are environmental microbiomes impacted by

changing environmental factors and how can this promote
diseases within an ecosystem?
2. How could various environmental microbiomes be utilized to
improve sustainable practices and technology?
3. How are environmental and human microbiomes

interconnected?
Attributions:
• Article 1 – Coffee Microbiota and Its Potential Use in

Sustainable Crop Management. A Review by Duong et al., 2020
License (CC BY).
• Article 2 – Soil fungal communities differ between shaded and
sun-intensive coffee plantations in El Salvador by Rao et al.,
2020 licensed under the Creative Commons CC0 public
domain dedication.
• Article 3 – https://www.frontiersin.org/articles/10.3389/
fpubh.2018.00235/full by Trinh et al., 2018 licensed under the
terms of the Creative Commons Attribution License (CC BY).
• Video 1 – Teaching coffee farmers about the birds and the bees
by VOA Learning English licensed under a Creative Commons
References:
1. Hindorf, H., & Omondi, C. O. (2011). A review of three major

fungal diseases of Coffea arabica L. in the rainforests of
Ethiopia and progress in breeding for resistance in Kenya.
Journal of Advanced Research, 2(2), 109–120. https://doi.org/
https://doi.org/10.1016/j.jare.2010.08.006
2. Lamelas, A., Desgarennes, D., López-Lima, D., Villain, L.,
Alonso-Sánchez, A., Artacho, A., Latorre, A., Moya, A., &
Carrión, G. (2020). The Bacterial Microbiome of Meloidogyne-
Based Disease Complex in Coffee and Tomato. Frontiers in

Plant Science, 11. https://www.frontiersin.org/article/10.3389/
fpls.2020.00136
3. Duong, B., Marraccini, P., Maeght, J.-L., Vaast, P., Lebrun, M., &
Duponnois, R. (2020). Coffee Microbiota and Its Potential Use
in Sustainable Crop Management. A Review. Frontiers in
Sustainable Food Systems, 4. https://www.frontiersin.org/
article/10.3389/fsufs.2020.607935
4. Fulthorpe, R., Martin, A. R., & Isaac, M. E. (2019). Root
Endophytes of Coffee (Coffea arabica): Variation Across
Climatic Gradients and Relationships with Functional Traits.
Phytobiomes Journal, 4(1), 27–39. https://doi.org/10.1094/
PBIOMES-04-19-0021-R
5. dos Santos DG, Coelho CCdS, Ferreira ABR, Freitas-Silva O.
Brazilian Coffee Production and the Future Microbiome and
Mycotoxin Profile Considering the Climate Change
Scenario. Microorganisms. 2021; 9(4):858. https://doi.org/
6. Caldwell AC, Silva LCF, da Silva CC, Ouverney CC (2015)
Prokaryotic Diversity in the Rhizosphere of Organic, Intensive,
and Transitional Coffee Farms in Brazil. PLoS ONE 10(6):
e0106355. doi:10.1371/journal.pone.0106355
7. Rodríguez, A. C.-, R. T.- Calzada, C. G.-D. la Peña, J. G. A.- Ávila,
E. N.- Reyna, F. V.- Paniagua, C. D.- Velásquez, and C. A. M.-
Herrera. “A Metagenomic Approach in the Evaluation of the
Soil Microbiome in Coffee Plantations under Organic and
Conventional Production in Tropical
Agroecosystems”. Emirates Journal of Food and Agriculture, Vol.
32, no. 4, Apr. 2020, pp. 263-70, https://doi.org/10.9755/
ejfa.2020.v32.i4.2092.
8. Rao, M., Rice, R., Fleischer, R., & Muletz Wolz, C. (2020). Soil
fungal communities differ between shaded and sun-intensive
coffee plantations in El Salvador. PLOS ONE, 15, e0231875.
9. Mejía-Alvarado, F. S., Ghneim-Herrera, T., Góngora, C. E.,
Benavides, P., & Navarro-Escalante, L. (2021). Structure and

Dynamics of the Gut Bacterial Community Across the
Developmental Stages of the Coffee Berry Borer,
Hypothenemus hampei. Frontiers in Microbiology, 12.
fmicb.2021.639868
10. Veloso, T. G. R., da Silva, M. de C. S., Cardoso, W. S., Guarçoni,
R. C., Kasuya, M. C. M., & Pereira, L. L. (2020). Effects of
environmental factors on microbiota of fruits and soil of Coffea
arabica in Brazil. Scientific Reports, 10(1), 14692.
11. Trinh, P., Zaneveld, J. R., Safranek, S., & Rabinowitz, P. M. (2018).
One Health Relationships Between Human, Animal, and
Environmental Microbiomes: A Mini-Review. Frontiers in Public
Health, 6. https://www.frontiersin.org/article/10.3389/
fpubh.2018.00235

20. Case Study #3 - Synthesis
(Create Your Own)
Develop your own case study related to

microbiomes!
Group members (4-5) will develop and write an original case study
involving a microbiome and health or environmental condition.
Topics must be approved by the instructor beforehand. The case
study should consist of 3 parts: (i) background and problem/issue,
(ii) approach to solve, implementation, and reasoning, (iii)
discussion of progression of the case study. The final part, (iv)
resolution and conclusion will be completed by another group and
presented over. The case study should have an interesting title,
include references, images, figures, etc. from relevant resources (be
sure to use in-text citations and include references at the end), and
3 critical thinking questions concluding each part (9 total).
574 | Case Study #3 - Synthesis (Create Your Own)

PART VIII
ADDITIONAL RESOURCES
Additional Resources | 575

576 | Additional Resources
21. The Integrative Human
Microbiome Project
The Integrative Human Microbiome Project is an open access
article published in Nature under the Creative Commons: By
Attribution 4.0 License (http://creativecommons.org/licenses/by/
4.0/).
The Integrative Human Microbiome Project
The Integrative HMP (iHMP) Research Network Consortium
Abstract
The NIH Human Microbiome Project (HMP) has been carried out
over ten years and two phases to provide resources, methods, and
discoveries that link interactions between humans and their
microbiomes to health-related outcomes. The recently completed
second phase, the Integrative Human Microbiome Project,
comprised studies of dynamic changes in the microbiome and host
under three conditions: pregnancy and preterm birth; inflammatory
bowel diseases; and stressors that affect individuals with
prediabetes. The associated research begins to elucidate
mechanisms of host–microbiome interactions under these
conditions, provides unique data resources (at the HMP Data
Coordination Center), and represents a paradigm for future multi-
omic studies of the human microbiome.
The Integrative Human Microbiome Project | 577

Main
Although the ’omics era has accelerated all aspects of biological

research, its effects have been particularly apparent in studies of
microbial communities and the human microbiome. In the 18 years
since the publication of the first human genome, studies of the
microbiome have grown from culture-based surveys of the oral
cavity and gut to molecular profiles of microbial biochemistry in all
1,2,3
ecological niches of the human body . Epidemiology and model
systems have been used to identify associations between changes in
4 5,6,7
the microbiome and conditions ranging from autism to cancer ,
and microbial and immunological mechanisms have been identified
that affect, for example, the efficacy of drugs used to treat cardiac
8 9
conditions or survival during graft-versus-host disease .
Contemporary studies of the human microbiome have also been
a source of basic biological and translational surprises, exposing
a compelling range of novel findings and open questions. Every
human being appears to carry their own, largely individual, suite
10,11 12,13,14
of microbial strains , which are acquired early in life , differ
15,16
between environments and populations , and can persist for
17 18
years or undergo relatively rapid transitions . Microbial diversity
manifests differently in different ecological niches of the body; for
example, greater diversity is generally expected in the gut, but can
be associated with dysbiotic states and risk of adverse events in
the female reproductive tract. The microbiome can be perturbed by
conditions such as inflammatory bowel disease and diabetes, but a
variety of microbiome-linked health states, and the underpinnings
of these links, remain unexplored. How dynamic is the microbiome
during processes such as pregnancy or viral infection? Which
changes in the microbiome represent causes rather than effects
of changes in health? Which molecular elements of a personalized
microbiome might be responsible for health outcomes, and how do
they integrate with and maintain physiological processes such as
the immune system and metabolism? And what ecological elements
dictate the success of a microbiota transplant, and why are they
578 | The Integrative Human Microbiome Project

successful in treating some individuals and conditions, but not
others?
The National Institutes of Health Human Microbiome Project was
one of the first large-scale initiatives to address a subset of these
19 20
questions (Fig. 1). Launched in 2007 , the first phase of the
program sought to determine whether there were common
elements to ‘healthy’ microbiomes, in the absence of overt disease.
21,22,23
Studies of both a baseline adult population and
‘demonstration’ populations with specific disease states established
typical ranges (for some populations) of microbial membership and
enzymatic repertoires across the body, combinations of metabolic
functions that were either prevalent or strain-specific, and some
of the host factors (such as race or ethnicity) that determine this
variation. Studies of targeted populations identified ecological
24,25 26,27,28
states of niches such as the vagina , skin , and
29,30,31,32,33
gut , among many others (https://www.hmpdacc.org/
health/projectdemos.php). This first phase of the HMP (HMP1) thus
yielded a wealth of community resources: nucleotide sequences of
microorganisms and communities from a large number of isolates,
34,35,36,37
individuals, and populations (http://hmpdacc.org) ;
protocols to support reproducible body-wide microbiome sampling
38,39,40
and data generation ; and computational methods for
41,42,43,44,45,46,47
microbiome analysis and epidemiology .

Fig. 1 The first and second phases of the Human Microbiome Project. The
ten-year NIH Human Microbiome Project (HMP) program, organized into two
phases (HMP1 and HMP2), developed reference sequences, multi-omic data
sets, computational and statistical tools, and analytical and clinical protocols
as resources for the broader research community. The HMP1 focused on the
characterization of microbial communities from numerous body sites (oral,
nasal, vaginal, gut, and skin) in a baseline study of healthy adult subjects, and
included a set of demonstration projects that focused on specific diseases or
disorders. The HMP2 expanded the repertoire of biological properties
analysed for both host and microbiome in three longitudinal cohort studies of
representative microbiome-associated conditions: pregnancy and preterm
birth (vaginal microbiomes of pregnant women), inflammatory bowel diseases
(gut microbiome) and prediabetes (gut and nasal microbiomes). These studies
followed the dynamics of these conditions through multi-omic analyses of
multiple measurement types over time, including changes in microbial
community composition, viromics, metabolomic profiles, gene expression and
protein profiles from both host and microbiome, and host-specific properties
such as genetic, epigenomic, antibody, and cytokine profiles, along with other
study-specific features. All sequences and multi-omic data, clinical
information, and tools from both HMP1 and HMP2 are housed in the HMP
Data Coordination Center (DCC) or referenced public or controlled-access
repositories to serve as a central resource for the research community.
One of the main findings of the HMP1 was that the taxonomic
composition of the microbiome alone was often not a good
correlate with host phenotype—this tended to be better predicted
by prevalent microbial molecular function or personalized strain-
21
specific makeup . This finding served as the foundation for the

development of the second phase of the HMP, the Integrative HMP
48
(iHMP or HMP2) , which was designed to explore host–microbiome
interplay, including immunity, metabolism, and dynamic molecular
activity, to gain a more holistic view of host–microbe interactions
over time. This multi-omic program sought to expand the resource
base available to the microbiome research community, to begin
to address the relationship between host and microbiome
mechanistically, and to address the questions introduced above.
Disease-targeted projects within the HMP2 were therefore
encouraged to use multiple complementary approaches in order
to assess the mechanisms of human and microbial activity
longitudinally and to provide protocols, data, and biospecimens for
future work. These projects included three studies that followed
the dynamics of human health and disease during conditions with
known microbiome interactions, thus addressing important health
outcomes directly while also serving as models of ‘typical’
microbiome-associated conditions of broad interest to the research
community. These comprised pregnancy and preterm birth (PTB);
inflammatory bowel diseases (IBD); and stressors that affect
individuals with prediabetes. These studies, which have now
49,50,51
reached the first stage of completion , together provide a
wealth of information and insights about not only microbial
dynamics, but also associated human host responses and microbial
inter-relationships. A collection of more than 20 manuscripts to
date describe some of these results at https://www.nature.com/
collections/fiabfcjbfj, and together they provide a rich multi-omic
data resource to be mined by future work
(http://www.ihmpdcc.org).
The vaginal microbiome, pregnancy and preterm birth
Preterm birth can have devastating consequences for newborn

babies, including death and long-term disability. In the United
52
States, approximately 10% of births are premature , and the

incidence is even greater in lower-resource countries.
Environmental factors, including the microbiome of the female
reproductive tract, are important contributors to prematurity.
Notably, these factors have a greater effect in women of African
53
ancestry, who also bear the highest burden of PTB . Infant
mortality has been reduced in recent decades, but the incidence
54
of PTB has not decreased , and progress in predicting individual
risk of PTB has stalled. During pregnancy, the maternal immune
system maintains a delicate balance of pro- and anti-inflammatory
55
effectors , and contributors to PTB include breakdown in
maternal–fetal tolerance, vascular disorders, stress, cervical
insufficiency, premature rupture of the fetal membranes, and intra-
56
amniotic infection . Microbial ascension into the uterus is thought
to precipitate PTB by disrupting the maternal immune balance,
leading to spontaneous preterm labour, and/or by the release of
microbial products (for example, collagenases, proteases or toxins)
that compromise the integrity of fetal membranes and lead to
57
premature rupture of the membranes .
The Multi-Omic Microbiome Study: Pregnancy Initiative (MOMS-
PI) research group, as part of HMP2, characterized the microbiomes
of pregnant women to gauge their effects on risk of PTB (Fig. 2). The
project followed 1,527 women longitudinally through pregnancy and
involved the collection of 206,437 specimens, including maternal
vaginal, buccal, rectal, skin and nares swabs, blood, urine, and birth
products, as well as infant cord and cord blood, meconium and first
stool, buccal, skin and rectal swabs. Subsets of these specimens
underwent 16S rRNA gene taxonomic analysis, metagenomic and
metatranscriptomic sequencing, cytokine profiling, lipidomics
analysis, and bacterial genome analysis. The MOMS-PI team
analysed 12,039 samples from 597 pregnancies to investigate the
dynamics of the microbiome and its interactions with the host
50
during pregnancy leading to PTB .

Fig. 2. The vaginal microbiome and its relationships with host factors in
pregnancy and preterm birth. The MOMS-PI project followed 1,527
pregnancies longitudinally and involved the collection of 206,437
biospecimens for analysis of host and microbial factors (16S amplicon,
metagenomic, and metatranscriptomic sequencing; cytokine profiling;
metabolomics; proteomics; genomics; and microbial isolate culture). Around
600 pregnancies were analysed in depth to assess features that lead to
preterm birth; this analysis identified both host ( for example, cytokine) and
microbial ( for example, ecological and specific strain) factors. As pregnancy
progresses, with predictable changes in systemic oestradiol levels, the uterine
and vaginal environments undergo various changes. The uterus switches
from an early pro-inflammatory condition to an anti-inflammatory condition
in the second trimester, and then back to a pro-inflammatory condition
before the onset of labour. Meanwhile, specific changes in the microbiome of
the vaginal lumen can be associated with preterm birth, possibly through
mechanisms involving microorganisms travelling from the vagina to the
uterus. The figure depicts an overview of longitudinal changes in the vaginal
mucosal ecosystem and uterus during pregnancy.
These multi-omic investigations identified temporal changes in the

vaginal microbiome associated with full-term pregnancies. Women
who often began pregnancy with a vaginal microbiome of greater
ecological complexity generally converged towards a more
homogeneous Lactobacillus-dominated microbiome by the second
58
trimester . Interestingly, this trend was most pronounced in

women of African ancestry with lower socioeconomic
profiles. Although the overall MOMS-PI cohort was
demographically diverse, most women who experienced
spontaneous PTB at less than 37 weeks of gestation were of African
ancestry. The MOMS-PI team (http://vmc.vcu.edu/momspi) also
identified signatures of higher risk for PTB in women who
experienced spontaneous preterm birth at less than 37 weeks of
50
gestation . Women who went on to experience spontaneous PTB
were less likely to exhibit a vaginal microbiota dominated
by Lactobacillus crispatus, as previously reported in other
59,60,61,62
populations , and were more likely to exhibit an increased
proportional abundance of several taxa including Sneathia
amnii, Prevotella-related clades, a Lachnospiraceae taxon known as
BVAB1, and a Saccharibacteria bacterium known as TM7-H1. Notably,
63
these taxa were also associated with low levels of vitamin D ,
suggesting that the vaginal microbiome might mediate a link
64
between PTB risk and vitamin D deficiency . The signatures of
PTB were also reflected in metagenomic and metatranscriptomic
measurements, and vaginal pro-inflammatory cytokines (including
IL-1β, IL-6, MIP-1β and eotaxin-1) were positively correlated with
PTB-associated taxa. Conveniently for future possible interventions,
the vaginal microbiomes of mothers who experienced PTB were
most distinct from those of control mothers early in pregnancy, and
a preliminary model to predict risk of PTB was most sensitive and
specific using vaginal microbiome profiles from samples collected
before 24 weeks of gestation.
The MOMS-PI research group identified intriguing associations
between the vaginal microbiota, host response, and pregnancy
outcomes that are consistent with the involvement of
microorganisms ascending from the vagina in at least some cases
of spontaneous PTB. As an essential next step, the contribution
of racial and demographic background to the vaginal microbiome
in pregnancy with relation to pregnancy outcomes must be fully
50
explored through harmonized, large-scale studies . It is clear that
56
PTB has a complex aetiology . The relative contributions of fetal

and maternal genetics and epigenetics, particularly as related to
genetic variation of the innate immune system, should be explored.
Harmonized large-scale studies would permit the development of
population-specific risk assessment algorithms using vaginal
microbiome profiles, features from genetic and prenatal (fetal)
genetic screens, biomarkers such as cytokines and metabolites, and
key clinical features from classic markers of risk including maternal
age, body mass index, pregnancy history (including history of PTB),
cervical length, and measures of stress and other environmental
exposures. With the addition of new data from the microbiome,
other environmental factors, and multi-omic inputs, new algorithms
promise to improve our ability to predict risk of PTB early in
pregnancy, to facilitate clinical trials by identifying high-risk
patients, and ultimately to stratify patient populations into
treatment groups.
The gut microbiome and inflammatory bowel disease
Studies of the gut microbiome in gastrointestinal disease have a

particularly long and detailed history, especially in complex chronic
conditions such as the inflammatory bowel diseases (IBD). IBD,
including Crohn’s disease and ulcerative colitis, affects millions of
individuals worldwide, with increasing incidence over the past 50
years or more coinciding with multiple factors such as
westernization, urbanization, shifts in dietary patterns,
antimicrobial exposure, and many more that could influence
65
host–microbiome homeostasis . The microbiome has long been
66,67
implicated in IBD, potentially as a causative or risk factor , as an
explanation for heterogeneity in treatment response (that is, some
individuals respond well to relatively benign aminosalicylates or
corticosteroids whereas others still experience severe inflammation
68
even after surgical intervention) , or as a novel point of therapeutic
intervention (for example, by transplantation of faecal
69,70
microbiota ). Although meta-omic techniques have been used

to identify functionally consistent microbial responses that help to
explain the gut microbiome’s role as part of a pro-inflammatory
71
feedback loop in the gut during disease , and a few strains of
72
microorganisms have been shown to be IBD-specific , no
comprehensive model of specific microbial, molecular, and immune
interactions yet exists to explain the disease’s onset and dynamic
progression.
Therefore, to better characterize mechanisms of
host–microbiome dysregulation during disease, the Inflammatory
Bowel Disease Multi’omics Database (IBDMDB) project followed 132
individuals from five clinical centres over the course of one year
each as part of HMP2 (Fig. 3). Integrated longitudinal molecular
profiles of microbial and host activity were generated by analysing
1,785 stool samples (self-collected and sent by mail every two
weeks), 651 intestinal biopsies (collected colonoscopically at
baseline), and 529 quarterly blood samples. To the extent possible,
multiple molecular profiles were generated from the same sets of
73
samples, including stool metagenomes, metatranscriptomes ,
74,75
metaproteomes, viromes, metabolomes , host exomes,
epigenomes, transcriptomes, and serological profiles, among
others, allowing concurrent changes to be observed in multiple
types of host and microbial molecular and clinical activity over time.
Protocols and results from the study, further information about its
76,77
infrastructure, and both raw and processed data products are
available through the IBDMDB data portal (http://ibdmdb.org), from
the HMP2 Data Coordination Center (DCC; http://ihmpdcc.org),
49
and in the accompanying manuscript .

Fig. 3. Host-microbiome dynamics in IBD. The IBDMDB followed more than
100 participants with IBD (Crohn’s disease or ulcerative colitis), as well
control individuals without IBD, for one year each to assess host and
microbial molecular activity during changes in disease activity and
gastrointestinal inflammation. Nearly all measured host–microbiome
properties showed changes in either activity or stability during disease,
including those shown here—not only microbial taxa and microbial
transcription, but also host- and microbiota-derived small molecules in the
gut, epithelial transcriptional responses at multiple points along the colon,
and circulating antibody levels in peripheral blood serology.
This unique study design allowed the IBDMDB to identify a variety of

differences in the microbiome and host immune response over time
during the course of the disease. Indeed, these dynamic changes
were of much greater magnitude than were cross-sectional

differences among clinical phenotypes, which have been
67,71,78,79
emphasized by previous studies . This was due in part to
the prospective nature of the cohort, which recruited patients with
Crohn’s disease or ulcerative colitis during both active and
quiescent periods of disease, showing that microbial compositions
in patients with IBD often revert to more control-like, ‘baseline’
configurations when the disease is not active. By identifying the
gut microbial configurations that were most different from
baseline—regardless of specific disease state—the study defined a
dysbiosis score that called out highly divergent microbial
compositions, which share many features common to an overall
inflammatory response (for example, tolerance to oxidative stress).
This dysbiosis was not unique to the microbial response to
inflammation, however, and was associated with other host and
biochemical alterations, pointing to new potential directions for
management of systemic dysregulation in IBD. These included large
shifts in acylcarnitine pools and bile acids, increased serum
antibody levels, and alterations in transcription for several microbial
species. Concurrent transcriptomics and 16S amplicon mucosal
community profiling from biopsies also identified potential host
factors that might be able to shape the microbial community, in
particular several chemokines, highlighting these as being involved
in a potentially dysregulated interaction during periods of disease
49
activity .
The study’s longitudinal multi-omic profiles further allowed
researchers to characterize the stability and dynamics of
host–microbiome interactions during disease, in particular
highlighting ways in which community state and immune responses
are distinctly less stable in participants with IBD than in control,
healthy individuals. In numerous cases, the microbiome of a
participant with IBD changed completely over the course of only
weeks (measured as maximal Bray–Curtis dissimilarity to earlier
samples from the same subject), whereas such shifts were rare in
individuals without IBD. The main microbial contributors to these
large-scale shifts from one time point to the next largely mirrored

the differences observed in dysbiosis, and the shifts frequently
marked the entrance into or exit from periods of dysbiosis. Finally,
the study’s long-term, complementary molecular measurements
enabled the construction of a network of more than 2,900
significant host and microbial cellular and molecular interactors
during IBD, ranging from specific microbial taxa to human
transcripts and small molecule metabolites. This network of
mechanistic associations identified several key components that
are central to the alterations seen in IBD, highlighting octanoyl
carnitine, several lipids and short-chain fatty acids, the
taxa Faecalibacterium, Subdoligranulum, Roseburia, Alistipes,
and Escherichia, some at both the metagenomic and
49
metatranscriptomic levels, and host regulators of interleukins .
Networks of mechanistic associations such as this may provide the
key to disentangling the complex system of interactions that results
in chronic inflammation in IBD and in other systemic microbiome-
linked immune diseases.
Multi-omics profiling in prediabetes
Type 2 diabetes mellitus (T2D) affects more than 10% of the adult
US population, and another 30% show early signs of the disease
80
(referred to as prediabetes) ; 70% of the latter will develop diabetes
in their lifetime. T2D is characterized by complex host–microbiome
81,82
interactions , but little is known about systemic alterations
during prediabetes, their effects on biological processes, or the
critical transition to full-blown T2D. Prediabetes and T2D are often
associated with insulin resistance, and thus studies of individuals
with prediabetes or insulin resistance offer unique opportunities to
investigate the earliest stages of diabetes. It is essential to create
a global and simultaneous profile of both host and microbial
molecules in individuals with prediabetes over time, in order to fully
understand the molecular pathways that are affected in people with
prediabetes and/or insulin resistance and how these conditions

affect both biological responses to environmental challenges (for
83,84
example, viral infections ) and the onset of T2D.
To better understand T2D at its earliest stages, as part of iHMP,
85
the Integrated Personal ’Omics Project (IPOP) followed 106 healthy
and prediabetic individuals during quarterly periods of health,
respiratory viral infection (RVI) and other perturbations over about
51
four years (Fig. 4). In one such perturbation, a subset of 23
individuals underwent a directed weight gain followed by weight
86
loss . In total, 1,092 collections across all participants were
profiled. For each visit, blood was assayed for host molecular ’omics
profiling and two types of samples, nasal swabs and faeces, were
collected for microbial profiling. Each participant’s exome was
sequenced once; otherwise, for each visit, 13,379 transcripts were
profiled from peripheral blood mononuclear cells, 722 metabolites
and 302 proteins from plasma, and 62 cytokines and growth factors
from serum. In addition, thousands of gut and nasal microbial taxa
and computationally predicted genes were profiled using 16S rRNA
amplicons. All visits were also intensively characterized by 51 clinical
laboratory tests. In addition, because of the focus on T2D, a number
of glucose dysregulation tests were performed, including
measurements of fasting glucose and haemoglobin A1C levels, oral
glucose tolerance tests, and tests of insulin resistance.

Fig. 4. Differential host and microbial responses to dietary perturbations and
infectious disease in individuals with prediabetes. For integrated personal
’omics profiling of the microbiome during prediabetes, 106 participants were
followed for up to four years, with samples (primarily blood and stool)
collected quarterly and additional samples collected during periods of RVI and
other stresses. The genomes of the participants were sequenced and
measurements of transcriptomes, proteomes, metabolomes, and microbiomes
taken at each visit, in addition to clinical details. Insulin-resistant individuals
showed differences from insulin-sensitive participants in various
measurements, both at baseline and in response to the stresses such as weight
loss and RVI.
Baseline measurements were generally stable within individuals,

even for long periods of time, with only some analytes changing
51
significantly over time . However, many analytes, such as clinical
laboratory measurements, cytokine profiles, and gut microbial taxa
(mostly those of low abundance) were highly variable between
individuals. Participants who were ultimately insulin-resistant had
distinguishable molecular and microbial patterns at baseline from
those who were ultimately insulin-sensitive, and an analyte test
was devised as part of the study in order to differentiate them.
Notably, individuals undergoing RVI or changes in weight showed
thousands of specific molecular and microbial changes during these
perturbations, and insulin-resistant and insulin-sensitive
individuals responded very differently to perturbations. For
example, during RVI, insulin-resistant participants showed
substantially decreased and delayed inflammatory responses (for
example, the acute phase response and IL-1 signalling) and altered
gut microbial changes when compared with insulin-sensitive
participants (for example, in Lachnospiraceae and Rikenellaceae but

not bacilli). Accordingly, there were fewer changes in nasal
microbiota in insulin-resistant participants, and both the richness
and the diversity of nasal microorganisms decreased during RVI
in insulin-sensitive but not insulin-resistant participants.
Furthermore, global co-association analyses among the thousands
of profiled molecules revealed specific associations in insulin-
resistant individuals that differ from those seen in insulin-sensitive
participants and vice versa, indicating different patterns of
51
host–microbiome interactions in the two groups .
Another important goal of the study was to assess how
host–microbiome multi-omics and related emerging technologies
can be used to better manage patients’ health. We found that taking
millions of measurements per individual over time enabled the early
51,87
detection of potential disease states . These included early
detection of T2D, which developed differently among participants
and was better detectable with varied assays; for example, some
individuals first exhibited measurements in the diabetic range on
tests of fasting glucose, whereas others did so on tests of
haemoglobin A1c, oral glucose tolerance tests, or even continuous
glucose monitoring. These results, together with detailed
characterization of glucose dysregulation over time, illustrate the
heterogeneity of T2D development. Overall, the data led to
microbially linked, clinically actionable health discoveries in a
number of diseases in addition to T2D, including metabolic disease,
cardiovascular disease, haematological or oncological conditions,
and other areas; these signs were often present before symptom
onset, demonstrating the power of using big data, including the
microbiome, to better manage human health.
Resources from the HMP2
Together, the HMP1 and HMP2 phases have produced a total of 42

terabytes of multi-omic data, which are archived and curated by
the DCC at at http://ihmpdcc.org and in public and/or controlled-

access repositories such as the Sequence Read Archive
(SRA; https://www.ncbi.nlm.nih.gov/sra), the Database of
Genotypes and Phenotypes (dbGaP; https://www.ncbi.nlm.nih.gov/
gap/), Metabolomics Workbench
(https://www.metabolomicsworkbench.org/), and others (Fig. 5).
All data on the DCC is available for unrestricted use, with a subset of
project metadata also being shared when permitted by institutional
review boards (IRBs), and other restricted data (for example, human
genome sequences and protected metadata) available through
controlled access at dbGaP (projects PRJNA398089, PRJNA430481,
PRJNA430482, PRJNA326441, phs001719, phs000256, phs001626,
phs001523, and others). The formal data models and associated
entity relationship schemas produced by all phases of the HMP
are freely available at https://github.com/ihmpdcc/osdf-schemas.
The DCC website allows users to find, query, search, visualize, and
download data from thousands of samples with associated
metadata. Once a user has identified a set of files, conditions,
subjects, or phenotypes of interest, he or she can add this set to
a shopping cart for further operations. Files can then be directly
downloaded for use at the user’s local site or in the cloud. The
HMP DCC efforts are thus by design consistent with the NIH’s
stated goals to make all data generated from NIH funding findable,
88
accessible, interoperable, and reusable . The success of these
efforts is evidenced by a consistently high rate of user access to
the web resources, with 9,000–12,000 user sessions each month,
and a greater throughput anticipated after the publication of these
resources here.

Fig. 5. Resources from HMP1 and HMP2 available at the DCC. The HMP DCC
(http://ihmpdcc.org) hosts raw and processed data from both phases of the
Human Microbiome Project, comprising in total more than 42 Tb of

multi-omic data. From the HMP1, these include 16S rRNA gene amplicons and
metagenomes from the healthy human subjects (HHS) baseline cohort, as well
as resources from demonstration projects (https://www.hmpdacc.org/health/
projectdemos.php) and genomes of associated microbial isolates. From the
HMP2, these include data that IRBs gave permission to be made publicly
available from the pregnancy and preterm birth (MOMS-PI), inflammatory
bowel disease (IBDMDB), and prediabetes (IPOP) projects, with links to raw
data in other repositories. Additional data deposited elsewhere including
microbial reference genomes, HMP1 human genomes, and controlled access
data for all HMP2 projects is also linked from the DCC. Categories of data are
colour coded, and the number of items in each dataset is indicated by the size
of the circles.
Complementary host–microbiome interactions
Although each of the three HMP2 studies revealed new biology

within their respective areas of health and disease, a surprising
range of host–microbiome immune and ecological features were
common among them. The combination of shotgun metagenomics,
untargeted metabolomics, and immunoprofiling was particularly
effective, as in all projects this subset of molecular measurements
tended to efficiently capture interpretable host and microbial
properties that are linked to disease. Conversely, genetic variants
were generally difficult to link to the microbiome in such small
populations, which were necessary in order to deeply profile multi-
omics over time, and we anticipate host sequencing to be more
useful when integrated into larger cross-sectional surveys. Another
notable property was that, as in most microbiome studies, changes
that occurred within individuals, populations, or phenotypes were
often much smaller than baseline variation between individuals.
This is particularly true at microbiome-relevant time scales, for
which repeated measures as rapid as days to weeks were necessary
to capture the most specific host–microbiome interactions. Health-
associated microbiome interactions can thus manifest in extremely
diverse ways among individuals, making a combination of large-
scale population surveys with within-subject longitudinal profiles

essential for understanding the mechanisms of microbiome-linked
disease.
As a result, other aspects of host–microbiome interactions were
highly localized and subject-specific within each of the three
studies. In all three conditions, microbial changes and associated
host responses were strongest when captured at the time the
changes occurred, and often within the tissue of origin. It is thus
clear from these and other studies that host–microbiome
interactions have both localized and systemic effects. Strong local
perturbations initiated from either the host or microbial side can
induce subsequent spatiotemporal responses that can continue
over time and/or in other tissues, presumably with signals carried
spatially by circulating small molecules and/or temporally by gene
regulation or microbial growth, and involving regulatory circuits
with both host and inter-microbial components. Continued
coordinated efforts to measure the diverse host and microbial
properties involved in each condition will thus be important for
developing targeted and, when necessary, personalized therapies
for microbiome-associated conditions, as well as for uncovering
general principles that govern host–microbiome interactions. Other
dynamic interactions that were not measured in all studies, such as
an individual’s first microbial exposures near birth and subsequent
immune development, may also represent key contributors to
baseline microbiome personalization and help to explain disease-
linked dynamics based on events that took place years or even
decades earlier.
Next steps in microbiome multi-omics
The collective results of the NIH HMP projects, alongside many

other studies, show that the microbiome is an integral component
of human biology, with a major role in health and well-being. Inter-
individual variability and highly diverse host–microbiome responses
over time have driven the development of new methods for

population microbiome studies using multiple, complementary
longitudinal measurements, as well as highlighting the need to
follow such studies up with mechanistic models in order to validate
causative associations. The successful close of the HMP program
itself has left an enduring legacy of multiple scientific generations
of trained human microbiome investigators; provided the resulting
community with a wealth of data, analytical, and biospecimen
resources; and positioned the NIH and other funding agencies to
89
continue work in a broad range of microbiome-linked conditions .
Funding for microbiome science, human and otherwise, is now
being coordinated among NIH Centers and Institutes
(https://www.niaid.nih.gov/research/trans-nih-microbiome-
working-group); other US government agencies including the
National Science Foundation, Environmental Protection Agency,
Department of Energy, National Institute of Standards and
Technology, Department of Agriculture, National Oceanographic
and Atmospheric Administration, National Aeronautics and Space
Administration, and Department of Defense
(https://commonfund.nih.gov/hmp/programhighlights);
philanthropic organizations including the Bill and Melinda Gates
Foundation, the March of Dimes, the Burroughs Wellcome Fund,
the Sloan Foundation, the Keck Foundation, the Juvenile Diabetes
Research Foundation, the Crohn’s and Colitis Foundation, and
others; and industry and public–private partnerships. Moreover, as
complex global projects are launched to tackle aspects of
personalized medicine, it is now obvious that it is informative to
include components focused on the effect of the human
microbiome.
As with any large study, the HMP2 has raised more new questions
than it has answered. The aetiologies of baseline inter-individual
differences in the microbiome, and of its dynamic changes over
time, were not apparent even from the wide range of measurement
types incorporated into these three studies and populations. Many
immune and biochemical responses appear to be associated with
specific strains that are unique to one or a few individual hosts,

but it is not clear whether such strains are sufficient or necessary
for their associated disease phenotypes. A few mechanisms were
identified by which signals in the gut can be transmitted to systemic
conditions such as diabetes, but not the specific small molecules
or immune cell subsets by which they are likely to be
transmitted—particularly in other health conditions that have not
yet been studied in such detail. Finally, each HMP2 study was
necessarily carried out within a geographically and genetically
constrained population, and global differences in early life events,
infectious disease exposure, or diet may change how microbiome
dynamics contribute to human disease. Human-associated
microbiology now clearly extends beyond infectious and
gastrointestinal diseases to areas barely imaginable a few decades
ago, including metabolism, neoplasia, maternal and child health, and
central nervous system function. As the NIH HMP comes to an end,
it is clear that its results have revealed a multitude of new avenues
of research and technologies for future investigation, and we look
forward to new discoveries based on resources from the program
and exciting findings yet to come.
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Acknowledgements
We thank the NIH Common Fund (particularly M. E. Perry), the

Trans-NIH Microbiome Working Group (TMWG) and the HMP
Science Advisors (iHMP advisors: J. Davies, F. Ouellette, E. Chang,
and the late S. Falkow) for their support throughout the HMP
program, and additional project principal investigators K. Jefferson
and R. Xavier. We acknowledge funding from NIH grants UH2/
UH3AI083263 and U54HD080784 (G.A.B., J.F.S., K. Jefferson)
supported with funds from the Common Fund, the National Center
for Complementary and Integrative Health, and the Office of
Research on Women’s Health, grant U54DK102557 (C.H., R. Xavier),
including funds from the Common Fund, the National Institute of
Diabetes and Digestive and Kidney Diseases, the National Center for
Complementary and Integrative Health, and the Office of Dietary
Supplements and grant U54DK102556 (M.P.S., G.M.W.), with funds
from the Common Fund and the National Institute of Diabetes and
Digestive and Kidney Diseases.

Reviewer information
Nature thanks Frederic Bushman and the other anonymous

reviewer(s) for their contribution to the peer review of this work.
Author information
A list of participants and their affiliations appears at the end of the

paper.
These authors contributed equally: Lita M. Proctor, Heather H.
Creasy, Jennifer M. Fettweis, Jason Lloyd-Price, Anup Mahurkar,
Wenyu Zhou.
Affiliations
1. National Human Genome Research Institute, National Institutes

of Health, Bethesda, MD, USA
Lita M. Proctor
2. Institute for Genome Sciences, University of Maryland School

of Medicine, Baltimore, MD, USA
Heather H. Creasy, Anup Mahurkar & Owen White
3. Department of Microbiology and Immunology, School of

Medicine, Virginia Commonwealth University, Richmond, VA,
USA
Jennifer M. Fettweis & Gregory A. Buck
4. Department of Obstetrics and Gynecology, School of Medicine,

Virginia Commonwealth University, Richmond, VA, USA
Jennifer M. Fettweis & Jerome F. Strauss III
5. Center for Microbiome Engineering and Data Analysis, Virginia

Commonwealth University, Richmond, VA, USA

Jennifer M. Fettweis, Gregory A. Buck & Jerome F. Strauss III
6. Department of Biostatistics, Harvard T.H. Chan School of Public

Health, Boston, MA, USA
Jason Lloyd-Price & Curtis Huttenhower
7. Broad Institute of MIT and Harvard, Cambridge, MA, USA

Jason Lloyd-Price & Curtis Huttenhower
8. Department of Genetics, Stanford University School of

Medicine, Stanford, CA, USA
Wenyu Zhou & Michael P. Snyder
9. Department of Computer Science, College of Engineering,

Virginia Commonwealth University, Richmond, VA, USA
Gregory A. Buck
10. Stanford Center for Genomics and Personalized Medicine,

Stanford, CA, USA
Michael P. Snyder
11. Stanford Diabetes Research Center, Stanford, CA, USA

Michael P. Snyder
12. The Jackson Laboratory for Genomic Medicine, Farmington, CT,

USA
George M. Weinstock
13. Department of Immunology and Infectious Diseases, Harvard

T.H. Chan School of Public Health, Boston, MA, USA
Curtis Huttenhower
Consortia
The Integrative HMP (iHMP) Research Network Consortium
• Lita M. Proctor, Heather H. Creasy, Jennifer M. Fettweis, Jason

Lloyd-Price, Anup Mahurkar, Wenyu Zhou, Gregory A.
Buck, Michael P. Snyder, Jerome F. Strauss III, George M.
Weinstock, Owen White & Curtis Huttenhower
Contributions
All authors contributed manuscript text and created or edited

figures. Individual HMP2 projects discussed were implemented and
managed by J.M.F., G.A.B. and J.F.S. (PTB); J.L.-P. and C.H. (IBD); W.Z.,
M.P.S. and G.M.W. (T2D); H.H.C., A.M. and O.W. (DCC).
Corresponding authors
Correspondence to Gregory A. Buck, Michael P. Snyder, Jerome F.

Strauss III, George M. Weinstock, Owen White or Curtis
Huttenhower.
Ethics declarations
Competing interests
M.S. is a cofounder of Personalis, Qbio, Sensomics, January,

Filtricine and Akna and advisor for Genapsys. The other authors
declare no competing interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations.

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• Video 2 – Human Science (Part I) – The Gut Brain Axis,
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• Figure 1 – Targeted amplicon and metagenomic sequencing

approaches to microbiome analysis by Bharti and Grimm, 2021.
• Figure 2 – In Vitro Model Schematic by Ventura et al., 2020.
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analysis by Jiang et al., 2019. Licensed under Creative
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• Video 2 – New technique explores gut microbiome one
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• Figure 1 – Possible definitions of a healthy microbiome:

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The Gut Microbiome
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Thinking. Licensed under Creative Commons: By Attribution
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• Video 2 – Diet and gut microbiome interactions in irritable
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• Video 3 – Microbiome and Obesity – Martin Blaser by National
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• Video 5 – Rob Knight: Fecal Transplants? The Disgusting is Par
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The Oral Microbiome
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• Figure 1 – Oral and systemic diseases associated with the oral
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• Images of body sites and organs in Figure 1 by Servier Medical
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• Figure 2 – Examples of metagenomic studies of associations
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The Skin Microbiome
• Video 1 – A mixed community of skin microbiome

representatives influences cutaneous processes by Research
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Hong by National Human Genome Research Institute. Licensed
• Video 3 – Expansion of known skin microbes could aid skin
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• Hot Spot Quiz Image – Skin Microbiome by Jane Ades, NHGRI
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• Figure 1 – Ecological Determinants of the Respiratory

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• Video 1 – The Lung Microbiome: Challenging Old Paradigms
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• Video 1 – VALENCIA: A nearest centroid classification for

vaginal microbial communities by Research Square. Licensed
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(right, Nugent score = 9) and without (left, Nugent score = 1) BV
by Lewis and Gilbert, 2020. Licensed under Creative Commons
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• Figure 2 – Schematic illustrating vaginal bacteria with potential
to impact the urinary tract by Lewis and Gilbert, 2020.
Licensed under Creative Commons Attribution 4.0 License
• Video 2 – Beyond bacterial vaginosis: vaginal lactobacilli and
HIV risk by Research Square. Licensed under Creative
• Video 3 – A temporal model of cervicovaginal microbiota
identifies targets to promote reproductive health by Research
Image Credits | 709

Interactions
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gut and skin for a healthy state by De Pessemier et al., 2021.
Licensed under the Creative Commons Attribution (CC BY)
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al., 2021. Licensed under the Creative Commons Attribution
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• Figure 3 – Development of the gut-brain over time by Liang et
al., 2018. Licensed under the Creative Commons Attribution
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• Video 2 – Functional GI Disorders: The role of the gut-brain
interaction, microbiome and nutrition by UMMCVideos.
Licensed under Creative Commons Attribution license (reuse
allowed).
• Figure 4 – Direct and indirect mechanisms of infecting the
brain by Olsen and Hicks, 2019 licensed under under the terms
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• Figure 5 – Chronic wounds and skin-brain axis by Hadian et al.,
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Health
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mineralization and alters the rhizosphere
microbiome by Research Square. Licensed under Creative
• Hotspot Image – Urban multispecies health by Robinson et al.,
2021 licensed under the Creative Commons Attribution License
• Figure 1 – Illustration and common names of representative

ocean animal life within their approximate depth-defined
ecological habitats by Apprill, 2017. Licensed under the terms
• Figure 2 – Conceptual diagram of a host-microbiome
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• Figure 3 – Photographs of marine animals and their associated
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Soil Microbiomes
• Video 1 – Understanding Our Soil: The Nitrogen Cycle, Fixers,

and Fertilizer by Jimi Sol. Licensed under Creative Commons:
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Plant Microbiomes
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assembly and functional adaptation by Research Square
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• Video 4 – [microbiome] The Plant Microbiome: Managing the
plant microbiome (3.3) by iMooX at licenced under Creative
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• Figure 1 – A summary of possible sources of forensically

relevant microbiota identified by the literature review by
Robinson et al., 2021 licensed under the terms of the Creative
• Video 1 – Raising temperatures: the immunology of influenza

by British Society for Immunology under a Creative Commons
• Article 1: Respiratory Viral Infection-Induced Microbiome
Alterations and Secondary Bacterial Pneumonia by Hanada et
al., 2018 under CC BY 4.0 license
• Article 2 – Allergic inflammation alters the lung microbiome
and hinders synergistic co-infection with H1N1 influenza virus
and Streptococcus pneumoniae in C57BL/6 mice by
LeMessurier et al., 2019 under CC BY 4.0 license
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• Video 1 – Influenza Virus – Viral entry and fusion inhibitor by

iCAP Université Claude Bernard Lyon 1 under a Creative
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influenza virus infection by Lee et al., 2019 under CC BY 4.0
license
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Pneumonia by Manohar et al., 2020 under CC BY 4.0 license
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microbiome evolution related to ventilator-associated
pneumonia by Sommerstein et al., 2019 under CC BY 4.0
license
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across farm environments in conventional and organic dairy
herds in Pennsylvania by Pitta et al., 2020 under CC BY 4.0
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fecal, environmental, and slurry resistomes of pig farms by
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Microbiota: Key Role of Airborne Microbial Communities by
Kraemer et al., 2018 under CC BY 4.0
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a Highly Transformable Haemophilus parasuis Strain SC1401 by
Dai et al., 2019 under CC BY 4.0
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• Article 2 – The Gut-Lung Axis in Health and Respiratory
Diseases: A Place for Inter-Organ and Inter-Kingdom
Crosstalks by Enaud et al., 2020 under CC BY 4.0
• Article 3 – Translating Lung Microbiome Profiles into the Next-
Investigator’s Perspective by Kitsios, 2018 under CC BY 4.0
• Figure 1 – Coffee exports by country 2019-20 by Dylan Parks.

Data source: International Coffee Organization
• Figure 2 – A world map of countries by coffee production, 2019
by Cbahrs licensed under CC BY-SA 4.0
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Disease Complex in Coffee and Tomato by Lamelas et al., 2020
License (CC BY).
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Arabica L. in the rainforests of Ethiopia and progress in
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Derivative Notes
Minor revisions not noted below include editing language for clarity,
length, and flow as well as corrections to and additions of hyperlinks
and citations. Other revisions not listed below include removing
first person language and references to sections of the text that
were not included in the book/article.
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Creative Commons CC0 public domain dedication:
Environmental Metagenomics
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Commons Attribution 3.0 License.

Health
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Ocean Microbiome and Marine Life
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Soil Microbiomes
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Plant Microbiomes
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• Embedded article by Jaiswal and Shukla, 2020 licensed under

the terms of the Creative Commons Attribution License (CC
BY).
◦ Grammar and syntax edits made under the section

‘Ecological Safety & Risk Assessment’.
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The Integrative Human Microbiome Project
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Microbiomes: Health and the Environment

Microbiomes: Health and the

About the Publisher vii

About This Project ix

Part I. An Introduction to Microbiomes

Part II. Analyzing Microbiomes

Part III. Human Health and Disease

4. Human Health and Disease 57

11. Environmental Nutrient Cycling and Human 215

Part V. Other Microbiome Applications

16. Forensic Microbiomes 479

Part VI. Journal Club

17. Journal Club Articles 543

Part VII. Case Studies

18. Case Study #1 - Human Health 547

Part VIII. Additional Resources

21. The Integrative Human Microbiome Project 577

Image Credits 705

Derivative Notes 719

About Mavs Open Press

Creation of this resource was supported by Mavs Open Press,

About the Publisher | vii

Pressbooks is an open source, web-based authoring tool based on

Information about open education at UTA is available online.

viii | About the Publisher

Microbiomes: Health and the Environment was created to provide

This project was completed by thorough review of published

About This Project | ix

About the Author

Dr. Dylan Parks is an Assistant Professor of Instruction at the

x | About This Project

UTA CARES Grant Program

Lead Author/Editor/Project Manager

Dylan Parks, PhD – Assistant Professor of Instruction, The

Stephanie Lucas, MS – Alumni, The University of Texas at Arlington

Cover art design by Westley Harwart

Microorganisms represent the fundamentals of life and interact

A microbiome can be best described as a collective polymicrobial

Figure 1. A schematic highlighting the composition of the term microbiome

An interactive H5P element has been excluded

Our fascination with microorganisms began before we even fully

The Human Microbiome

It was evident that the human microbiome and its involvement in

One or more interactive elements has been excluded

Co-evolution of the host-microbiota symbiosis can be considered

Traditionally, it has been difficult to characterize complete

Considering the abundance of microbiomes that no doubt play role

Check Your Understanding

• What is the difference between a microbiome and

• Video 1 – What is the human microbiome? by

1. Apprill, A. (2017). Marine Animal Microbiomes: Toward

Choosing the correct approach when characterizing a microbiome

Depending on the type of research questions and the particular

What is the Difference between Culture-Dependent and Independent

-Excerpt taken from Findley and Grice, 2014 available

Up until the 1980s, microbiologists routinely relied on

Culture-independent methods of microbial identification

Within the culture-independent targeted approach, PCR