Prevalence of blaCTX-M and blaTEM Genes in Cefotaxime-Resistant Escherichia coli Recovered from Tertiary Care at Central Nepal: A Descriptive Cross-Sectional Study

Urinary tract infections (UTIs) are highly prevalent globally, and various antibiotics are employed for their treatment. However, the emergence of drug-resistant uropathogens towards these antibiotics causes a high rate of morbidity and mortality. This study was conducted at the Microbiology Laboratory of Grande International Hospital from November 2021 to May 2022 and aimed to assess the prevalence of UTI caused by Escherichia coli and their antibiotic susceptibility pattern with a focus on extended-spectrum beta-lactamases (ESBLs) and the prevalence of two genes (blaCTX-M and blaTEM) in cephalosporin-resistant E. coli. Altogether, 1050 urine samples were processed to obtain 165 isolates of E. coli. The isolates were identified by colony morphology and biochemical characteristics. Antimicrobial susceptibility tests (ASTs) were determined by the Kirby–Bauer disk diffusion method, and their ESBL enzymes were estimated by the combined disk method (CDM). Two ESBL genes (blaCTX-M and blaTEM) were investigated by polymerase chain reaction (PCR) in cefotaxime-resistant E. coli. Among the 1050 urine samples that were processed, 335 (31.9%) were culture-positive with 165 (49.2%) identified as E. coli. The age group ≥60 years (30.3%) had greater susceptibility to bacterial infections. AST revealed that meropenem was highly effective (95.7% susceptibility), while ampicillin showed the least sensitivity (42.4%). Among the E. coli isolates, 86 were multidrug resistant (MDR) and 10 were extensively drug resistant (XDR). Of these, 46 MDR (96%) and 2 XDR (4%) were ESBL producers. The prevalence of ESBL genes (blaCTX-M and blaTEM) was 49.3% and 54.8%, respectively. The overall accuracy of CDM as compared to PCR for the detection of the blaCTX-M gene was 55.26%. The prevalence of MDR E. coli harboring the blaCTX-M and blaTEM genes underscores the imperative role of ESBL testing in accurately identifying both beta-lactamase producers and nonproducers.


Introduction
Te presence of bacteria in the urine can range from asymptomatic to a serious kidney infection with sepsis [1].In community medicine, urinary tract infections (UTIs) are the second most frequent infection [2].UTI has emerged as a signifcant and urgent public health issue.An estimated 150 million cases of UTI, which have a signifcant risk of morbidity and mortality, are detected each year [3].Males and females of all ages are both afected by UTIs.Females are more prone to experience UTIs than males, which is probably due to anatomical variations, hormonal infuences, and behavioral factors [4].
UTI cases range from 23.1% to 37.4% among Nepalese patients visiting general hospitals.More than 95% of UTI instances are due to bacteria, which is a prevalent cause.Escherichia coli causes more than 80% of UTIs and is the most frequent bacteria [5].It is well-recognized that UTIs can result in kidney scarring permanently as well as shortterm morbidities such as fever, dysuria, and lower abdominal pain (LAP) [6].Altogether, 50% of all nosocomial infections and 70% to 95% of uropathogenic E. coli (UPEC) are considered the source of communityacquired UTIs [7].
E. coli predominates in a higher extent of UTI cases, followed by Proteus species, Staphylococcus saprophyticus, Klebsiella species, and other Enterobacteriaceae families [8].A signifcant public health concern is the development of antibiotic resistance in the treatment of UTIs.Counterfeit and spurious pharmaceuticals of uncertain quality are widely available, notably in underdeveloped nations where hunger, illiteracy, and poor hygiene habits are highly prevalent [9].
Te most researched class A beta-lactam enzymes in Gram-negative bacteria are the TEM and CTX-M betalactamases, which are mainly plasmid-borne [10].Tey are thought to be the most typical beta-lactam-resistant mechanism in Gram-negative bacilli and are spreading quickly throughout the world [11].Based on the antibiotic resistance profle of the urinary pathogens from the most recent surveillance data, treatment for UTI cases is frequently initiated empirically.Tus, the purpose of the current investigation was to ascertain the prevalence of the bla CTX-M and bla TEM genes in cefotaxime-resistant E. coli isolated from urine samples of UTI-diagnosed patients who had visited Grande International Hospital in Kathmandu, Nepal.

Methodology
2.1.Study Design, Site, and Criteria.Tis hospital-based descriptive cross-sectional study was carried out at Grande International Hospital, and molecular assays were conducted at National College, Kathmandu, at the Department of Microbiology, Kathmandu, Nepal, on the duration of November 2021 to May 2022.

Inclusion Criteria.
Samples from patients of all age groups for evaluation of urinary tract infections referred by the clinician were accepted, and the isolated Escherichia coli isolates were included in our study.

Exclusion Criteria.
Isolates other than Escherichia coli were excluded.

Sample Size and Sampling Technique.
For routine culture and antibiotic susceptibility testing, 1050 urine samples referred by clinicians were processed, and altogether, 165 Escherichia coli isolates were selected using a convenience sampling method.

Sample Collection and Transportation.
Patients were instructed to fll a sterile, dry, wide-necked, leak-proof container with 10-20 ml of the clean-catch midstream region of their urine.Te container was correctly labelled with the sample code, name, date, and collection time, and it was immediately transported to the microbiology laboratory.For delayed processing, the urine samples were subjected to 10% boric acid [12].

Laboratory Processing of the Specimen
2.4.1.Urine Culture.Te urine samples were inoculated on the surface of cystine lactose electrolyte defcient (CLED) agar plates, using a standard calibrated loop (∼4 mm diameter).Te semiquantitative bacteriuria count was determined to estimate a signifcant UTI.On the surface of the culture medium, a loop of urine was streaked, and it was then incubated for 24 hours at 35 °C under aerobic conditions.Te total count of colonies was used to calculate the colony-forming unit (CFU) per milliliter of urine.Te bacterial count was reported as signifcant for >10 5 CFU/ml.If the specimen was found to be contaminated, then a repeat sample was requested [13].2.6.Antibiotic Susceptibility Test.Isolated organisms were preceded by an antibiotic susceptibility test (AST) following CLSI guidelines recommendation.Te antibiotics used were ampicillin (10 μg), amikacin (30 μg), cotrimoxazole (25 μg), ciprofoxacin (5 μg), nitrofurantoin (300 μg), norfoxacin (10 μg), ceftazidime (30 μg), gentamicin (30 μg), cefotaxime (30 μg), and meropenem (10 μg).Te Kirby-Bauer disk difusion method was used to conduct the in-vitro susceptibility test.In this approach, the test organism's broth culture was uniformly spread out across the surface of Mueller Hinton agar (equivalent to McFarland (0.5; inoculum density: 1.5 × 10 8 organisms/ml)).Te medium was covered with the appropriate antibiotic disks, which were then incubated for 18 hours at 37 °C.Following incubation, the inhibition zone was measured using a measuring scale in mm, and the zones were compared using established interpretive criteria based on CLSI guidelines recommendations to determine whether they were susceptible, intermediate, or resistant.E. coli ATCC 25922 was utilized to standardize the drug susceptibility test and to check antibiotic disk quality control.[15].

Screening of Multidrug Resistant (MDR), Extensive Drug Resistance (XDR), and Pandrug Resistance (PDR).
In the study, isolates were classifed as MDR if they were resistant to at least three classes of antibacterial agents, whereas the isolates resistant to at least one agent of all antimicrobials but susceptible to only one or two antibacterial groups were classifed as XDR.Finally, isolates that were still resistant to

DNA Extraction of E. coli
Isolates.DNA extraction of bacterial isolates was carried out from cefotaxime (CTX)resistant E. coli using boiling lysis.For this, the preserved bacteria were subcultured on nutrient agar (NA) and were incubated for 24 hrs at 37 °C.A Luria Bertani (LB) broth was used to inoculate the isolated colony from the NA, and it was then incubated at 37 °C.Te DNA was extracted and then suspended in 50 μL of TE bufer, which was later maintained at deep freeze (−20 °C) for preservation [17,18].

Diagnostic Comparison of Phenotypical Method with
Molecular Method.Te following formula was used to compute the sensitivity (SE), specifcity (SP), positive predictive value (PPV), negative predictive value (NPV), and accuracy (Ac) to proceed with the evaluation of phenotypical detection when compared with PCR methods: where TP is the True Positive, TN is the True Negative, FP is the False Positive, and FN is the False Negative [23].

Data Processing and Statistical Analysis.
All the raw data of experiments was entered in an MS Excel sheet.Te data analysis was carried out using SPSS version 20 (Statistical Package for Social Science).All the numerical data were presented as simple descriptive data.Te comparison of antimicrobial-resistant data with beta-lactamases and non-betalactamase producers as well as the comparison of the drugresistant pattern (MDR and XDR) with ESBL producers and nonproducers was performed using the chi-square or Fisher exact test.To determine the signifcance of the outcome, a p value less than or equal to 0.05 was considered statistically signifcant.

Distribution of Bacteria in a Urine
Sample.Among 1050 urine specimens processed, the number of samples containing bacterial growth in culture media was found to be 335/1050 (31.9%).Among 335 bacterial growth, there were 165/335 (49.2%) growth of Escherichia coli, whereas 170/335 (50.7%) growth was observed for bacteria other than E. coli and the remaining 715 (68%) out of a total of 1050 samples were found to be sterile.

Age-Wise and Gender-Wise Distribution of Cases.
Out of 1050 samples, 400 (38%) received from male patients, while 650 (61.9%) were from female patients.Te female participants of all age groups showed the highest number of participants belonging to the age group of ≥60 years was 240 (22.9%), whereas the lowest number of participants falling under <10 years was 4 (2.3%) (Figure 1).

Antibiotic Susceptibility Pattern of E. coli Isolates.
Antimicrobial susceptibility tests of Escherichia coli obtained from urine were carried out using the Kirby-Bauer disk difusion method.Among E. coli isolates, Canadian Journal of Infectious Diseases and Medical Microbiology 95.7% were sensitive to meropenem, followed by gentamicin (86.7%), and nitrofurantoin (84.8%).Similarly, 64.2% and 55.7% of the isolates were sensitive to amikacin and cefotaxime, respectively.However, 57.6% and 53.4% of E. coli isolates showed resistance against ampicillin and cotrimoxazole.Te sensitive and resistant rates are elucidated in Table 1.4).

Antibiotic Resistance
To determine the prevalence of the bla CTX-M and bla TEM genes, bacterial DNA amplifcation was carried out using the conventional PCR method.Te amplifcation of bla CTX-M and bla TEM genes is shown in Figures 2 and 3.Among 73 cefotaxime-resistant E. coli isolates, the bla CTX-M , bla TEM , and both (bla CTX-M + bla TEM ) genes were found to be 49.3%, 54.8%, and 32.87%, respectively.Te prevalence of bla TEM gene was subsequently greater than that of bla CTX-M genes and both co-producer genes (bla CTX-M + bla TEM ).Te order of magnitude based on the comparison in the prevalence of these genes is represented as bla TEM > bla CTX-M > bla CTX-M + bla TEM which is depicted in Figure 4.

Discussion
Urinary tract infection (UTI) severity varies, ranging from asymptomatic bacteria ascending to the kidneys, which can subsequently lead to sepsis [24].In a community, UTIs are the second most common infection; elder people are more prone to it because of less immunity, decreased secretions of diferent hormones, poor sanitation, and so on [25].
In our study, 165 (15.7%) urine samples isolated E. coli in culture.E. coli were the highest prevalent bacteria from the urine sample which was well represented by a study conducted in Nepal by Poudel et al., in which the prevalence of E. coli was estimated to be 42.4% [26].Many other researchers conducted in Nepal have reported a higher prevalence of E. coli [19,21,22,24].A comparable study conducted by Batra et al. showed 23.6% culture growth among various specimens [27].However, in our study, the growth was found to be inferior to the study carried out by Kateregga et al., where the growth was 46.9%.Administration of previous antimicrobial therapy may hinder the ability of the organism to fourish in culture media [28].
During antibiotic susceptibility testing, antibiotics of diferent classes were tested against the isolates.Among 165 E. coli isolates, 95.7% and 86.7% were sensitive toward meropenem and gentamicin, respectively.Tis fnding synchronizes with the fndings of Sah et al. and Parajuli et al., who reported that their susceptibility towards meropenem was more than 80% [29,30].Another study reported that 86.4% of E. coli was found to be sensitive to carbapenem Canadian Journal of Infectious Diseases and Medical Microbiology drugs such as meropenem, followed by gentamicin (72.8%), and amikacin (66%) which was nearly similar to our study because carbapenems are utilized as a supplementary therapy option for infections brought on by MDR Gramnegative pathogens [31,32].Our study, compared to other research that difers in sample types, sample sizes, organism growth rates, and antibiotic resistance patterns, may account for the proportion of sensitive isolates in both investigations.
In our study, out of 165 E. coli isolates, 85 (51.5%) showed multidrug resistant (MDR), and 10 (6%) showed extensive drug resistance.Te present fnding of MDR was nearly similar to the previous fnding done at Bir Hospital in Nepal, where the rate of MDR was equivalent to 67.4% [33].However, the present fndings of MDR were comparatively lower than the fndings compared to studies [30][31][32][33][34]. Patients who misuse, overuse, and inappropriately use antimicrobial therapy raise the cases of MDR.Te primary contributing factor to the greater multidrug-resistant pattern may be incorrect antibiotic treatments from general practitioners, nurses, or over-the-counter medications, typically given in inappropriate doses, before presenting to the hospital [34].Te antibiotic-resistant patterns of Escherichia coli have been signifcantly associated with the presence of various beta-lactamase genes and the resistance traits for quinolones and aminoglycosides in the plasmid.Antibiotic resistance is primarily caused by switching the target sites, drug inactivation, modifcation enzymes, and the pump efux system [35,36].
In our study among E. coli isolates, 30.3% were ESBL producers which are similar to the fndings by Shilpakar et al., who reported 35.5% of ESBL [37].Another study by Uc-Cachon et al. [38] reported 83.13% ESBL among E. coli isolates which was higher than our study.Similarly, in the report by Poudyal et al., the ESBL-producing E. coli was as high as 80% [39].Another study in Nepal showed 34.5% ESBL, where 33.3% were observed in E. coli which was a similar fnding with our study [40].According to a study by Shristi et al., the prevalence of E. coli that produces ESBLs was just 18.2% [41].Te several patients enrolled in the research, including outpatients, inpatients, patients in intensive care units (ICUs), and patients with various underlined diseases, could be one explanation for the apparent variation in diferent research studies that have been conducted internationally.Tese characteristics of hospitalized patients allow for signifcant factors that contribute to antibacterial resistance.Tese elements may involve intrusive methods and technologies, the administration of broad-spectrum antibiotics regularly, more patients who frequently have co-morbid conditions, and extended hospital stays [42].Our study revealed the major common ESBL genes were TEM and CTX-M in E coli isolates that were resistant to cefotaxime, and bla TEM (54.8%), was in strong concordance with an Indian study [43].Te most prevalent ESBL gene among Enterobacteriaceae has also been identifed as the bla CTX-M gene [43][44][45].Multiple gene occurrences within the same organism were also seen, with bla TEM + bla CTX-M (32.87%) being the most prevalent [19].Tese genes are typically found on plasmids or chromosomal DNA [43,44].Te presence of the gene in plasmids further facilitated its transfer to diferent species of bacteria.Te TEM betalactamase was the frst plasmid-mediated enzyme, from which many of the ESBL have been derived as TEM can hydrolyze the third generation of cephalosporins, particularly ceftazidime.At the beginning of the 21st century, a new class of ESBLs on plasmids dubbed bla CTX-M that preferentially hydrolyze cefotaxime became predominant in European countries and started to spread in Southeast Asia [44].Monitoring of ESBLs should not be limited to phenotypic screening since there have been reports illustrating the disparity between phenotypic and genotypic detection [45].Our study is focused on the isolation of plasmids from ESBLproducing and non-ESBL-producing E. coli isolates and PCR detection of plasmid-borne beta-lactamase genes (bla TEM and bla CTX-M ) that confer antibiotic resistance phenotype.Te relative distribution of each gene among the E. coli isolates studied was analyzed and the presence of ESBL-associated genes which phenotypically non-ESBL isolates was evaluated.Te presence of ESBL genes (CTX-M) in phenotypically undetected cases among tested E. coli isolates may confer ESBL phenotype to pathogens after acquiring the needed mutation.It is therefore necessary to consider prevalent ESBL genes (bla CTX-M ) in phenotypically undetected cases making appropriate steps to address problems associated with the increasing frequency and rapid spread and horizontal gene transfer of ESBL-producing bacteria.Consequently, the new information can be useful in developing accurate protocols for detecting ESBLs and treating infections.While comparing the efciency of phenotypical test keeping PCR detection as a reference method, our study revealed a sensitivity of 69.4% and a specifcity of 42.5%, with an overall accuracy of 55.2%.Te proportion of sensitivity difers based on study, sample size, burden of ESBL, and nature of genes that are recognized by phenotypical methods.In our study, the specifcity might have been lowered compared to sensitivity, and we assume it is because of the presence of ESBL genes in phenotypical undetected strains of Escherichia coli that were resistant to cefotaxime.
Tis study revealed data on the growing dominance of MDR-E. coli and ESBL in Nepal.A previous study conducted by Regmi et al. in Nepal also revealed that 63.04% of MDR was found in E. coli [46].Another study conducted by Manandhar et al. estimated 62% of MDR in E. coli and 16 ESBL-producing E. coli out of 19 ESBL-producing Gramnegative bacteria in Kathmandu, Nepal [47] that determined increasing trends of MDR and ESBL producing in Escherichia coli.According to our study, 50.6%, 54.8%, and 32.8% of the 73 cefotaxime-resistant E. coli isolates had the genes bla CTX-M, bla TEM , and bla CTX-M + bla TEM , suggesting that these genes could transfer from one healthcare to other hospitals while shifting the patients.It aids in the ongoing monitoring and surveillance of both the genes coding for antibiotic resistance and other traits for it for better treatment options, stops the use of unnecessary antibiotics to stop the spread of antibiotic-resistant E. coli, and increases the number of antibiotics available for their efective use in protecting future generations [48].Te primary causes of prolonged infection, increased hospitalization, a higher cost of medication, and increased morbidity and mortality rates are often identifed as bacteria that are becoming increasingly resistant to regularly used antibiotics [49].Antibiotic usage guidelines at the national level should be developed and implemented.Te use of antibiotics should be addressed only when required by medical professionals.

Strengths and Limitations
Te prevalence of E. coli, their antibiogram, and the status of MDR, XDR, and PDR in urine samples from our study in a single hospital setting can be useful additional reference data for the existing literature and treating physicians.Tis study's examination of the incidence of resistant genes in ESBL-producing E. coli emphasizes the value and necessity of molecular diagnostic facilities for a more accurate identifcation of infectious illnesses.However, there are some limitations in our study as well.Te prevalence of Canadian Journal of Infectious Diseases and Medical Microbiology AMR cannot be generalized, as our study's methodology was exclusive to a single institution and used small clinical samples.We were unable to characterize the resistance genotypes of other Gram-negative organisms because of the limited laboratory resources and fnancing.Furthermore, our study did not encompass the characterization of genotypes for other Ambler class beta-lactamases, and we did not examine the presence of other ESBL gene members, including those within the SHV family.

Conclusions
E. coli was the most predominant in our current investigation.Females were highly infected as compared to males.Meropenem and gentamicin were found to be more sensitive towards E. coli isolates, while ampicillin was least efective.Te multidrug-resistant and extensively drugresistant E. coli increases complications in treating UTIs.Te application of the PCR method in our study determined that even non-ESBL producers by the phenotypical method harbor ESBL genes such as TEM, CTX-M, and both coproducers that was very crucial information which concluded that the molecular methods also should be performed simultaneously with phenotypical methods on cefotaximeresistant strains.Nevertheless, the phenotypical methods were easy to perform, cheap, and have good sensitivity for detection of ESBL and are still recommended by the CLSI Guidelines which could be useful for detection in the least developed countries like ours.To accurately estimate the burden of AMR, it is advised that future research involving a larger sample size and more detection of a larger number of diferent genes can sufciently improve detection of missed cases of ESBL by the phenotypic method.

Figure 1 :
Figure 1: Age and gender-wise distribution of cases.
Isolates.According to Bergey's Manual of Systemic Bacteriology, standard microbiological methods such as the study of colony morphology, Gram's staining, and other biochemical tests (catalase test, oxidase test, Triple Sugar Iron (TSI) test, Sulphide Indole Motility test (SIM), Urease test, Citrate utilization test, Methyl Red test, Voges-Proskauer test, Sorbitol tests, and so on) were used to identify E. coli [14].

Table 2 :
Comparison of antibiotic resistance pattern with phenotypically detected ESBL cases in E. coli isolates.

Table 3 :
ESBL status among MDR and XDR E. coli isolates.

Table 4 :
Molecular prevalence of bla CTX-M and bla TEM genes among ESBL and non-ESBL E. coli isolates.

Table 5 :
Diagnostic evaluation of the phenotypical method on comparison with detection of bla CTX-M genes associated with ESBL in cephalosporin-resistant E. coli.True Positive, FP � False Positive, TN � True Negative, FN � False Negative, PPV � positive predictive value, NPV � negative predictive value.