Journal of Advances in Medicine and Medical Research

Volume 35, Issue 10, Page 1-8

DOI: 10.9734/jammr/2023/v35i10113
ISSN:2456-8899
Original Research Article
PREVALENCE AND PATTERN OF EXTENDED SPECTRUM BETA-
LACTAMASES (ESBL) PRODUCING Klebsiella pneumoniae IN
BIOLOGICAL SPECIMEN AT A TERTIARY HOSPITAL
*JONAH A A., OBUNGE O K., WARISO K. T.
Department of Medical Microbiology, University of Port Harcourt Teaching Hospital, Port Harcourt, Rivers state.
*correspondence: [email protected]

ABSTRACT

Aim: The study was carried out to assess the distribution and pattern of ESBL producing
Klesbsiella pneumoniae in specimen analysed at the Microbiology laboratory of a tertiary
healthcare center.
Study Design: Cross-sectional study
Place and Duration of Study: Department of Medical Microbiology, University of Port
Harcourt Teaching Hospital, Rivers state, Nigeria carried out between January 2019 to
June 2019
Method: Clinical specimen including blood, wound biopsy/aspirate, urine and sputum
specimens were collected and processed according to standard methods. Blood agar,
Cysteine Lactose Electrolyte Deficient agar (CLED) and MacConkey agar were used to
culture the specimens to isolate K. pneumoniae. A confirmatory test was carried out on all
suspected ESBL isolates using combination disks according to the CLSI guidelines.
Results: The study reports a prevalence rate of 34.7% (118/340) for ESBL-production
among K. pneumoniae isolates in UPTH, Port Harcourt. Blood culture was found to have
the highest proportion of ESBL K. pneumoniae (57.1%) while urine samples had the least
proportion of ESBL K. pneumoniae growth (32.5%). There was no significant distribution
of ESBL K. pneumoniae growth by age groups or gender among the patients sampled.
However, the occurrence of ESBL K. pneumoniae was higher among in-patients (37.0%)
compared to outpatients (28.7%).
Conclusion: The growth of ESBL K. pneumoniae was observed to be higher among
inpatient subject indicative of the likelihood of high prevalence ESBL K. pneumoniae in
hospital acquired infection. Therefore, there is a great need for urgent interventions in the
areas of antimicrobial usage and infection prevention and control in our settings.

Keywords: antibiotics, prevalence, extended spectrum beta-lactamase, klebsiella pneumoniae

1.0 INTRODUCTION
Extended-spectrum beta-lactamases (ESBLs) are a heterogeneous group of plasmid-mediated
bacterial enzymes that confer significant resistance to oxyimino-cephalosporin and
monobactam antimicrobials[1]. They are however inhibited by beta-lactamase inhibitors such
as clavulanic acid[2]. There is a global variation even in closely related regions of the
prevalence of ESBLs. Reports have shown that ESBL-producing bacteria are prevalent in
clinical and community settings with serious public health consequences[3, 4]. A study
conducted by Mohammed et al in Maiduguri Nigeria, recorded a prevalence of 23.8% and
30.0% ESBL- producing Escherichia coli and Klebsiella spp, respectively.[5] Klebsiella
pneumoniae, a gram negative bacillus, is one of the common pathogens associated with
multiple drug resistance and an important source of transferable antibiotic resistance.[6]
There has been a significant increase of ESBLs among clinical isolates caused by the
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Volume 35, Issue 10, Page 1-8
DOI: 10.9734/jammr/2023/v35i10113
ISSN:2456-8899
Enterobacteriaceae in the last three decades[7–9]. Consequently, the occurrence of ESBL has
been a major challenge in both the healthcare settings and local communities. The
consequences of ESBL (Extended-Spectrum Beta-Lactamase) Klebsiella infections in
hospital settings can be significant and potentially life-threatening and are associated with
treatment difficulties, spread of infection, especially in high-risk areas such as intensive care
units, Increased morbidity and mortality, while increasing the Length of stay and costs and
exacerbating the current challenge of antimicrobial resistance[1, 3, 7, 10]. Therefore, it is
important to implement effective infection control measures, use antibiotics judiciously, and
promote the development of new treatments for ESBL Klebsiella infections to reduce their
impact in hospital settings. The current study was carried out to assess the distribution and
pattern of ESBL producing Klesbsiella pneumoniae in specimen analysed at the
Microbiology laboratory of a tertiary healthcare center.[11]

2.0 METHODS
2.1 Study Area
This study was conducted at the University of Port Harcourt Teaching Hospital (UPTH)
located at 4o53’59,4N, 6o 55’45.6E in Rivers State, Nigeria. The State is a cosmopolitan area
full of industrial activities especially in the oil and gas sector. The State has a population of 5
million people and is bounded on the South by the Atlantic Ocean, to the North by Imo, Abia
and Anambra States, the East by Akwa-Ibom State and to the West by Bayelsa and Delta
States. It is home to 3 major indigenous ethnic groups: Ijaw, Ikwerre and Ogoni. People from
diverse cultural and ethnic backgrounds also live and work in the State. The University of
Port Harcourt Teaching Hospital has an estimated bed capacity of 830 and an estimated
200,000 patients are seen annually. It is a tertiary healthcare institution with specialist
consultants in various medical specialties that serves as a referral centre in Rivers State and
neighbouring States including Bayelsa, Imo and Abia.
2.2 Study Population
The study population included all individuals attending the outpatient clinic and those on
admission at the different wards of the study centre from whom various clinical specimens
were collected. The individuals selected for the study included those that were recommended
for microbiological investigations by the attending physicians.
2.3 Sample size
The sample size was determined using the Fischer’s formula[12], n= (Z2pq)/d2, where Z is
standard deviation corresponding to a specific confidence interval = 1.96 (at confidence
interval 95%), p= the prevalence of ESBLs from a similar study[1] = 28.0% (0.331), q= 1.0-
p, and d is degree of accuracy desired (usually set at 0.05). Therefore, n = 3.84 x 0.28 x
0.72/0.0025 = 309.78 + 10% attrition for incomplete data = 340.76.
2.4 Sampling Method
Patients from whom Klebsiella pneumoniae were isolated from their clinical specimens
(urine, wound biopsies/aspirates, blood and sputum) at the UPTH Medical Microbiology and
Parasitology Laboratory between January and June 2019, were selected by systematic random
sampling based on sampling interval k = N/n where N= 1600 (Estimated number of
individuals referred for microbiological examination at the study center) and n = 340
(required sample size).

2.5 Specimen collection and analysis

Clinical and basic demographic information were obtained from selected patients using a
PROFORMA data collection sheet. The information collected included age, gender,

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DOI: 10.9734/jammr/2023/v35i10113
ISSN:2456-8899
ward/clinic and specimen types. The specimen collected included wound aspirate, urine,
blood and sputum specimen and all specimen were cultured on processed according to
standard procedures as previously described. For blood samples were inoculated medium was
aerobically incubated at 35 – 37oC for 7 days, urine samples were inoculated on Blood agar
and Cysteine Lactose Electrolyte Deficient (CLED) agar and incubated aerobically at 35 –
37oC for 18-24hours, sputum samples and wound/aspirate samples were cultured on
MacConkey agar plate and incubated aerobically at 35 – 37oC for 18-24hours [13] The
ESBL confirmatory test was carried out on all the suspected ESBL isolates using the
combination disks according to the CLSI guidelines[14].
2.6 Data analysis
The data were analyzed using the Epi Info v 7 software and presented in tables or charts as
appropriate. Variables such as sex, age group, wards, and departments were expressed as
frequencies and proportions. The Chi-square statistic was used to compare the distribution of
ESBL and non ESBL- producing isolates by gender, age groups and patient types. All
analyses were done at a 95% confidence interval and a p-value of less than 0.05 was
considered significant.
2.7 Ethical Consideration
Ethical approval to carry out the study was obtained from the Ethical committee of University
of Port-Harcourt teaching hospital. Willing informed consent was obtained from each of the
patients before they were included into the study.

3.0 RESULTS
A total of 340 isolates were obtained from specimen collected from patients within a six-
month period (January to June 2019). Table 1 shows the demographic distribution of the
patients with Klebsiella pneumoniae positive specimen. Of the 340 K. pneumoniae, 127
(37.4%) were from male subjects and 213 (62.6%) were from female subjects. The age
distribution showed that 6 (1.8%) were between 40 – 49 years old, 10 (2.9%) were at least 60
years old, 14(4.1%) were at least 50 – 59 years old, 26 (7.6%) were less than 20 years old,
127 (37.4%) were between 30 – 39 years old and 157 (4.2%) were between 20 – 29 years old.

Table 1: Demographic characteristics of subjects with confirmed Klebsiella pneumoniae isolates

Variable Frequency (n = 340) Percentage

Gender
Male 127 37.4
Female 213 62.6
Age Groups (years)
< 20 26 7.6
20 – 29 157 46.2
30 – 39 127 37.4
40 – 49 6 1.8
50 – 59 14 4.1
>60 10 2.9

Figure 1 shows the distribution of the different specimen positive for Klebsiella pneumoniae
growth. There were 7 (2.1%) sputum, 7 (2.1%) Blood culture, 83 (24.4%) wound
aspirate/biopsies, and 243 (71.5%) urine samples.

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Volume 35, Issue 10, Page 1-8
DOI: 10.9734/jammr/2023/v35i10113
ISSN:2456-8899

Figure 1: Distribution of specimen positive for K. pneumoniae growth

Figure 2 shows that a 34.7% (n = 118) prevalence of ESBL producing K. pneumoniae isolates
among the 340 K. pneumoniae isolates identified in the study.

Figure 2: Prevalence of ESBL -producing Klebsiella pneumoniae

Table 2 shows the distribution of ESBL Klebsiella pneumoniae producing isolates by the
different specimen was not statistically significant in the different samples (2 = 2.80, p =
0.4203). The table showed that the highest proportion of ESBL producers was found in blood
culture specimen (57.1%), followed by sputum samples (42.9%), wound aspirates/biopsies
(38.6%) and urine samples (32.5%).
Table 2: Distribution of ESBL producing Klebsiella pneumoniae from different specimen.

Specimen ESBL positive ESBL negative Total Chi-square

(n, %) (n, %) (n, %) (p-value)
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DOI: 10.9734/jammr/2023/v35i10113
ISSN:2456-8899
Sputum 3(42.9) 4(57.1) 7(100.0)
Blood Culture 4(57.1) 3(42.9) 7(100.0)
Wound aspirate/biopsies 32(38.6) 51(61.4) 83(100.0) 2.80 (0.4203)
Urine 79(32.5) 164(67.5) 243(100.0)

The distribution of the ESBL-producers by gender was not statistically significant (2 = 0.23,
p = 0.6248). the data showed that ESBL K. pneumoniae distribution was higher in female
subjects (35.75) compared to male subjects (33.1%). The findings also showed that the
distribution of ESBL K. pneumoniae was highest in persons less than 20 years (42.3%),
followed by persons between 30 – 39 years (36.2%), while the age group with the least
distribution of ESBL K. pneumoniae was the persons between 50 – 59 years (28.6%), with
persons between 40-49 years not having any growth of ESBL K. pneumoniae. However, The
distribution of ESBL producing K. pneumoniae in the different age groups was not
statistically significant (2 = 4.31, p = 0.546).

Table 3: Gender and Age-related distribution of Klebsiella pneumoniae

ESBL-positive ESBL-negative Total Chi-square
Variable (n, %) (n, %) (n, %) (p-value)
Gender
Male 42(33.1) 85(66.9) 127(100.0) 0.23 (0.6248)
Female 76(35.7) 137(64.3) 213(100.0)
Age Groups (years)
< 20 11(42.3) 15(57.7) 26(100.0)
20 – 29 54(34.4) 103(65.6) 157(100.0)
30 – 39 46(36.2) 81(63.8) 127(100.0) 4.31 (0.546)
40 – 49 0(0.0) 6(100.0) 6(100.0)
50 – 59 4(28.6) 10(71.4) 14(100.0)
>60 3(30.0) 7(70.0) 10(100.0)

Table 4 shows the distribution of the ESBL-producing isolates and patient types, 37% of the
inpatient subjects had ESBL K. pneumoniae compared to 28.7% in the outpatient subjects.
However, the distribution of ESBL K. pneumoniae was not statistically significant (2 = 2.05,
p = 0.152).
Table 4: Distribution of ESBL and Patient Types

Source ESBL Positive ESBL Negative Total Chi-Square

n, (%) n, (%) (n, %) (p-value)
Inpatient 91(37.0) 155(63.0) 246(100.0)
2.05 (0.152)
Outpatient 27(28.7) 67(71.3) 94(100.0)

DISCUSSION
Urine specimen had the highest proportion of Klebsiella growth (71.5%), followed by
wounds/aspirates (24.4%), while sputum samples (2.1%) had the least proportion of
Klebsiella growth. This is consistent with reports of studies that isolated Klebsiella
pneumoniae in clinical settings, as similar studies report that urine samples and
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Volume 35, Issue 10, Page 1-8
DOI: 10.9734/jammr/2023/v35i10113
ISSN:2456-8899
wound/aspirates were the most common sources of Klebsiella pneumoniae isolated in a
variety of clinical settings.[2, 9, 15, 16]
There was a 34.7% prevalence of ESBL klebsiella among the 340 klebsiella isolates observed
in the current study. The prevalence of ESBL producing K. pneumoniae was observed to be
34.7%. This was consistent with the findings of previous studies done by Adeyankinnu et.
al,[17] and Kiratisin et. al.[18] The clinical samples were varied in distribution with urine as
the highest in frequency 216 (63.5%) and blood culture as the least 3 (0.9%). This is not
surprising because previous work done by Kiratisin et. al observed that ESBL- producing
isolates were recovered majorly from urine specimens. The prevalence of ESBL-production
among K. pneumoniae isolates in this study is 34.7%. This is comparable to the rates of
39.8% reported in Enugu,[19] 33.6% in Abuja,[20] 31.6% in Lagos, 35.3% in Zaria[21] and
30.0% in Maiduguri. Studies however have shown higher prevalence of ESBL producers as
reported by Ogefere et. al.,[19] and Egbebi e.t al., in Benin, Southwest Nigeria, Saraswathi
e.t al.,[22] Hyderabad in India with rates as high as 44.3%, 56.0%, and 50.0%, respectively
among Gram-negative bacteria isolated in clinical settings. The observed difference may be
attributed to the different study population and specimen used such as urine and wound swabs
by Ogefere et al. The study by Egbebi et al., used a double-disk synergy testing method for
ESBL testing, while Saraswathi et al., assessed only post-operative infected wounds. The
comparability of high prevalence in many developing nations throughout the world compared
to industrialized countries in Europe and North America is especially remarkable. Klebsiella
spp. are of significant medical value among other Enterobacteriaceae because they exhibit the
ESBL mechanism and are commonly identified in hospital and community infections that
require medications.
Blood culture was found to have the highest proportion of ESBL K. pneumoniae while urine
samples had the least proportion of ESBL K. pneumoniae growth. This is in agreement with
reports of similar studies showing a relatively high occurrence of ESBL producing K.
pneumoniae in clinical specimen ranging from 24.6% - 60%.[2, 23–25]
Similarly, the findings of the current study showed the occurrence of ESBL K. pneumoniae
was higher among in-patients (37.0%) compared to outpatients (28.7%). A comparison of
studies in developed nations report a 10% - 30% occurrence of ESBL K. pneumoniae growth
in blood specimen[2, 15, 26, 27] compared to developing countries like Nigeria which report
between 40% - 55% occurrence of ESBL K. pneumoniae growth in blood culture in hospital
settings.[1, 28–30] The contrasting reports of ESBL K. pneumoniae growth in developed
nations compared to developing nations is an indication of the effectiveness of antimicrobial
prescription policies in developed countries compared to developing nations. Similarly, many
hospital settings in developed nations tend to have better management and control of
antimicrobial resistance in comparison to developing countries.
CONCLUSION
The study showed that blood culture specimens had the highest burden of ESBL K.
pneumoniae growth. It was also observed that ESBL K. pneumoniae growth was not
significantly associated with gender of age groups. The growth of ESBL K. pneumoniae was
observed to be higher among inpatient subject indicative of the likelihood of high prevalence
ESBL K. pneumoniae in hospital acquired infection. Therefore, there is a great need for
urgent interventions in the areas of antimicrobial usage and infection prevention and control
in hospital-based settings.
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