Urinary tract infections (UTIs) are among the most frequent bacterial infections encountered globally, affecting both hospitalized and community-based populations. Escherichia coli accounts for up to 80% of uncomplicated UTIs and remains the principal causative agent, especially among females due to anatomical predispositions [1].

The global rise of antimicrobial resistance has severely complicated UTI management. One of the critical contributors to this crisis is the emergence and spread of extended-spectrum β-lactamase (ESBL)-producing organisms, notably E. coli. ESBLs are enzymes capable of hydrolyzing penicillins, third-generation cephalosporins, and aztreonam, rendering many β-lactam antibiotics ineffective [2,3]. These enzymes are often encoded by transferable plasmids, facilitating rapid dissemination among Enterobacteriaceae [4].

Among the different types of ESBLs, the most frequently detected genes are TEM (Temoneira), SHV (Sulfhydryl Variable), and CTX-M (cefotaximase), with CTX-M variants currently dominating the epidemiological landscape worldwide [5,6]. The detection and characterization of these enzymes are crucial for epidemiological surveillance and guiding antimicrobial therapy.

India has reported a high prevalence of ESBL-producing organisms, particularly in tertiary care hospitals, raising serious concerns about treatment failures, prolonged hospital stays, and increased healthcare costs [7,8]. The overuse and misuse of antibiotics in the community and hospital settings are key drivers behind this resistance pattern [9,10].

Phenotypic detection methods like the combined disk test and double-disk synergy test (DDST) have been widely used in clinical laboratories. However, these methods lack the specificity and sensitivity offered by molecular techniques. Polymerase Chain Reaction (PCR)-based detection of bla_TEM, bla_SHV, and bla_CTX-M genes offers accurate and rapid results, enabling effective infection control and epidemiological monitoring [11-14].

The dissemination of ESBL genes has also been linked to mobile genetic elements like integrons, transposons, and insertion sequences, increasing their public health relevance [15,16].

In rural India, where empirical therapy is common, delayed or inaccurate detection of resistance genes further aggravates the problem. Compounding this challenge is the limited availability of molecular diagnostic tools in secondary care centers [17].

Therefore, the present study was undertaken to determine the prevalence of ESBL-producing E. coli in urinary isolates and to characterize the presence of blaTEM, blaCTX-M, and blaSHVgenes using PCR, alongside their antibiotic susceptibility patterns.

MATERIALS AND METHODS

This was a cross-sectional, observational study conducted over a 12-month period at a tertiary care hospital in the Department of Microbiology. A total of 836 non-repetitive urine samples were collected and processed for microbiological analysis.

Inclusion Criteria

1. Patients of all age groups with clinically suspected urinary tract infections.
2. Indoor and outdoor patients attending the hospital.
3. Patients who provided informed consent.

Exclusion Criteria

1. Patients who received antibiotics within 48 hours before urine sample collection.
2. Duplicate isolates from the same patient.
3. Non- coli urinary isolates.

Sample Collection and Processing

Urine samples were collected in sterile containers using midstream clean-catch technique. Samples were cultured on MacConkey and Blood agar and incubated aerobically at 37°C for 24 hours. Isolated organisms were identified using standard biochemical methods.

Phenotypic Screening of ESBL

Isolates of E. coli were subjected to the phenotypic confirmatory test using the Combined Disk Test (CDT) with ceftazidime (30 µg) and ceftazidime-clavulanic acid (30/10 µg) as per CLSI guidelines (2021). A ≥5 mm increase in zone diameter with the combination disk was interpreted as ESBL positive.

Antimicrobial Susceptibility Testing

Antibiotic susceptibility testing was done using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. Antibiotics tested included ampicillin-sulbactam, gentamicin, cefoxitin, amikacin, ciprofloxacin, meropenem, ceftazidime, ceftriaxone, nitrofurantoin, tigecycline, piperacillin-tazobactam, fosfomycin, and ceftazidime-clavulanate.

Molecular Characterization by PCR

DNA was extracted from ESBL-positive isolates using the boiling method. PCR was used to detect the presence of blaTEM, blaCTX-M, and blaSHVgenes using specific primers.

PCR cycling conditions for each gene were standardized as follows:

TEM: Initial denaturation at 98°C for 5 min; 35 cycles of denaturation at 98°C for 30s, annealing at 51°C for 30s, extension at 72°C for 30s; final extension at 72°C for 5 min.

CTX-M and SHV: Similar conditions with annealing at 55°C.

Amplicons were visualized on 1.5% agarose gel electrophoresis using ethidium bromide staining.

RESULTS

Throughout the learning period a total 836 urine samples were collected and  processed. Out of 836 which 366 (49%) were showing Isolates of E. coli isolates. In which 82 (22.5%) were phenotypically identified as ESBL producers and 284 (77.6%) were Non-ESBL (Fig. 1).  Among 82 ESBL producing strains, 26 (31.7%) belong to Outdoor patients and 56 (68.3%) belong to Indoor patients (Fig.2).

Figure 1 Distribution of E.coli Isolates among ESBL / Non-ESBL

Figure 2: Distribution of E.S.B.L producing E.coli Isolates among IPD/OPD Patients

Table 1 shows that Out of 366 patients who were included in this study 108 (29.5%) were Male & 258 (70.5%) were Female patients.

Table 1 Distribution of patients according to Gender (Male/ Female)

Table 2 shows that the largest percentage of cases (24.1%) occurs among patients aged 21 to 30 years, while the lowest percentage (0.9%) occurs among patients aged 80 and above.

Table 3: Distribution of Patients according to Age in tubular form

Table 3 shows the distribution of patients according to their residence 36.4% of the patients were from urban areas, while 63.6% of the patients were from rural areas.

Table 4 Distribution of Patients according to their residence status in tubular form

Table 4 shows the distribution of patients according to their educational status. It was found that majority (74.4%) patients were illiterate followed by, 15.3% patients pre primary, 6% patients primary school, 2.7% patients high school and 1.6% patients were found to be graduate or above.

Table 5:  Distribution of patients according to their educational status

Table 5 shows the distribution of patients based on socioeconomic status. A total of 6% of the 366 patients were upper class, followed by middle class patients (25.2%) and lower class patients (68.8%).

Table 6: Distribution of Patients according to their Socio-economical status

Table 7 shows the distribution of patients according to their Susceptibility of ESBL Isolates to various Antibiotics (N=82) status.

Table 7: Distribution of Antimicrobial susceptibility of ESBL Isolates (N=82) status

Table  8: Association of Antimicrobial Susceptibility with Demographic Characteristics.

MOLECULAR RESULTS  

POLYMERASE CHAIN REACTION (PCR)

PCR was used to amplify the TEM, CTX-M, and SHV gene sequences.

       Table 9: The PCR cycling conditions to amplify TEM gene fragments.

bla-TEM gene Results\

                           Figure 3: Amplified DNA with PCR for blaTEM gene

L corresponds to the DNA Ladder.

L1 corresponds to the positive.

Control: L2 corresponds to Negative control blaTEM gene.

L3-L7 are the sample positive for blaTEM  gene.

Table 10: The PCR cycling condition to amplify CTX-M gene fragments.

bla-CTX gene Results

           Figure 4: Amplified DNA with PCR for CTX-M gene.

Lane: 1-9 show positive for CTX  gene

Lane: 10 DNA Ladder

Line: 11-16 and 19, 20 CTX genes positive

Lane: 17 Negative control for CTX gene

Lane: 18 show positive control for CTX GENE

Table 11: The PCR cycling condition to amplify SHV gene fragments.

bla SHV Results

                                        Figure 5: Amplified DNA with PCR for bla SHV gene

L1 corresponds to the DNA Ladder.L2 corresponds to the positive Control.L3 Corresponds to the Negative Control to blaSHV gene;L4-L6 sample positive for blaSHV gene

Table  and figure shows that out of 82 ESBL positive patients, gene TEM was seen in 53.7% patients, gene CTX-M was seen in 35.4% patients &  gene SHV was observed in 10.9% patients.

Table 12: Detection of different genes in ESBL producing E.coli isolates.

Figure 6: Detection of different genes in ESBL producing E.coli isolates.

DISCUSSION

Urinary tract infections (UTIs) remain one of the most common bacterial infections, with Escherichia coli as the predominant uropathogen. The emergence of extended-spectrum beta-lactamase (ESBL)-producing E. coli complicates the empirical management of UTIs due to their resistance to cephalosporins and penicillins. In the present study, 22.5% of the 366 E. coli isolates were found to be ESBL producers, which aligns with findings from studies conducted across India and other parts of the world. For instance, Grover et al. reported an ESBL prevalence of 24% among E. coli isolates in their North Indian cohort [18m, while Taneja et al. found a rate of 20.1% in hospitalized patients [19].

The present study highlights the prevalence and molecular profile of ESBL-producing E. coli in urinary tract infections. A total of 22.5% of E. coli isolates were ESBL positive, which is in concordance with other Indian studies. Gupta et al. [14] reported 26%, and Jain et al. [8] reported 29.3% ESBL prevalence among uropathogens.

The demographic profile of patients in our study reveals a higher prevalence of infections among females (70.5%) compared to males (29.5%), which is consistent with previous findings by Colodner et al., who highlighted female predominance due to shorter urethra and closer proximity to the anal canal [20]. Furthermore, the majority of patients were from rural backgrounds and belonged to the lower socioeconomic class, which has also been associated with higher UTI risks due to poor hygiene and limited healthcare access [21].

The predominance of female patients (70.5%) is consistent with findings by Foxman et al. [1] and Rawat and Nair [11], who observed higher UTI incidence in women due to anatomical and hormonal factors. The highest prevalence was in the 21–30-year age group, similar to findings by Sharma et al. [13] and Singh et al. [7].

Our antimicrobial susceptibility results revealed that ESBL-producing E. coli showed 100% susceptibility to carbapenems (meropenem), nitrofurantoin, fosfomycin, and ceftazidime-clavulanic acid. This is in concordance with previous reports by Paterson and Bonomo, who emphasized the high efficacy of carbapenems against ESBL-producing strains [22]. Similarly, Manoharan et al. and Rodrigues et al. also observed similar susceptibility trends with nitrofurantoin and fosfomycin [23, 24].

Antibiotic resistance profiling revealed 100% resistance to ceftriaxone and ceftazidime in ESBL strains. Similar resistance patterns were observed by Paterson and Bonomo [2] and Bradford [3], who emphasized ESBLs’ role in hydrolyzing third-generation cephalosporins. However, complete susceptibility to carbapenems (meropenem), nitrofurantoin, and fosfomycin was noted, consistent with findings by Canton and Coque [5] and Jain and Mondal [8].

On the contrary, resistance to ceftriaxone and ceftazidime was universally observed in ESBL producers, which is expected as these are third-generation cephalosporins commonly hydrolyzed by ESBL enzyme [25]. Ciprofloxacin resistance was seen in 48.8% of the isolates, comparable to a study by Gupta et al., where fluoroquinolone resistance exceeded 50% [26]. This growing resistance limits the options for oral outpatient treatment and necessitates stringent antimicrobial stewardship.

Molecular characterization in our study detected the blaTEM gene in 53.7% of isolates, blaCTX-M in 35.4%, and blaSHV in 10.9%. This gene distribution is consistent with several studies across India. For example, Naseer and Sundsfjord documented the predominance of the TEM gene globally [27] . Similarly, Shahid et al. also reported TEM as the most frequently detected gene in their isolates from North India [28] . However, CTX-M genes have been increasingly reported in Western and South Indian studies, suggesting regional genetic variation [29,30].

PCR-based detection showed blaTEM (53.7%) as the predominant gene, followed by bla(35.4%) and blaSHV(10.9%). Similar trends were reported by Bonnet [6], who highlighted the dominance of CTX-M in Europe, but TEM still prevails in South Asia. A study by Shaikh et al. [12] also reported TEM dominance in E. coli from Indian clinical samples.

Interestingly, we observed a statistically significant association of ciprofloxacin and cefoxitin resistance with male gender (p<0.05), which has not been extensively explored in prior studies but may reflect gender-based pharmacokinetic or behavioral differences requiring further investigation.

Other molecular studies from India, including that by Singh et al. [17] and Gupta and Datta [14], showed a high prevalence of TEM and CTX-M genes, affirming our findings. Carattoli [15] emphasized the role of plasmids in gene dissemination, which may explain the high occurrence in both community and hospital settings.

The presence of multiple ESBL genes in a single isolate has been noted in various studies including Poirel et al. [16], who found co-expression of TEM and CTX-M in 34% of isolates, possibly due to integrons or transposons. In our study, some strains were positive for more than one gene.

Gender-wise distribution showed statistically significant differences in resistance to cefoxitin and ciprofloxacin, which was also observed in a study by Kumarasamy et al. [9]. They hypothesized that biological and treatment-seeking behavior may influence resistance patterns.

Our results also support the use of ceftazidime-clavulanic acid and piperacillin-tazobactam as empirical therapy, as resistance rates were low (0–14.6%). Similar empirical recommendations were made by the CLSI (2021) and supported by Sharma et al. [13]

Overall, the integration of molecular techniques alongside phenotypic testing enhances diagnostic precision, which is echoed by studies from Shaikh et al. [12], Carattoli [15], and Paterson et al. [2]. PCR not only confirms the presence of genes but also aids in epidemiological surveillance. Thus, this study emphasizes the need for continuous surveillance, rational antibiotic usage, and mandatory molecular characterization in tertiary care centers.

Our results reinforce the urgent need for continuous surveillance of ESBL prevalence and gene patterns. Jain et al. emphasized the critical role of molecular typing in understanding the local epidemiology of antimicrobial resistance [31] . Moreover, Nathisuwan et al. proposed that integrating molecular diagnostics into routine labs can guide appropriate therapy and infection control measures[32].

Our findings underscore the importance of using antibiotics like nitrofurantoin and fosfomycin in outpatient UTI management and reserving carbapenems for complicated or resistant cases. The high susceptibility rates to these drugs are supported by findings from Kaur et al. and Karunasagar et al. [33,34].

Overall, this study adds valuable data to the regional antimicrobial resistance database and supports the need for rapid phenotypic and genotypic screening of ESBL-producing uropathogens to prevent treatment failures and limit resistance spread.

CONCLUSION

This study highlights a significant proportion of ESBL-producing E. coli isolates in UTIs, with the blaTEMgene being the most prevalent, followed by blaCTX-M and blaSHV. These strains exhibit high resistance to third-generation cephalosporins but retain susceptibility to meropenem, nitrofurantoin, fosfomycin, and ceftazidime-clavulanate. Regular screening, molecular typing, and effective antibiotic stewardship are essential to curb the spread of resistant strains.

LIMITATIONS

1. This was a single-center study; results may not be generalizable to other regions.
2. Only three ESBL genes were tested; other ESBL or AmpC genes were not included.
3. Genotypic analysis was limited to PCR; sequencing could provide deeper insights.
4. The study lacked follow-up on clinical outcomes post-treatment.

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