Jonah et al.

, | Article 8191
J Pure Appl Microbiol. 2023;17(1):20-28. doi: 10.22104/JPAM.17.1.12

Research Article

PATTERN of CTX-M and SHV GENES IN ESBL PRODUCING Klebsiella

pneumoniae
JONAH A A., AWOPEJU A T O., GEORGE F., WARISO K. T.
Department of Medical Microbiology, University of Port Harcourt Teaching Hospital, Port Harcourt, Rivers state.

ABSTRACT
Background: The introduction of extended-spectrum beta-lactams marked a positive
advancement in combating resistance posed by beta-lactamases in the antibiotic era. Despite
this, mutations in the beta-lactamase gene present a notable challenge to effectively addressing
infectious diseases on a global scale. This study specifically investigated the distribution and
patterns of CTX-M and SHV genes in Klesbsiella pneumoniae that produce extended-spectrum
beta-lactamases, isolated from patients within a tertiary healthcare institution.
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. Phenotypic and gentotypic
methods were used to identify isolates with CTX-M and SHV genes.
Results: The study reports a prevalence rate of 34.7% (118/340) for ESBL-production among
K. pneumoniae isolates in UPTH, Port Harcourt. It was observed 83.1% of the samples tested
positive for the CTX-M gene, while 89.8% tested positive for the SHV gene. The chi-square
test showed no statistically significant difference between the presence of CTX-M and SHV
genes (p-value = 0.128).
Conclusion: The considerably high prevalence and distribution of CTX-M and SHV beta-
lactamase enzymes in ESBL-producing isolates reveal a significant variation in the genetic
mechanisms contributing to antibiotic resistance. The study highlights a significant prevalence
of ESBL-producing K. pneumoniae, indicating a pressing need for proactive measures to
address antibiotic resistance.

Keywords: Antibiotics, ESBL, klebsiella pneumoniae, SHV, CTX-M

1.0 INTRODUCTION
The introduction of the extended spectrum cephalosporins into clinical use in the early 1980s
was a welcome development as it provided effective therapy especially for healthcare
associated infections (HCAI) caused by multi-drug resistant gram-negative bacilli including
the Enterobacteriaceae. However, this breakthrough was short-lived as a result of the
production of extended spectrum beta lactamases (ESBL) which brought about the
emergence of antimicrobial resistance.[1] This has continued to be an issue of major concern
considering its continual spread amongst many microorganisms globally both in healthcare
facilities as well as in communities[2] thereby causing serious infections and posing a grave
global public health threat.[3]
Extended-spectrum beta-lactamases (ESBLs) are a heterogeneous group of plasmid-mediated
bacterial enzymes that confer significant resistance to oxyimino-cephalosporin and
monobactam antimicrobials.[4] They are however inhibited by beta-lactamase inhibitors such
as clavulanic acid.[5] There is a global variation even in closely related regions of the

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prevalence of ESBLs. Reports have shown that ESBL-producing bacteria are prevalent in
clinical and community settings with serious public health consequences.[6, 7]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.[8]
There are over 300 different ESBL variants known, with Temoniera (CTX-M) and Sulfhydryl
variable-1 (SHV) variants being the commonest ESBLs.[9] However, the past decade has
observed an emerging strain of ESBLs called Cefotaxime-Munich (CTX-M). This new strain
of ESBL is currently the most frequent non-CTX-M, non-SHV ESBL type.[10] Selective
hydrolysis of cefotaxime rather than ceftazidime generally characterizes the CTX-M,
however, CTX-M-15 hydrolyses ceftazidime.[10] A study to detect bla-SHV and bla-CTX-M
genes in ESBL-producing Klebsiella pneumoniae isolates from patients with suspected
nosocomial infections, conducted in Egypt by Ahmed et al showed 53.3% of the isolates were
positive for bla-CTX-M and 10% for bla-SHV [10]. The current study assessed the distribution and
pattern of CTX-M and SHV genes in ESBL producing Klesbsiella pneumoniae isolated from
patients in a tertiary healthcare institution.
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 Klesbsiella pneumoniae isolated from patients attending the
University of Port Harcourt Teaching Hospital within a 6-month period 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.
2.4 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
The specimen collected included wound aspirate, urine, blood and sputum specimen and all
specimen were processed according to standard procedures as previously described.[11] The
ESBL confirmatory test was carried out on all the suspected ESBL isolates using the
combination disks according to the CLSI guidelines.
2.5.1 Phenotypic Detection of ESBL
Characterization was conducted on cephalosporin-resistant K. pneumoniae isolates, and their
ESBL production was assessed using the double disk synergy test following the CLSI 2015
guidelines. ESBL-producing strains were identified as isolates exhibiting synergy zones
between Amoxicillin/clavulanic and one or more third generation cephalosporins [16].

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The presence of an inhibition zone with a distorted or enlarged appearance, forming a keyhole
shape between the cephalosporin discs and the Amoxicillin/Clavulanate disc, was interpreted
as indicative of an ESBL enzyme production phenotype. Positive and negative controls were
established using K. pneumoniae K6 ATCC 700,603 (ESBL producer) and Escherichia coli
ATCC 25,922 (non-ESBL producer), respectively.
2.5.2 Genotypic Detection of ESBL
The boiling method was used to extract DNA of Purified colonies of ESBLs producing K.
pneumonia suspended in TE buffer. SHV and CTX-M genes were detected as described
previously [12]. PCR amplification for selected ESBL genes was done in 20 μl volumes
containing 4 μl of 5× master mix of 0.4 μl concentrations of each primer, 12.2 μl of PCR water,
BSA 1 μl and 2 μl of DNA template. A programmable thermo cycler was used with initial
denaturation at 94˚C for 5 min; followed by 35 cycles at 94˚C for 30s, annealing was done
between 30 second and 1 min depending on the primer temperature, then a short extension step
at 72˚C for 1 min and a final extension at temperature of 72˚C for 10 min for short fragments
and 20 min for longer fragments. Electrophoresis was done to the amplified PCR products,
with a 1 kb DNA ladder as a standard in 1.0% agarose gel in 1× TBE buffer and stained with
ethidium bromide. This was visualized under the UV trans illumination. Positive control strains
were used for the different test genes and distilled water used as a negative control.
2.6 Data analysis
The data was analyzed using the Epi Info v 7 software and presented in tables or charts as
appropriate. The Chi-square statistic was used to compare the distribution of CTX-M and SHV
genes in the ESBL isolates. 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.

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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.

80
71.4
70

60
Percent (%)

50

40

30 24.4
20

10
2.1 2.1
0
Urine Wound aspirate/biopsy Blood culture Sputum
Specimen

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

Of the 340 K. pneumoniae isolates analyzed in this study, 118 were extended spectrum beta-
lactamases (ESBL)-producing isolates, giving a prevalence rate of 34.7%, while 222 (65.3%)
were non-ESBL-producing K. pneumoniae isolates (Figure 2).

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 118, 34.7%

ESBL POSITIVE

222, 65.3% ESBL NEGATIVE

Figure 2: Prevalence of ESBL -producing Klebsiella pneumoniae.

Table 2 show the distribution of the CTX-M and SHV genes in the examined ESBL producing
isolates. The results indicate that 83.1% of the samples tested positive for the CTX-M gene,
while 89.8% tested positive for the SHV gene. The chi-square test showed no statistically
significant difference between the presence of CTX-M and SHV genes (p-value = 0.128).
Additionally, 16.9% of samples were negative for the CTX-M gene, and 10.2% were negative
for the SHV gene.

Table 2: Distribution of CTX-M and SHV genes in ESBL producing isolates

CTX-M SHV Chi-square

n (%) n (%) (p-value)
Positive 98(83.1) 106(89.8) 2.31 (0.128)
Negative 20(16.9) 12(10.2)
Total 118(100.0) 118(100.0)

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4.0 DISCUSSION

The study reveals a considerable prevalence (34.7%) of extended-spectrum beta-lactamase

(ESBL)-producing K. pneumoniae isolates among the 340 samples analyzed. This is a matter
of significant concern as ESBL-producing strains are known for their resistance to a broad
range of antibiotics, making the treatment of infections caused by these strains more
challenging[13]. 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,[14] 33.6% in
Abuja,[15] 31.6% in Lagos, 35.3% in Zaria[16] and 30.0% in Maiduguri. Studies however have
shown higher prevalence of ESBL producers as reported by Ogefere et. al.,[14] and Egbebi e.t
al., in Benin, Southwest Nigeria. However, our result of ESBL production of 34.7% K.
pneumoniae was in contrast to similar studies done by Akujobi et. al,[17] and Altayb et. al.[18]
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 Klebsiellae in different regions of the world.[19] 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.
The findings of the present study underscore the importance of antibiotic stewardship programs
to optimize the use of antibiotics and curb the spread of antibiotic-resistant strains. Clinicians
should be informed about the prevalence of ESBL-producing K. pneumoniae and the specific
genetic mechanisms involved, allowing for more targeted and effective treatment guidelines.
A similar study by El Sherif et. al, reported that 45.5% and 44% of Klebsiella spp SHV and
CTX-M genes were detected among 77 K. pneumoniae isolates respectively. Other studies done
in India by Jemina et. al. observed that Klebsiella spp isolates contained 45% of bla-SHV like
genes and 40% bla-CTX-M genes. Similarly, Tofteland et. al., reported that three out of the
twenty-five clinical cases of ESBL-producing K. pneumoniae had the bla-CTX-M phenotype, and
fifteen had bla-SHV cases, while one case had both bla-SHV and bla-TEM. Many of the studies done
showed that CTX-M gene is now the most common ESBL type in different parts of the world
thus replacing TEM and SHV genes but the present study still showed SHV gene as the most
common in the area of study.[20],[21] This finding could be attributed to the fact that the
observed changing pattern of CTX-M gene seen in Europe and Asia are just being reported in
Nigeria and more studies are beginning to evolve in Nigeria. Further studies done in Nigeria to
buttress the fact that SVH gene are more predominant, which is also established by the present
study, include studies by Yahaya et. al., from North eastern, and Egbebe et. al, and Aibunu et.
al.[22] from South western Nigeria. In two of these studies, SHV gene was the most
predominant with 36.4% and 53.0% respectively.

5.0 CONCLUSION
The considerably high prevalence and distribution of CTX-M and SHV beta-lactamase enzymes
in ESBL-producing isolates reveal a significant variation in the genetic mechanisms
contributing to antibiotic resistance. The study highlights a significant prevalence of ESBL-
producing K. pneumoniae, indicating a pressing need for proactive measures to address
antibiotic resistance. The genetic basis of resistance, as evidenced by the high presence of CTX-
M and SHV genes, emphasizes the importance of targeted interventions in both clinical and

25
public health settings. Recommendations for addressing the significant prevalence of ESBL-
producing K. pneumoniae include enhancing surveillance systems to monitor distribution
across various clinical specimens. In addition, there is a crucial need to develop and enforce
antibiotic stewardship programs aimed at guiding appropriate antibiotic use, thus minimizing
the risk of further resistance development.

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