Streptobacillus moniliformis

• Fastidious, Gram (-), pleomorphic

bacillus, biochemically inactive

• Normally found in the oropharynx of

rodents

• Identification is serologic -
identification of serum agglutinins;
fourfold increase b/n acute and
convalescent titers is significant to
diagnosis
• RAT BITE FEVER
– infection in man acquired by bite, scratch or
direct contact w/ infected animal;
– Characterized by relapsing fever, chills,
muscle aches, vomiting, skin rash on palms
and soles of feet; lymphadenitis
– Complications: arthritis, hepatic and splenic
disorder, endocarditis and meningitis
– Difficult to diagnose since it resembles other
diseases

• HAVERVILL FEVER Streptobacillus

– Acquired by ingestion of contaminated food moniliformis
– Resembles Rat Bite Fever but GIT signs are
more pronounced
 Cardiobacterium hominis
• Nonmotile, oxidase and indole (+),
catalase (-)

• Grows on BAP, enhanced @ increased

CO2

• Ferments glucose and maltose but

negative for lactose

• Normally found in the upper RT Cardiobacterium hominis

on BAP
• Rare cause of endocarditis; C in HACEK
SPIROCHETES
 Genera and Species to be considered:
• Treponema pallidum subsp pallidum
• Treponema pallidum subsp pertenue
• Treponema pallidum subsp endemicum
• Treponema carateum
• Borrelia recurrentis
• Leptospira interrogans
 General Characteristics
• Long, slender, helically curved, gram (-
) bacilli

• Unique morphology: axial fibrils and

an outer sheath

• Axial fibrils/axial filaments:

– flagella-like structures hat wrap around
the cell wall
– Enclosed with an outer sheath
– Attached w/in the cell wall by platelike
structures called INSERTIUM DISKS
located near the ends of the cells
– Responsible for the corkscrew-like
motility of spirochetes
 Differentiation of genera
Based on:
• Number of axial fibrils
• Number of insertion disks
• Biochemical and metabolic features
GENUS AXIAL FILAMENTS INSERTION DISKS MORPHOLOGY

Treponema 6 -10 1 Slender w/ tight coils

Borrelia 30 – 40 2 Thicker, w/ fewer and looser coils

Leptospira 2 3-5 Resemble borrelia except for their

hooked ends
Treponema pallidum

Borrelia recurentis

Leptospira interrogans
 TREPONEMA
• Microaerophilic, stain poorly w/ Gram’s or Giemsa’s and
are best observed w/ DARK-FIELD or PHASE-
CONTRAST MICROSCOPY.

• Major pathogens infect only humans and have not been

cultivated;

• Other treponemes are normal inhabitants of the oral

cavity or the genital tract; culturable anaerobically

• Pathogenesis: T. pallidum can penetrate through intact

membranes or through breaks in the skin – invades the
bloodstream – diff body sites

• T. pallidum has a remarkable tropism to arterioles and

infection ultimately leads to ENDARTERITIS and
progressive tissue destruction
 Epidemiology and Spectrum of Disease
AGENT MOT LOCATION DISEASE SIGNS/SYMP AGE GROUP
TOMS
T. pallidum Sexual Worldwide Venereal Chancre All ages
subsp pallidum Congenital Syphilis
T. pallidum Traumatized Humid, warm Yaws Skin: papules, Children
subsp pertenue skin comes in climates: Africa, nodules and
contact w/ South an Central ulcers
infected lesion America, Pacific
T. pallidum Mouth – to- Arid, warm Endemic Skin/mucous Children
subsp mouth by climates: North nonvenereal patches,
endemicum utensils Africa, SE Asia, syphilis papules,
Middle East macules, ulcers,
scars
T. carateum Traumatized Semiarid, warm Pinta Skin papules, All ages but
skin comes in climates: Central macules primarily
contact w/ and South children and
infected lesion America, Mexico adolescents
 Vincent’s Disease

• Acute,
necrotizing,
ulcerative
gingivitis char by
destructive
lesions of the
gums
• Caused by oral
spirochetes
Syphilis: C.A. Treponema pallidum
1. Primary Syphilis
– Appearance of chancre (painless ulcer) at the site of inoculation, most commonly
the genitalia
– Chancre heals w/in 3-6 wks
– Dissemination of the organism occurs
– Highly infectious phase

2. Secondary Syphilis
– Occurs 2-24wks after primary syphilis when the orgs have reached a sufficient
number
– Px is ill; systemic symptoms occur: fever, weight loss, malaise, loss of appetite
– Skin is most affected: widespread rash (EXANTHEM)
– Condyloma latum – painless, wart-like lesion in vulva or scrotum; packed w/
spirochetes; ulcerates and is therefore EXTREMELY CONTAGIOUS

LATENT STAGE
– The disease then becomes subclinical but not really dormant
– Serologic tests can be done on this latent stage
3. Late/Tertiary Syphilis
– Tissue destructive phase that appears 10-25yrs after initial infection
in up to 35% of untreated pxs
– Complications occur:
• CNS disease
• Cardiovascular abnormalities
• Eye disease
• Gummas (granuloma-like lesions) in any organ
PRIMARY SYPHILIS SECONDARY TERTIARY SYPHILIS
 Laboratory Diagnosis
• Direct Detection
– Skin lesions by dark-field/ phase-contrast microscopy
– Serous exudate using PCR

• Serodiagnosis – measure 2 types of antibodies

– Treponemal Abs –produced against treponemal Ags
– Non-treponemal Abs/Reaginic Abs
• produced by infected pxs against components of mammalian cells T. pallidum in dark-field mx
• Also produced by pxs w/ infectious diseases: leprosy, TB, chancroid,
leptospirosis, malaria, rickettsial disease, trypanosomiasis, LGV, measles,
chicken pox and IM
• Non-infectious diseases: drug addiction, autoimmune disorders, old age,
pregnancy, and recent immunization

– Tests for Reaginic Abs – flocculation/agglutination test; (+) result is

agglutination; SCREENING TESTS
• VDRL (Venereal Disease Research Laboratory)
• RPR (Rapid Plasma Reagin)

– Specific Serologic Tests/ CONFIRMATORY TESTS

• FTA-ABS (Fluorescent treponemal antibody absorption)
• TP-PA (Treponema pallidum particle agglutination) test
T. pallidum in FTA-ABS
 VDRL

RPR

(-) (+)
 AST; Treatment; Prevention
• AST – not performed
• Treatment – penicillin G
• Prevention – no vaccines; best accomplished by
early treatment; safe sex practice
 BORRELIA
• w/ 3 -10 loose coils and are actively
motile

• In contrast to treponemes, Borrelia

spp stain well w/ Giemsa

• Culturable; microaerophilic

• Causative agent of:

– Relapsing Fever (tickborne and
louseborne disease)
– Lyme disease (tickborne disease)

• MOT: vector borne, transmitted Ornithorodos (soft tick) hard tick

through bite of louse or tick
 Relapsing Fever
• Caused by Borrelia recurrentis and transmitted by the
louse Pediculus humanus subsp humanus

• Char by abrupt onset of fever, headache, and myalgia that

lasts 4-10 days.

• Incubation period – 2-15 days

• As the host produces specific Abs, organisms disappear

from the bloodstream becoming hidden in different body
organs (AFEBRILE PERIOD)

• Organisms reemerge w/ newly modified Ags and multiply

(FEBRILE PERIOD)

• More relapses assoc w/ tickborne relapsing fever, but

louseborne relapsing fever is more severe

• They have the ability to vary surface antigens as well as

avoid complement attack
 Lyme Disease
• Caused by Borrelia burgdorferi, B. garinii, B. afzelii and B. valaisiana

• Transmitted by Ixodes ticks (hard ticks)

– Natural hosts are deer and rodents but can attach to pets and humans
– All stages of ticks can harbor the spirochete and transmit disease
– Require 24hrs of attachment before disease transmission

• Most common vector-borne disease in N. America and

Europe

• 3 STAGES
1. First
• presence of char red, ringed-shaped skin lesion w/ a central clearing
that 1st appears at the site of tick bite but may develop to distant sites
erythematous migrans (EM)
• Headache, fever, muscle and joint pain, malaise
2. Second
• Weeks – months after infection
• Arthritis
• Most impt features: NEUROBORRELIOSIS neurologic disorders
(meningitis and neurologic deficits) and carditis
3. Third
• Chronic arthritis
 Laboratory Diagnosis
Specimen Collection, Transport and Processing
• Peripheral blood smear – specimen of choice for
direct detection of borreliae
• Specimens for stain/culture: blood, biopsy, joint and
CSF

Direct Detection
• Relapsing Fever – orgs can be seen in:
– wet preparations of peripheral blood under dark or
bright-field illumination, in w/c spirochetes move
rapidly, often pushing the red blood cells around
– Thick and thin films w/ Wright’s or Giemsa stains using
procedures similar to those used to detect malaria
• Lyme Disease
– Tissue section in Warthin-Starry silver stain
– PCR

Borrelia recurrentis in DFI

 Serodiagnosis
• Relapsing Fever
– Not reliable because of the many antigenic shifts of Borrelia
– Pxs may exhibit increased titers to Proteus O,X,K antigens (up
to 1:80)
• Lyme Disease
– IFA – screening test
– ELISA – screening test
– Western Blot – confirmatory test
 AST, Treatment and Prevention
• AST – not performed
• Treatment
– tetracyclines; doxycycline, amoxicillin, cefuroxime and
parenteral cephalosporins (drugs of choice for 1st stage of
Lyme Disease)
– Broad-spectrum cephalosporins like ceftriaxone or
cefotaxime (drugs of choice for those who fail initial
treatment; later stages)
• Prevention
– Surface protein A vaccine
 LEPTOSPIRA
• Include both free-living and parasitic forms.

• Pathogenic Leptospira spp are currently classified in 7

spp; main species as Leptospira interrogans, causative
agent of Leptospirosis

• LEPTOSPIROSIS is a zoonosis w/ a worldwide

distribution but is most common in warm climates
where there is contact w/ infected animals or water
contaminated w/ their urine or blood of infected
animals.

• Leptospires enter the human host thru breaks in the

skin, mucous membranes, or conjunctivae; orgs are
maintained in nature by virtue of persistent
colonization of renal tubules of carrier animals

• Pathogenic leptospires rapidly invade the bloodstream

after entry and enter the CNS and kidneys.

• Virulent strains show chemotaxis toward hemoglobin as

well as the ability to migrate through host tissues
 Spectrum of Disease
• Incubation period – 2 to 20 days
• ANICTERIC LEPTOSPIROSIS
– Fever, headache , myalgia
– Self-limiting illness w/ septicemic
stage
• Severe headache, very high fever (3-7
days)
• IMMUNE STAGE/ICTERIC
LEPTOSPIROSIS/WEIL’S
DISEASE
– Most severe illness, w/ symptoms
caused by liver, kidney, and/or
vascular dysfunction w/ lethal
pulmonary hemorrhage
 Laboratory Diagnosis
SPECIMEN COLLECTION, TRANSPORT
AND PROCESSING
• During the 1st 10 days of illness, leptospires
are present in the blood and CSF
• Urine can be obtained beginning in the 2nd
week of illness up to 30days after the onset
of symptoms
Direct Detection
• Dark-field microscopy – blood, CSF, and
urine
• PCR
• Fluorescent Antibody Staining
Cultivation
• Culture orgs from blood, CSF, urine. A few drops of heparinized or Na
oxalate-anticoagulated blood are inoculated into tubes of semi-solid
media enriched w/ rabbit serum (Fletcher’s or Stuart’s) or bovine serum
albumin.

• Urine shd be inoculated soon after collection because acidity may harm
the spirochetes

• 1-2 drops of undiluted urine are added to 5ml of Fletcher’s/Stuart’s

• The addition of 200ug/mL of 5-fluorouracil may prevent bacterial

contamination

• All cultures are incubated @ RT or 30C in the dark for up to 6-8wks

• Growth will be seen below the broth’s surface, presence shd be

examined weekly using wet mount under dark-field mx. Leptospires will
exhibit corkscrew-like motility.
 Treatment
• Ceftriaxone, penicillin, amoxicillin, doxycycline, and
tetracycline
ANAEROBIC
BACTERIOLOGY
 Anaerobic Bacteria
Gram (+) Spore Forming Rods Gram (+) Cocci
• Clostridia  Peptococcus

Gram (+) Non-Spore Forming Rods

 Peptostreptococcus
• Actinomyces  Anaerobic Streptococcus
• Bifidobacterium  Ruminococcus
• Eubacterium  Coprococcus
• Propionibacterium
Gram (-) Cocci
Gram (-) Rods  Acidoaminococcus
• Bacteroides  Megasphaera
• Prevotella (Saccharolytic)  Veillonella
• Porphyromonas (Asaccharolytic)
• Fusobacterium
 General Characteristics
• These organisms will not grow in the presence of
oxygen; require reduced oxygen tension  Indications of Involvement
of Anaerobes in Infectious
Process
• Some are:
– Microaerophilic – when they require 5% O2 such as
Actinomyces spp, Bifidobacterium spp and 1. Infection is in close proximity to a
Clostridium spp mucosal surface

– Aerotolerant anaerobe - when they require reduced 2. Presence of foul odor

concs of O2 (anaerobic and a microaerophilic
environment) eg most strains of Propionibacterium,
Lactobacillus, some Clostridium spp 3. Presence of large quantity of gas

– Obligate anaerobe – when they require a strict 4. Presence of black color or brick-red
anaerobic environment: 0% O2. eg Most Bacteroides, fluorescence
many Clostridium, Eubacterium, Fusobacterium,
Peptostreptococcus and Porphyromonas 5. Presence of sulfur granules

• Are frequently involved in polymicrobial infections 6. Distinct morphologic characteristics in

(infection is caused by more than 1 bacterial spp) Gram stained preparations

• Predominant normal flora in the lower intestine

Endogenous Anaerobes of Various  Conditions that Predispose
Anatomical Sites individuals to Anaerobe-
associated
Site Anaerobes Infections/Diseases

Skin Propionibacterium, Peptostreptococci 1. Human or animal bite wounds

2. Aspiration of oral contents into the lungs

URT Peptostreptococci, Actinomyces, after vomiting
Propionibacterium, Fusobacterium, Prevotella,
Veilonella
3. Tooth extraction, oral surgery, traumatic
puncture of the oral cavity

Oral Cavity Actinomyces, Eubacterium, peptostreptococcus,

4. GIT surgery or traumatic puncture of the
Fusobacterium, Prevotella, Bifidobacterium,
Porphyromonas, Veilonella bowel

5. Genital tract surgery or traumatic

Bifidobacterium, Eubacterium, Clostridium, puncture of the genital tract
Intestine
peptostreptococci, Bacteroides fragilis group,
Bilophila, Fusobacterium, Porphyromonas, 6. Introduction of soil into a wound
Prevotella, Sutterella, Veilonella

GUT Peptostreptococci, Bifidobacterium, Fusobacterium,

Lactobacillus, Mobiluncus, Prevotella, Veilonella
 Anaerobic Media and
Incubation
• Contain supplements such as:  ANAEROBIC SYSTEMS for
– Hemin, Blood, Vitamin K incubation include:
– Na bicarbonate (source of CO2) 1. Anaerobic jar method
– Thioglycollic acid, Na thioglycollate and 2. Evacuation-replacement systems
cystine (absorb oxygen) 3. Glove-box method
4. Disposable anaerobic bags
• For primary isolation of anaerobes:
– nonselective blood agar plate,  Anaerobic cultures are incubated @ 35-
– anaerobic selective media, and an 37deg C for at least 48hrs. After
examination, plates shd be reincubated
– enrichment broth should be used (eg FT). for another 2-4 days to ensure that some
of the slower-growing organisms (eg
Actinomyces), are not missed.
• They do not need to be stored
anaerobically, but they should be held in a  Broth cultures shd be held for 7 days
reduced form 8 to 16 hours before before reported if there is (+) or (-)
inoculation. growth.
 Anaerobic Systems
1. ANAEROBIC JAR METHOD
– oxygen is removed from the system
through rxn w/ hydrogen. This rxn takes
place in the presence of a Palladium
catalyst. Generation of H2 and CO2 takes
place by the addition of water: 2H2 + O2 --
---- 2H2O

• Methylene blue strips (blue) are used to

determine the effectiveness of the system:
if the color of the MB strip:
– Becomes white – minimal anaerobic
condition
– Remains blue/reverts to blue –
anaerobic environment is achieved
The GasPak Anaerobic System

Purpose: to create an oxygen-free environment

for the growth of anaerobic microorganisms.

•Inoculated plates or tubes are placed inside the

chamber. Anaerobic conditions are created by
adding water to a gas generator envelope that is
placed in the jar just before sealing.

•The envelope contains two chemical tablets,

sodium borohydride and sodium bicarbonate.
Water reacts with these chemicals, producing
hydrogen gas and carbon dioxide. The hydrogen
gas combines with free oxygen in the chamber to
produce water, removing all free oxygen from
the chamber.

•This reaction is catalyzed by the element

palladium (attached to the lid of the jar). The
carbon dioxide replaces the removed oxygen,
creating a completely anaerobic environment.
GasPak System
 Anaerobic Systems
CANDLE JAR
• The candle jar can be used to decrease the
concentration of oxygen in the culture
environment. Bacterial plates or tubes are
placed in a jar along with a lighted candle.

• The lid is sealed. As the flame burns,

oxgyen decreases and the flame goes
out. Oxygen is present, but at a lower
percentage than in the atmosphere.

• The concentration of carbon dioxide

increases as a result of the flame; this is
also desirable for many microaerophiles.
 Anaerobic Systems
2. EVACUATION-
REPLACEMENT SYSTEM–
an airtight container, such as Brewer Jar or
GasPak jar is used.

• The air in the jar is removed and replaced

with a mixture of gas containing 85% of
nitrogen, 10% of hydrogen and 5% of
carbon dioxide

• A redox indicator system (MB strips) shd

be used to monitor the effectiveness of
the system.

• This method has largely been replaced by

more modern techniques.
 Anaerobic Systems
3. GLOVE-BOX METHOD a self-
contained anaerobic environment of 85%
of nitrogen, 10% of hydrogen and 5% of
carbon dioxide is provided through
external gas sources.

• Specimens and other materials are passed

into the chamber through an entry lock
system. Cultures can be incubated in an
incubator inside the glove box.

• This system also permits the med tech to

perform most techniques necessary to
isolate and identify anaerobes w/in the
box.

• This is useful for laboratories that handle a

great volume of anaerobic cultures.
GLOVE-BOX METHOD
 Anaerobic Systems
4. DISPOSABLE ANAEROBIC
BAGS provide a convenient anaerobic
incubation system. The BioBag system consists
of a plastic bag w/ a H2CO3 gas generator that
is released on the addition of water.

• Palladium pellets act as catalysts while a

resazurin indicator monitors the system’s
effectiveness. The bag is sealed after activation
of the gases.

• Three-jar system:
– 1st jar – holds uninoculated plates
– 2nd jar – holds plates w/ colonies for subculture
– 3rd jar – holds freshly inoculated plates
 AEROTOLERANCE TESTS
• Performed on any colony type found growing anaerobically.

• The colony is subcultured:

– onto an AEROBIC BLOOD agar plate (incubated in increased CO2/ candle jar)
– onto an ANAEROBIC BAP (incubated anaerobically/anaerobic jar)

COLONY TYPE GROWTH ON AEROBIC GROWTH ON ANAEROBIC

BLOOD BLOOD
Obligate aerobe Growth No growth
Microaerophile
Obligate anaerobe No growth Growth
Facultative anaerobe Growth Growth
 ANAEROBIC MEDIA
MEDIUM CONTENTS PURPOSE
Anaerobic blood agar (ANA) TSA w/ 5% blood Isolation of all anaerobes (obligate and
Supplements: yeast, hemin, Vitamin K reducing facultative)
agent: L-cystine

Anaerobic PEA blood agar ANA plus phenylethyl alcohol Inhibits swarming by Proteus and growth of
facultative anaerobes. Isolation of gram +
and gram – obligate anaerobes

Anaerobic kanamycin-vancomycin blood ANA plus antibiotics kanamycin and VA VA inhibits facultative and obligate gram +
agar (KV) anaerobes
Kanamycin inhibits facultative gram – rods

Isolation of gram (-) anaerobes esp

Bacteroides, Prevotella and some
Fusobacterium

Anaerobic paromomycin-vancomycin Similar to KV; alternate of KV Same as KV; pigment prodn by Prevotella is
laked blood (PVLB) agar “Laked” means blood is frozen and thawed detected earlier because of laked blood

Thioglycollate Enrichment broth w/ Vitamin K and hemin Supplement to plating media for slower-
growing bacteria; supports growth of most
anaerobes

Bacteroides bile-esculin (BBE) Detects organism’s ability to grow in 20%

bile and to hydrolyze esculin (brown-black
color)
IDENTIFICATION OF ANAEROBES
• Key factors in the identification of anaerobes
include:
– Colonial characterizations
– Pigment production
– Hemolysis
– Pitting of the agar surface
– Gram stain morphology
– Presence and location of spores
– Susceptibility to high levels of antibiotics
– Biochemical reactions

1. GRAM STAIN
2. COLONIAL MORPHOLOGY
3. GAS-LIQUID CHROMATOGRAPHY
4. BIOCHEMICAL REACTIONS
5. AST
 Gram Stain
• Very important in the identification of  Presence or absence of spores and
anaerobes their location. Particularly important
• Methanol fixation works better than heat to Clostridia spp.
fixation for anaerobes
 Terminal spores – end of the
• Bacteroides and Prevotella – pale, organism
pleomorphic, gram (-) bacilli w/ bipolar  Central spores – middle
staining  Subterminal spores – b/n the end
and the middle

• Fusobacterium – long, thin, filamentous,

gram (-) bacilli w/ tapered ends; often
arranged end to end

• Fusobacterium necrophorum – pale,

irregularly staining, highly pleomorphic,
gram (-) bacilli w/ swollen areas

• Actinomyces – branching, gram (+) bacilli

VARIATIONS IN ENDOSPORE MORPHOLOGY:
•(1, 4) central endospore
•(2, 3, 5) terminal endospore
•(6) lateral endospore
Bacteroides and Prevotella Fusobacterium

Fusobacterium necrophorum
Actinomyces
 Colonial morphology

• Actinomyces israelli – gram (+), non-

spore forming rod appears as
rough, heaped, white colonies that
resemble a “molar tooth” on ANA

• Prevotella melaninogenica – brown

to black pigment on ANA after 7
days of incubation
Gas-Liquid Chromatography (GLC)

• Analysis of metabolic end products The following VOLATILE ACIDS can be

into broth during anaerobic growth. identified:
 Acetic
 Propionic
• Fermentative products like glucose  Butyric and Isobutyric
can be used to identify anaerobes.  Isovaleric
 Caproic and Isocaproic
• Certain organisms characteristically  NONVOLATILE ACIDS identified
produce one or a pattern of end include:
products.  Pyruvic
 Lactic
 Succinic
• Short chain volatile fatty acids can
 Phenylacetic
be identified through a gas
chromatograph.
 Biochemical Reactions

• Lombard Dowell Presumpto

Plate System offers a series of 12
biochemical rxns that can be used to identify
anaerobes.

• Presumpto Plate I
• Presumpto Plate II
• Presumpto Plate III
• Presumpto Plate IV

• Other tests include: catalase, nitrate, urease,

and indole

• Pages 276-278; 286-287 Delost Presumpto Plate II

 AST Pattern

• The susceptibility of anaerobes to high-level

antibiotic disks (1mg kanamycin) can be used
as a preliminary tool in identification

• Most anaerobes have a characteristic

susceptibility pattern to:
– 10 ug colistin
– 5 ug vancomycin
– 1 mg kanamycin

• Delost pages 288-289

CLOSTRIDIA
 Species to be considered:
• Clostridium botulinum
• Clostridium difficile
• Clostridium perfringens
• Clostridium tetani

• Clostridium septicum
• Clostridium sordellii
 General Characteristics
• Anaerobic or aerotolerant gram (+) bacilli that will form spores
anaerobically

• Normally found in soil, water, and dust. Some spp are normal inhabitants
of the intestinal tract of many animals including humans

• Most are catalase (-); an important differentiation from Bacillus spp w/c
are catalase (+) and form spores aerobically

• Clostridia produce true exotoxins. Examples:

– Tetanospasmin – Clostridium tetani
– Botulism toxin – Clostridium botulinum
– 12 different toxins (including enterotoxins) – Clostridim perfringens
C. botulinum C. tetani

C. perfringens C. difficile
 Clostridium Groups
GROUPS Name Species
Group 1 Gas Gangrene C. perfringens Myonecrosis or gas gangrene of
C. septicum endogenous or exogenous nature
C. noyvi (Type A)
C. bifermentans
C. histolyticum
C. sordellii

Group II Tetanus C. tetani Tetanus (tetanospasmin – toxin

produced) ; exogenous wound infection
allows the organism to enter usually
through a puncture wound

Group III Botulism C. botulinum Food, wound and infant botulism

Group IV Antibiotic-Associated C. difficile Produces both an enterotoxin and

Pseudomembranous cytotoxin. The organism multiplies in the
colitis GIT following antibiotic therapy.

Group V Miscellaneous C. perfringens Frequently encountered in infections: brain

C. ramosum abscess, pneumonia, intraabdominal
C. bifermentans infections, gynecological infections,
bacteremia
 Epidemiology
• Most of the anaerobic bacteria that
can cause infections are also part of
man’s normal flora

• Other pathogenic anaerobes like

Clostridium botulinum and tetani
are soil and environmental
inhabitants and not considered as
part of normal flora
MODE OF ACQUISITION EXAMPLES
Endogenous strains of normal flora gain access Bacteroides fragilis, Clostridium perfringens
to normally sterile sites (surgery, accidental
trauma) or host defense mechanisms
(malignancy, diabetes, burns, aspiration)

Contamination of existing wound or puncture Tetanus, gas gangrene

by objects contaminated w/ Clostridia spp
Ingestion of preformed toxins in vegetable or Botulism
meat
Colonization of GIT w/ potent toxin-producing Infant botulism
organisms
Person – to – person spread Nosocomial spread of C. difficile – induced
diarrhea and pseudomembranous colitis, bite
wound infections
 Pathogenesis and Spectrum of Disease
ORGANISM VIRULENCE FACTORS SPECTRUM OF DISEASE AND
INFECTIONS
Clostridium Exotoxins: α-toxin is most impt, mediates GAS GANGRENE - life threatening, toxin -
perfringens destruction of host cell membranes mediated destruction of muscle and other
Enterotoxins: inserts and disrupts mucosal tissues
cell membranes FOOD POISONING – abdominal cramps,
diarrhea and vomiting; self - limiting

Clostridium Tetanospasmin- neurotoxic exotoxin that TETANUS (LOCKJAW) – generalized

tetani disrupts nerve impulses to muscles muscle spasms; could lead to respiratory failure

Clostridium Neurotoxins – extremely potent BOTULISM – nearly complete paralysis of

botulinum resp and other muscles; infant botulism – GIT of
infants; wound botulism – toxin on infected
wound

Clostridium Toxin A – enterotoxin PSEUDOMEMBRANOUS COLITIS –

difficile Toxin B - cytotoxin inflamed bowel is overlaid w/ a
pseudomembrane made up of necrotic debris,
wbcs and fibrin
 Clostridium perfringens
• Most common Clostridium isolated from  Has 12 known toxins
clinical specimens
• SPECTRUM OF DISEASE  CA also of necrotizing bowel
– Gas gangrene
– Food poisoning
disease – abdominal cramps,
– Postabortion sepsis vomiting, bloody diarrhea and
– Intraabdominal and pleuropulmonary infections acute inflammation
– Enterocolitis
– Antibiotic associated diarrhea
 FOOD POISONING – mild and
• Organism is VERY FERMENTATIVE. Gases self-limiting; foul smelling
formed accumulate in tissue restricting further
blood flow, increasing necrosis. stools, diarrhea but no
vomiting; caused by beta
• Surgical debridement or amputation may be
needed to remove necrotic tissue; the use of toxins; implicated foods: beef,
hyperbaric oxygen chamber can prevent the turkey, and chicken
spread of gas gangrene

• Gas gangrene is seen on pxs w/ poor

circulation like diabetics
Gas Gangrene/Myonecrosis
• life – threatening, toxin – mediated destruction
of muscle and other tissues; clinically manifests in
two forms:

• Cellulitis/ wound infection – necrotic skin is

exposed to C. perfringens w/c grows and
damages local tissue; palpation reveals a moist,
spongy, crackling consistency to the skin due to
pockets of gas ( crepitus) crepitus

• Clostridial myonecrosis – C. perfringens

inoculated w/ trauma into muscle, secretes
exotoxins that destroy adjacent muscles - release
enzymes that ferment CHO, - gas formation - Gas
pockets w/in the subcutaneous tissues and
muscles – thin, blackish fluid exudes from the
skin.
GAS GANGRENE
 Identification
• Cultivation on Ana BA
– Double zone of hemolysis on anaerobic blood agar;
inner zone is completely lysed due to theta
toxin, while outer zone is incompletely
hemolysed due to alpha toxin and
lecithinase

• Cultivation on Egg Yolk Agar

– Lecithinase produces an opaque yellow halo on colonies

• Cultivation on Nagler Plate

– ½ of plate is streaked w/ Type A antitoxin and then suspected
culture is streaked across the plate at right angle to the antitoxin
– Zone of precipitation around the colonies on the side w/o
antitoxin and little or no precipitation on the side w/ antitoxin –
indicates a (+) lecithinase test

• Reverse CAMP test – confirms the presence of Double zone of hemolysis on

organism.
– Suspected clostridia is streaked on BAP. Grp B strep (S. agalactiae) anaerobic blood agar
is streaked at right angle to the first streak. POSITIVE TEST:
arrowhead appearance at the intersection of the two streaks.
 Clostridium tetani
• CA of tetanus; tetanospasmin is a IDENTIFICATION
potent neurotoxin that travels along nerve
fibers from the wound to the anterior  Gram stain – drumstick/
horn cells of the spinal cord causing tennis racket appearance
convulsive contractions of voluntary  Biochemical – (+) indole and
muscles gelatinase, (-) lecithinase and lipase;
unable to ferment most CHOs; motile
 Usually not cultured from clinical
• Spasms involve the neck and jaw, thus, the specimens
name “lockjaw”

• DIAGNOSIS – clinical picture and

history of injury of the patient

• THERAPY & PREVENTION

– administration of neutralizing Abs
– Untreated cases has a mortality of >50%
– DPT vaccine
Tetanus/Lockjaw
– Classically follows a puncture wound by a rusty
nail or can follow skin trauma by any object
contaminated w/ C. tetanus spores (found in soil,
animal feces)
– Spores are deposited in the wound and
germinate as long as there is localized anaerobic
environment (necrotic tissue)
– From its location, C. tetani releases
tetanospasmin – inhibit inhibitory interneurons
w/c would send high frequency impulses to
muscle cells, resulting to sustained tetanic
contraction

– Severe muscle spasms, esp in jaw muscles

(trismus/lockjaw); grotesque grinning
expression (risus sardonicus) due to facial
muscles’ spasm

– Mortality is high once the stage of lockjaw has

been reached
Neonatal tetanus
 Clostridium botulinum
• CA of food, wound and infant botulism DIAGNOSIS – clinical s/s and px
• Botulism toxin – powerful neurotoxin that binds history
the synapse of the cholinergic nerve fibers
resulting to flaccid paralysis in the facial muscles,
head, throat, thorax, diaphragm, arms and legs TREATMENT – administration of
• DEATH usually occurs from respiratory paralysis antitoxin; infection is irreversible
once toxins bind to nervous tissues
• There are 7 types of botulism toxin: A, B, C, D, E, F
and G. A, B, E and F most commonly implicated in IDENTIFICATION
human botulism  Gram stain – tennis racket appearance
 Biochemical – (+) lipase and
• FOOD BOTULISM – ingestion of lecithinase, (-) indole; ferments only
contaminated home-canned foods glucose, motile

• WOUND BOTULISM – occurs when organism

grows in the wound; can result from improperly
sterilized surgical dressings and casts

• INFANT BOTULISM – most common type;

ingestion of spores fr soil, dust and honey that can
germinate in the GIT
Botulism
• rapidly fatal food poisoning caused by the
extremely lethal neurotoxin of C. botulinum

• Neurotoxin blocks the release of

acetylcholine causing flaccid muscle
paralysis

• Pxs are afebrile, initially developing nerve

palsies causing double vision (diplopia) and
difficulty swallowing (dysphagia) – general
muscle weakness – sudden respiratory
paralysis – death

• Pxs must be immediately treated w/

antitoxin w/c can neutralize only the
unbound neurotoxins in the bloodstream
 Infant botulism
• Occurs when infants ingest food
contaminated w/ C. botulinum
spores (eg. Fresh honey w/
contaminated spores)

• Spores germinate and C.

botulinum colonize infant’s
intestinal tract – toxin is released.

• Initially, infant is constipated for

2-3 days – difficulty in
swallowing, muscle weakness
Infant botulism
 Clostridium difficile
Pseudomembranous colitis
• Antibiotic-associated diarrhea, w/c can follow the use of
broad spectrum antibiotics (ampicillin,
clindamycin and cephalosporins)

• These antibiotics can wipe out part of the normal

intestinal flora, allowing C. difficile to superinfect the
colon
• On colonization – release of exotoxins:
– Toxin A (enterotoxin)- causes diarrhea;
– Toxin B (cytotoxin)– cytotoxic

• Characterized by severe diarrhea, abdominal cramps and

fever

• Red inflamed mucosa of the colon w/ an area of white

exudate (PSEUDOMEMBRANES) on the surface of
the large intestine. Necrosis of the mucosal surface occurs
underneath the pseudomembranes
Pseudomembranous colitis
 Identification
• Demonstration of cytotoxin through:
– Enzyme immunoassay
– Tissue culture – gold standard
– Latex agglutination
• Gram stain: spores are oval and subterminal

• Cultivation: cycloserine cefoxitin

fructose agar (CCFA)

• Biochemical: (+) gelatinase, (-) lecithinase, lipase,

and indole; ferments glucose and lactose

• Organism is motile and fluoresces under UV light

 Laboratory Diagnosis
Specimen for:
 Infant botulism – serum and stool
 Wound botulism – serum, stool, and tissue biopsy
 C. difficile culture – feces
 C. difficile toxin – liquid/unformed feces;
▪ solid, formed stool, rectal swab are adequate
to detect carriers but not the active cases of
enterocolitis!
Anaerobic transport system Anaerobic transport Anaerobic transport system
for liquid specimens system for swab for tissue specimens
specimens
Direct Detection Methods
 Antigen Detection
 Cytotoxin B of C. difficile can be detected using tissue
culture assay
 ELISA for Toxins A and/or B for C. difficile
 Gram Stain
• Standard Gram stain procedures and reagents are used except: safranin is
left on for 3-5 minutes; 0.5% aqueous basic fuchsin can be used as
counterstain

C. perfringens C. botulinum C. tetani C. difficile

Large, boxcar shape, Resemble Drumstick or May produce
w/ blunt ends chains up to 6
Spores are seldom
tennis racket tennis racket
appearance cells aligned
seen
end to end
 Cultivation and Incubation Conditions
(pg 461 Bailey and Scott)
• Bacteroides Bile Esculin agar (BBE)  Inoculated plates shd be immediately
• Laked Kanamycin-Vancomycin blood agar incubated under anaerobic conditions @
(LKV) 35-37deg C for 48hrs. In general, cultures
• Anaerobic phenylethyl alcohol agar (PEA) shd not be exposed to O2 until after
48hrs incubation (sensitive during their
• Anaerobic Blood Agar
log phase growth)
• Chocolate agar
 Colony morphology changes
dramatically between 24-48hrs; plates
• Cultures for C. difficile are plated on can be examined after 24hrs w/o O2
cycloserine cefoxitin fructose
exposure for B. fragilis and C.
agar (CCFA)
perfringens

• Special anaerobic blood bottles w/ various  Plates that show no growth at 48hrs shd
media including: be incubated for at least 5days before
– Thioglycollate broth; discarding
– Thiol broth;
– Schaedler’s broth
 C. difficile on CCFA C. botulinum on egg yolk agar

C. perfringens on Blood agar C. tetani on Blood agar

 AST and Treatment
• Penicillins for Clostridia, w/ or w/o beta-lactamase

• C. difficile-induced GI disease – metronidazole or

vancomycin

• C. botulism and C. perfringens food poisoning – AST not

recommended
 Prevention
• Tetanus – multiple dose vaccine (DPT)

• Individuals who have eaten food suspected

of containing botulinum toxin shd be purged
w/ laxatives, have their stomach pumped,
and be given high enemas
 Obligate Intracellular and
Nonculturable Bacterial Agents
 Genera and species to e considered:

• Chlamydia
– Chlamydia trachomatis
– Chlamydia psittaci
These organisms are
– Chlamydia pneumoniae obligate intracellular
• Rickettsia bacteria and are also
– Rickettsia rickettsii considered extremely
– Rickettsia prowazekii
difficult to culture or
– Rickettsia typhi
• Orientia tsutsugamushi unable to be cultured.
• Ehrlichia
• Coxiella burnetii
• Trophoryma whipplei
 CHLAMYDIA
• Obligate intracellular bacteria once regarded as viruses

• Have unique developmental life -cycle (pg 511 Bailey &

Scott)
– RB (reticulate body) – replicative form
– EB (elementary body) – extracellular, metabolically inert,
infective form
– PB (persistent body) – viable, noncultivable growth stage;
resulting in a stable relationship w/ host cell – chronic infection

• Resemble gram (-) bacilli but they do not have a

peptidoglycan layer in cell wall

• Human pathogens:
– Chlamydia trachomatis
– Chlamydia psittaci
– Chlamydia pneumoniae
 Chlamydia trachomatis
• Has 17 serovars associated w/ clinical syndromes including:
infertility, ectopic pregnancy

• Differentiation of serovars is based on MAJOR OUTER

MEMBRANE PROTEIN (MOMP) antigens

• most common sexually transmitted pathogen and a major

cause of PID and ocular trachoma

• Spread primarily by sexual contact; other modes: during

birth, through fomites (hand to eye), flies

• Natural habitat - MAN

Pathogenesis and Spectrum of Disease
• Chlamydiae grow in host cells including:
– Mucosal epithelium, vascular endothelium
– Smooth muscle cells
– Monocytes and macrophages

• Action: modulation of apoptosis of infected host cells; they can

either induce or inhibit programmed cell death

• Spectrum of Diseases:
– Trachoma
– Lymphogranuloma venereum
– Oculogenital infections
– Perinatal infections
 Trachoma
• Chronic inflammation of the
conjunctiva

• Major cause of preventable

blindness worldwide

• Conjunctiva can become scarred

leading to distorted eyelids,
eyelashes turn (SCAR TRACTION)
in and are misdirected; corneal
damage – ulceration, scarring and
visual loss
Lymphogranuloma venereum (LGV)
STAGES
1. 1st stage - primary small genital lesion
at the initial infection site

2. 2nd stage – acute lymphadenitis (groin

swelling or bubo); systemic infection,
granulomatous proctitis

3. 3rd stage – genital hyperplasia, rectal

fistulas, rectal stricture, draining
sinuses

• Diagnosis:
– isolation of causative agent fr a bubo/ other
infected site
– Frei’s test – intradermal skin test of LGV
Ag (sensitive only in the early stages)
3rd Stage LGV
 Oculogenital and Perinatal Infections
• Acute INCLUSION CONJUNCTIVITIS in adults –
w/ purulent discharge but does not lead to
blindness

• Genital infections:
– Urethritis,cervicitis, bartholinitis, proctitis
– Salpingitis, epididymitis, acute urethral
syndrome in females
– Infertility due to tubal occlusion resulting fr PID Females

• Both sexes can be asymptomatic

• Perinatal infections – conjunctivitis in

infants w/ infected mothers; incubation
period: 5-12 days – 6 weeks; pneumonia
in 10-20% of infected infants

Males
 Laboratory Diagnosis
• Specimen Collection and Transport:

– Specimens: urethra, cervix, nasopharynx,

rectum, aspirates fr fallopian tubes and
epididymis
– Screening specimens for women: endocervix
swab
– Urethral specimens shd be collected 2hrs after
voiding urine
– Keep specimens cold (heat labile) during
transport; if not cultured w/in 24hrs, shd be
frozen at -70deg C

• Cell Culture: cycloheximide - treated McCoy

Hela cells (most common); monkey kidney
cells; used for legal cases
 Direct Detection Methods
• Cytologic exam
– Cell scrapings fr conjunctiva using
Giemsa.
– Pap smears – endocervical and urethral
scrapings

• Ag detection and NA Hybridization

– DFA – using fluorescein-isothiocyanate
– ELISA
Chlamydia trachomatis in DFA
– Amplification assays
– Serodiagnosis
 AST, Treatment, Prevention

• AST not practical

• Treatment: erythromycin,
macrolides: tetracyclines
and fluoroquinolones

• Prevention: behavioral
change; safe sex practice
 Chlamydia psittaci
• Differs fr. C. trachomatis:
– not sulfonamide sensitive
– EB morphology
– Inclusion bodies

• Common in birds; Psittacine birds (parrots, parakeets) –

major reservoir for human disease (pneumonia)

• Outbreaks have occurred among turkey-processing

workers and pigeon aficionados

• MOT – inhalation of aerosols fr infected birds ; human – to

human MOT is very rare

• Organisms are deposited in the alveoli; alveolar

macrophages and then carried to regional lymph nodes –
dissemination to RES
Spectrum of Disease
• Respiratory: pneumonia, severe headache, mental status changes
and hepatosplenomegaly
• Infection ranges fr unapparent to life – threatening

Laboratory Diagnosis
• Serologic
• Culture is performed only in Biosafety Level 3 containment
facilities

Treatment
• Tetracycline
 Chlamydophila pneumoniae
(Chlamydia pneumoniae TWAR)
• TWAR strain(TW Taiwan/ AR acute respiratory) –
1st isolated fr the conjunctiva of a child in Taiwan
serologically related to AR-39 fr a college student
in US

• Difference fr C. trachomatis: pear-shaped

appearance of its EB

• Human pathogen (no animal reservoirs

identified); MOT-inhalation of infected aerosol Chlamydia pneumoniae

• Spectrum of Diseases: URT infections

• Lab Dx: throat swab, sputum for PCR

• Treatment: tetracycline and erythromycin

 Rickettsiaceae
• Rickettsiae are fastidious, obligate,
intracellular parasites

• Small (0.3um x 1-2um),

pleomorphic, Gram (-) bacilli

• multiply by binary fission in the

cytoplasm of host cells; release of
mature rickettsiae results in host
cell lysis

• Disease caused in humans is vector-

borne or can be contracted through
inhalation of spores
Pathogenesis and Spectrum of Disease
• Rickettsia do not undergo
intracellular developmental cycle
as opposed to Ehrlichia

• Orgs deposited directly into the Orientia tsutsugamushi

bloodstream – engulfed by
endothelial cells – multiply – cell
lysis upon release

• Damage to endothelial cells Rickettsia typhi

accounts for the changes
throughout the body: skin, brain,
lung, muscle

• Refer to page 519

Rickettsia rickettsii
3 GROUPS OF RICKETTSIA; differentiation is based on:
– arthropod MOT,
– rate of intracellular growth,
– rate of intracellular burden, and
– extent of intracellular growth

1. Spotted fever group – R. akari, R. conorii, R. rickettsii

2. Typhus group – R. prowazekii, R. typhi
3. Scrub typhus group – Orientia tsutsugamushi

Table 46-4 page 519 Bailey and Scott

• Primary clinical manifestation of
Rickettsial disease: triad of fever,
headache, rash

• Lab Dx : Immunohistology and PCR;

direct detection of Ehrlichia and
Anaplasma from PBS or CSF

• Specimen: biopsy of skin tissue

from the rash (optimum specimen)

• Treatment: tetracyclines
 Spotted Fever Group
AGENT DISEASE VECTOR DISTRIBUTION

Rickettsia akari Rickettsial pox Mites Worldwide

Rickettsia conorii •Mediterranean and Israeli Ticks Southern Europe,

Spotted Fevers Mideast, Africa
• Indian tick typhus;
•Kenya tick typhus

Rickettsia rickettsi Rocky Mountain spotted Ticks North and South

fever America
(southeastern
states and
Oklahoma)
 Typhus Group and Scrub Typhus Group
AGENT DISEASE VECTOR DISTRIBUTION

Rickettsia Endemic typhus Lice Worldwide

prowazekii Brill-Zinsser Disease None; Worldwide
recrudescent
disease

Rickettsia typhi Murine typhus Fleas Worldwide

Scrub typhus Group

Orientia Scrub typhus chiggers South and

tsutsugamushi Southeast Asia,
South Pacific
 Ehrlichia, Anaplasma, Neorickettsia
AGENT DISEASE VECTOR DISTRIBUTION

Ehrlichia chaffeenis Human monocytic Ticks Southeast, South

ehrlichiosis (HME) Central, and mid-
Atlantic US
Ehrlichia ewingii Granulomatous Ticks US
ehrlichiosis (immunocompromised
pxs)
Anaplasma Human granulocytic Ticks US, Europe
phagocytophilum ehrlichiosis (GME)

Neoricketssia Sennetsu fever Ticks Southeast Asia

sennetsu (E. particularly Japan
sennetsu)
 Weil-Felix Test
• Serologic assays for the dx of rickettsial infections include:
– IFA (indirect fluoresecence assay)
– EIA (enzyme immunoassay)
– Western immunoblotting (Weil-Felix Test) – agglutination of certain strains of
Proteus vulgaris by serum w/ pxs w/ rickettsial infections

DISEASE OX-19 OX-2 OX-K

Brill – Zinsser V V ____

Epidemic Typhus + V ____

Murine typhus + V ____

Rickettsialpox ___ ___ _____

Rocky Mountain Spotted Fever + + ____

Scrub typhus ___ ___ V

Q Fever ___ ___ ___

Ehrlichiosis ___ ____ ____

 Coxiella burnetti
• Causative agent of Q fever, an acute
systemic infection that primarily affects
the lungs

• Gram (-) coccobacillus, smaller than

Rickettsia and is more resistant to various
physical and chemical agents

• Can survive extracellularly, but can be

grown only in lung cells

• Has a spore-like life cycle and can exist in 2

antigenic states:
– Phase I form – when isolated fr animals;
highly infectious Micrograph of Coxiella
– Phase II form – when grown in cultured burnetti
cell lines; not infectious
 Epidemiology and Pathogenesis
• Most common reservoirs: cattle, sheep, goats

• Infected animals (asymptomatic) shed the bacteria

in urine, feces, milk, and birth products

• MOT: inhalation of contaminated aerosols

• Incubation period – 2wks- 1month

• Q fever is endemic worldwide except in

Scandinavia; clinical manifestations:
– Headache, fever, chills, myalgias
– Atypical pneumonia, granulomatous hepatitis,
endocarditis
– Neurologic manifestations: encephalitis,
meningoencephalitis
– No development of rash (in contrast to
Rickettsial infections)
 Laboratory Diagnosis, Treatment
• Done in Biosafety Level 3
containment facility

• Cultures (shell vial assay w/

human lung fibroblasts) are
incubated for 6-14days @37deg C
in CO2. Bacteria are then detected
through IFA (reference method
for both acute and chronic Q
fever)

• Treatment: tetracyclines Coxiella burnetti

 Tropheryma whipplei
• Gram (+) actinomycete w/c is the causative agent of
Whipple’s disease

• WHIPPLE’S DISEASE
– Found primarily in middle-age men
– Char by the presence of PAS-staining macrophages
(indicating mucopolysaccharide or glycoprotein) in almost
every organ system
– Diarrhea, weight loss, arhtralgia, lymphadenopathy
– Hyperpigmentation, long hx of joint pain
– Distended and tender abdomen
– Neurologic and sensory changes; endocarditis

• Treatment: trimethoprim/sulfamethoxazole,
tetracycline (serious relapses observed); colchicine –
controls symptoms
 presence of PAS-staining macrophages

Irregular nodular folds

characteristic of Whipple’s disease
 Calymmatobacterium granulomatis
• Encapsulated, pleomorphic, Gram (-) bacilli usually
observed in vacuoles of large mononuclear cells

• CA of GRANULOMA
INGUINALE/DONOVANOSIS

– STD, low infectivity

– Major cause of genital ulcers in India, Papua New

Guinea, Carribean, Australia and parts of South
America
– Char by subcutaneous nodules that enlarge and evolve
to form beefy, erythromatous, granulomatous, painless
lesions that bleed easily
– These genital lesions have been mistaken for neoplasms

– Pxs often have inguinal lymphadenopathy

beefy, erythromatous, granulomatous, painless lesions that
bleed easily
 Lab Dx, Treatment
• Seen in scrapings of lesions stained w/
Wright’s or Giemsa; subsurface infected cells
must be present

• grps of orgs are seen w/in mononuclear

endothelial cells – DONOVAN BODY:
– Stains blue w/ prominent polar granules
giving rise to a “safety pin” appearance
surrounded by a large, pink, capsule

• Capsular polysaccharide of C. granulomatis

cross reacts w/ Klebsiella species…strong
affinity to Klebsiella…present name:
Klebsiella granulomatis

• Treatment: tetracycline or
ampicillin
 CELL WALL - DEFICIENT
BACTERIA: MYCOPLASMA AND
UREAPLASMA
 Genera and Species to be considered
• Mycoplasma pneumoniaea
• Mycoplasma hominis
• Mycoplasma genitalium
• Ureaplasma urealyticum
 General Characteristics
• Smallest known free-living forms (0.3 –
0.8um); NO CELL WALL bacteria
• Belong to the class Mollicutes (soft skin
in Latin)
• Require sterols for membrane fxn and
growth
• Mollicutes appear most closely related Mycoplasma pneumoniae
to bacilli, streptococci and lactobacteria
• Most are aerobic and fastidious
 Epidemiology and Pathogenesis
• Mycoplasmas are part of the normal flora of the URT and
GUT

• Infants are commonly colonized w/ U. urealyticum and M.

hominis, but will not persist after 2yrs of age; on puberty
colonization results from sexual contact

• MOT: vertical, during birth

• M. pneumoniae – cause of community acquired

pneumonia

• Pathogenesis:
– colonize mucosal surfaces of URT and GUT;
– rarely produce invasive diseases except in immunocompromised pxs;
– have great affinity to ciliated and non-ciliated epi cells
– M. pneumoniae has P1 adhesin (causes interaction with host cells )
ORGANISM CLINICAL MANIFESTATIONS

Mycoplasma pneumoniae Asymptomatic infection

URT infection in young children
LRT infectionin adults

Genital mycoplasmas: Systemic infections in neonates: meningitis

Ureaplasma urealyticum and U. urealyticum- chronic lung disease
Mycoplasma hominis Invasive disease in immunocompromised pxs – bacteremia
UGT infections: prostatitis, PID, bacterial vaginosis

Mycoplasma genitalium Nongoncoccal urethritis in men; cervicitis and endometritis in

females
Erythema multiforme rash (Stevens-Johnson syndrome) associated
with Mycoplasma pneumoniae infection in a preadolescent girl.
 Laboratory Diagnosis
Specimen Collection, Transport and Processing
• Specimens:
– blood, joint fluid, amniotic fluid, urine, prostatic secretions, pleural
secretions, bronchoalveolar lavage
– Tissues and swabs of: throat, nasopharynx, urethra, cervix, vagina
– Blood for culture of genital mycoplasmas shd be collected w/o
anticoagulants and immediately inoculated into culture medium
– Transport media are necessary, mycoplasma are prone to drying (no
cell wall)

Direct Detection Methods

• PCR

• Take note of table 47-3 page 528 Bailey & Scott

Cultivation
• Media shd contain beef/soybean protein w/
serum, fresh yeast extract

• M. pneumoniae – glucose (dextrose) shd be

incorporated since this mycoplasma ferments
glucose to lactic acid – change in color indicates
presence of M. pneumoniae

• U. urealyticum and M. hominis – urea and/or

arginine shd be incorporated
 Approach to identification
Mycoplasma pneumoniae
• spherical, grainy, yellowish colonies embedded in
the agar w/ a thin outer layer;

• definitive demonstration: place 0.5% guinea pig

rbcs in PO4-buffered saline, after 20-30mins @ RT,
colonies are observed for adherence of rbcs: ONLY
M. PNEUMONIEA & ONE SEROVAR OF U.
UREALYTICUM HEMADSORB

Ureaplasma urealyticum and Mycoplasma

hominis
• A8 agar – urease prodn in the presence of CaCl3
indicator
• Colonies are dark brownish clumps (U. urealyticum)
• Urease negative w/ Fried egg appearance (M.
hominis)
Fried egg appearance
Mycoplasma pneumoniae Mycoplasma hominis

Mycoplasma genitalium Ureaplasma urealyticum

 Treatment
• M. pneumoniae infections are self-limiting, no treatment
required but to shorten disease duration, they are
susceptible to macrolides, tetracyclines, ketolides, and
fluoroquinolones (sparfloxacin)