International Journal of TROPICAL DISEASE & Health

Volume 44, Issue 6, Page 51-58

DOI:10.9173/IJDTH/2023/v35i11113

Original Research Article

COMPARATIVE ASSESSMENT OF ANTIBIOTIC SUSCEPTIBILITY IN ESBL

AND NON-ESBL Klebsiella pneumoniae IN A TERTIARY HOSPITAL

JONAH A A., AWOPEJU A. T. O.

Department of Medical Microbiology, University of Port Harcourt Teaching Hospital, Port Harcourt, Rivers state.
*correspondence:draanu@yahoo.com

ABSTRACT
Aim: The objectives of this investigation were to identify K. pneumoniae isolates
expressing ESBLs and to determine their antibiotic susceptibility pattern in a clinical
setting.
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: 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 with subsequent antibiotic susceptibility testing according to the CLSI
guidelines.
Results: The pattern of resistance of the ESBL producers isolated from the patient’s
specimen were to 44.1% (ciprofloxacin), 44.1% (ofloxacin) and 96.6%
(sulfamethoxazole/trimethoprim) respectively. Resistance to gentamicin, Ciprofloxacin,
Trimethoprim / Sulfamethoxazole and Cefotaxime was significantly higher in the ESBL
producing K. pneumoniae compared to the non-ESBL producing K. pneumoniae.
Conclusion: A poor activity of gentamicin and fluoroquinolones against ESBLs were
observed in this study and this could be as a result of the fact that the genes coding for
CTX-M type of ESBL is known to be associated with plasmids which encodes resistance
to tetracycline, aminoglycosides and quinolones

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

1. INTRODUCTION
Klebsiella pneumoniae (KP) is one of the leading causes of nosocomial infections seen
worldwide, causing pneumonia, bloodstream infections, urinary tract infections, surgical site
or wound infections and meningitis.[1] Increased rate of treatment failure and death
associated with infections caused by KP is a major concern for the clinicians especially in
intensive care units and pediatric patients.[2, 3] This could be attributed to the unprecedented
use of antibiotics in health care set up without proper antibiotic policy which has led to
increased prevalence of infections caused by extended-spectrum beta-lactamase producing
KP (ESBL-KP). In recent years, outbreaks of infection caused by multidrug-resistant ESBL-
KP have been reported throughout the world.[4, 5]

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Volume 44, Issue 6, Page 51-58
DOI:10.9173/IJDTH/2023/v35i11113

ESBL represent a major group of bacterial beta-lactamases that belong to Bush-Jacoby

functional subgroup 2be and Ambler class A, that retain the ability to hydrolyzes penicillin
and early cephalosporins such as cephaloridine and cephalothin and in addition hydrolyze one
or more oxyimino-cephalosporins such as cefotaxime, ceftazidime, and aztreonam at a rate
generally more than 10% that of benzylpenicillin.[4, 6] The subgroup has been derived by
amino acid substitutions in the enzymes TEM-1, TEM-2 and SHV-1 of functional subgroup
2b and has broadened the spectrum of the substrate. The subgroup 2be is characteristically
sensitive to clavulanic acid, and this feature is used in their detection in laboratories.[2, 3, 6]
The risk factors associated with infection due ESBL-KP are severe underlying illness, long-
term treatment with multiple antibiotics, prolonged duration of hospital stay, surgical
intervention, instrumentation and presence of indwelling intravenous catheters. Mechanical
ventilation and endotracheal intubation are the risk factors associated in infections seen in
intensive care units.[2, 4, 5] Knowledge of present scenario of prevalence and drug resistance
helps in the development of antibiotic policy for infection caused by ESBL-KP. Therefore,
this study was conducted to determine the prevalence of ESBL-KP and antimicrobial
sensitivity profile of the ESBL-KP to provide insights that could ai hospital infection control
program to prevent the spread of resistant strains.
2. 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[7], 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[8] = 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 2019 to 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).

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Volume 44, Issue 6, Page 51-58
DOI:10.9173/IJDTH/2023/v35i11113

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,
ward/clinic and specimen types. The specimen collected included wound aspirate, urine,
blood and sputum specimen and all specimen were processed according to standard
procedures as previously described.[9] The ESBL confirmatory test was carried out on all
the suspected ESBL isolates using the combination disks according to the CLSI guidelines as
previously described [10]. Bacterial suspensions of the various suspected ESBL isolates were
prepared in 2mls of peptone water and the turbidity of each adjusted to correspond to 0.5
McFarland’s standard. With the aid of sterile swab sticks lawns were made from the
suspensions on a Mueller Hinton agar (Oxoid, Cambridge UK) plate. Antibiotic disks
containing 30µg of ceftazidime alone and 30µg/10µg ceftazidime/clavulanic acid and 30µg
cefotaxime alone and in combination with 30µg/10µg cefotaxime/clavulanic acid was placed
30mm apart on the agar plate that has been streaked with the standardized inoculum of the
test organism (K. pneumoniae) and incubated overnight aerobically for 16-18 hours at 35-
370C after which the zone of inhibition for each antibiotic was measured. An increase in zone
diameter of ≥5 mm in the presence of cephalosporin-clavulanic acid over ceftazidime or
cefotaxime alone was interpreted as ESBL producers. Antibiotic susceptibility patterns of the
EBSL and non-ESBL producers were tested against Gentamycin, Ciprofloxacin, Ofloxacin,
Amoxiclav, Ceftazidime, Trimethoprim/Sulfamethoxazole, Cefotaxime and Meropenem
according to standard methods previously described.[10]
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. RESULTS
A total of 340 isolates were obtained from specimen collected from patients during the study.
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 44, Issue 6, Page 51-58
DOI:10.9173/IJDTH/2023/v35i11113

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

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Volume 44, Issue 6, Page 51-58
DOI:10.9173/IJDTH/2023/v35i11113

Table 1 shows the antibiotic susceptibility pattern of the ESBL and non-ESBL Klebsiella
pneumoniae. The table shows that resistance to gentamicin, Ciprofloxacin, Trimethoprim /
Sulfamethoxazole and Cefotaxime was significantly higher in the ESBL producing K.
pneumoniae compared to the non-ESBL producing K. pneumoniae.
Table 1: Antibiotic Susceptibility Pattern of ESBL-Producers and Non-ESBL producers
ESBL-Positive (n =118), % ESBL-Negative (n = 222), % Chi-square
Antibiotics (p-value)
Susceptible Intermediate Resistant Susceptible Intermediate Resistant
n (%) n (%) n (%) n (%) n (%) n (%)
Gentamycin 10 (8.5) 0 (0.0) 108 (91.5) 138 (62.2) 0 (0.0) 84 (37.8)
Ciprofloxacin 39 (33.1) 27 (22.8) 52 (44.1) 176 (79.2) 7 (3.2) 39 (17.6)
Ofloxacin 66 (55.9) 0 (0.0) 52 (44.1) 180 (81.1) 3 (1.3) 39 (17.6)
AMC 13 (11.0) 68 (57.6) 37 (31.4) 118 (53.2) 19 (8.5) 85 (38.3) 190.01 (0.0001)*
Ceftazidime 22 (18.6) 52 (44.1) 44 (37.3) 178 (80.2) 11 (4.9) 33 (14.9)
SXT 4 (3.4) 0 (0.0) 114 (96.6) 88 (39.6) 4 (1.8) 130 (58.6)
CTX 4 (3.4) 0 (0.0) 114 (96.6) 146 (65.7) 7 (3.2) 69 (31.1)
Meropenem 114 (96.6) 0 (0.0) 4 (3.4) 209 (94.1) 6 (2.7) 7 (3.2)
AMC: Amoxicillin, SXT: Trimethoprim / Sulfamethoxazole, CTX: Cefotaxime
All figures are presented in frequencies and percentage (n, %)
*Difference in susceptibility pattern is statistically significant (p < 0.05)
**Difference in susceptibility pattern is not statistically significant (p > 0.05)
4. DISCUSSION
It is worthy to note that the prevalence of ESBL producing K. pneumoniae observed in the
current study contrasted with similar studies done by Olowe et. al., Akujobi et. al,[11] and
Altayb et. al.[12] in Ogun State, Nnewi, and Sudan who recorded lower ESBL production
rates of 7.5%, 23.6%, and 26.6%, respectively. This could be attributed to the assessment of
Klebsiella species done in the study by Olowe et al., while the study by Altayb et al., was
observed to be a multi-center study. The observed differences in the prevalence recorded in
different studies reflect the differing burden of ESBL producing Klebsiella pneumoniae in
different regions of the world.[13] Differences in study design, patient selection, sample size,
patterns of antibiotic stewardship in the various centers, and geographical differences that
occurs in clinical isolates could account for the observed differences. Especially noteworthy
is the similarity of high prevalence in many developing countries of the world compared to
the developed countries in Europe and North America. It is a well-documented fact that
Klebsiella spp. are of high medical importance among other Enterobacteriaceae as
expressing ESBL mechanism and are frequently isolated in hospital and community
infections for which antibiotics are needed. Antibiotic misuse is a common practice in
developing countries which could have contributed to the relatively high prevalence of ESBL
producing Klebsiella pneumoniae observed in this study and previous ones. In many parts of
Nigeria, off-the-counter availability of many antibiotics including the penicillins and
cephalosporins is a common occurrence. The occurrence of these ESBL-producing K.
pneumoniae observed in the current study may be as a result of the spread of resistance
vertically between Klebsiella spp. The relatively high prevalence of ESBLs observed from
this study could be an indication of the significant occurrence of vertical or horizontal spread
of resistance genes among K. pneumoniae in the study setting.
The antimicrobial susceptibility patterns of all ESBL and non-ESBL producing K.
pneumoniae isolates were observed in the present study. Overall, it was observed that both
ESBL and non- ESBL producing K. pneumoniae isolates had a level of resistance to all the
conventional antimicrobial agents used including meropenem. However, the difference in

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Volume 44, Issue 6, Page 51-58
DOI:10.9173/IJDTH/2023/v35i11113

susceptibility pattern was statistically significant (p<0.05) with remarkably higher mean
percentage of resistance to commonly used antibiotics among the ESBL-producing K.
pneumoniae compared to the non-ESBL producing isolates. All the ESBL-producing K.
pneumoniae isolates from this study were multidrug resistant.
These very high resistance rates make their use as empirical chemotherapeutic agents
inappropriate. This is an expected phenomenon for ESBL producers. However, it is a
worrisome occurrence considering the development of resistance pattern observed with the
non ESBL-producers. This development could be an indication that first-line antimicrobial
agents for non-ESBL producing K. pneumoniae isolates in the present area of study needs to
be reviewed and studies done to draw up a new antibiogram for management of Gram-
negative bacilli in the area so as to avoid treatment failures. This explains that beyond beta
lactamase production, there are other mechanisms of resistance that needs to be investigated.
There may be a fraction of patients treated with these first- line drugs that may still
experience treatment failure even though they were not ESBL- producers. This calls for
urgent action with regard to education of the public against the misuse of antibiotics and strict
compliance to the antibiotic regimen. Though, there was a 3.4% resistance of the ESBL-
producing K. pneumoniae to meropenem in this study, meropenem still remains the most
active antibacterial agent that isolates were most susceptible. This agrees with patterns of
resistance observed in studies done by Ahmed et al. and Ullah et. al who recorded a 6.7%
resistance from Egypt, and 11.11% from North-west of Pakistan respectively. These were
however slightly higher than resistance recorded in the present study. However, several other
studies had 100% susceptibility to the carbapenems even though imipenem was the choice
carbapenem used as against meropenem used in this present study.[14],[15] The 3.2%
resistance recorded by the non ESBL-producers against meropenem strongly suggest the
production of carbapenemase enzymes by some of the isolates. ESBL-producing K.
pneumoniae are known to be susceptible to meropenem.
A poor activity of gentamicin and fluoroquinolones against ESBLs were observed in this
study and this could be as a result of the fact that the genes coding for CTX-M type of ESBL
is known to be associated with plasmids which encodes resistance to tetracycline,
aminoglycosides and quinolones.[6] The prevalence of CTX-M genes was well elaborated
genotypically in the study. In many clinical settings, infections caused by Enterobacteriaceae
of which K. pneumoniae belongs, are commonly treated with aminoglycosides,
fluroquinolones, and sulfamethoxazole/trimethoprim. However, the present study shows a
high resistance to these drugs of choice. It was observed that 44.1%, 44.1% and 96.6% of the
ESBL-producing isolates were resistant to ciprofloxacin, ofloxacin and
sulfamethoxazole/trimethoprim respectively. The widespread use of fluoroquinolones is an
identifiable risk factor worldwide for the emergence of fluoroquinolones-resistant ESBL-
producing strains and an association of ESBL-production and resistance to
sulfamethoxazole/trimethoprim has been reported.[4, 16] Although, amoxicillin-clavulanate,
ceftazidime and cefotaxime showed in vitro activity against some ESBL-producing isolates
with 13%, 22% and 4% susceptibility respectively, ESBL enzymes confer resistance to all
penicillin and cephalosporins. Hence, the in-vitro activity of amoxicillin-clavulanate,
ceftazidime and cefotaxime observed against some ESBL-producing isolates with 13%, 22%
and 4% susceptibility respectively were not reportable for treatment with the susceptibility
profiles of non-ESBL-producing isolates reveal a higher activity against the isolates. It is
however difficult to accurately ascertain contributory factors to the observed susceptibility
pattern of the community acquired ESBL- producing K. pneumoniae in this study. It has been
reported that the gut plays a prominent role in the development of antibiotic resistance and
the emergence of resistant microorganisms as observed in vulnerable patients.[1, 9, 17] A

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DOI:10.9173/IJDTH/2023/v35i11113

recent report from report showed 16% fecal carriage of ESBLs isolates with the majority
(over 80%) of these being E. coli, and in Saudi Arabia 12.7% isolates were ESBLs producers,
of which 95.6% were E. coli and 4.4% were K. pneumoniae.[8]
5. CONCLUSION
The findings of the study show a considerable prevalence of ESBL-producing K. pneumoniae
in the clinical isolates. While considerable resistance to First- and second-generation
antibiotics was observed among the ESBL-producing K. pneumoniae an indication of the
likelihood of vertical or horizontal spread of resistance genes among K. pneumoniae in the
study setting. The findings of the current study makes the immediate creation and
implementation of antimicrobial prescription policies a must-have in all clinical and
community settings to check the spread of antimicrobial resistance and it consequent
disadvantages.

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