Enzymes known as extended-spectrum beta-lactamases (ESBLs) have the ability to hydrolyze and confer resistance to cephalosporins from the first, second and third generations as well as monobactams like aztreonam and penicillin.[1]

In the early 1980s, Germany was the first nation to isolate ESBLs from Klebsiella pneumoniae strains, and it was discovered that these enzymes were a key factor in the bacteria’s resistance to third-generation cephalosporins.[2]

Gram-negative facultative anaerobes with a rod-like structure and no spores are known as Enterobacteriaceae. Escherichia and Klebsiella, which are both responsible for serious bacterial infections, especially in patients who stay in hospitals for extended periods of time, are included in this group.[3]

Essential antibiotics (β-lactams) and tiny hydrophilic solutes can passively diffuse through channel-forming proteins found in gram-negative bacteria's outer membrane. A class of proteins called porins is present in Shigella, Salmonella and E. coli, among other gamma-proteobacteria. In addition to acting as bacteriocin and bacteriophage receptors, these porins support the integrity of the cell membrane. By adhesion, invasion, and serum resistance, they also aid in the pathogenesis of bacteria. In gram-negative bacteria, different kinds of porins have been found and grouped according to their structural functionality, activity, regulation, and expression.[4]

Improving patient outcomes and maximizing the effectiveness of care required a proper diagnosis of ESBL infections. BacT/Alert 3D and other automated blood cultures are essential to this procedure.[5]

The design of this study was observational and this study was conducted in the department of bacteriology, Teerthanker Mahaveer University, Hospital in Moradabad.

SPECIMEN COLLECTION-

In our research blood were collected in blood culture bottle from the patient under strict aseptic precautions. In adult, about 5 to 10 ml of blood was collected and in children, the volume collected is adjusted based on age and weight, ranging from 1-5 ml then inoculated in to the 50 ml brain heart infusion broth. The bottles were incubated in the BACT/ALERT automated blood culture system for up to 16-18 hrs. Bottles flagged positive were subjected to Gram staining and sub-culture onto MacConkey agar and blood agar for isolation of Gram-negative bacilli.

Gram-negative isolates were identified based on colony morphology, Gram staining and standard biochemical tests (e.g., indole, citrate, urease, motility and TSI reactions).

ANTIMICROBIAL SUSCEPTIBILITY TESTING-

Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar as per Clinical and Laboratory Standards Institute (CLSI) 2024 guidelines. The antibiotic discs used included third-generation cephalosporins such as ceftazidime (30 µg) and cefotaxime (30 µg).

PHENOTYPIC CONFIRMATION OF ESBL PRODUCTION-

Phenotypic confirmation was done using the Combined Disc Diffusion Test (CDDT). Mueller-Hinton agar plates were inoculated with standardized bacterial suspensions (0.5 McFarland standard). Discs of ceftazidime (30 µg) and ceftazidime + clavulanic acid (30/10 µg) were placed 25 mm apart (center to center). An increase in zone diameter of ≥5 mm around the combination disc compared to the ceftazidime disc alone confirmed ESBL production.

Statistical analysis

Tables and figures were created using Microsoft Word and Excel.

Ethics statement

The study was approved by Institutional Ethical Committee TMU Moradabad with Ref no. TMU/IEC/2024-25/PG/134. Prior to the collection and processing of samples, each subject provided their informed consent. Participants were given a general explanation of the study’s purpose and nature, as well as the freedom to decline participation or to withdraw at any moment without compromising their ability to obtain other health services. The data gathered was kept private

Out of total 93 samples, 30 were ESBL producer and there was a higher prevalence of ESBL production in males 19(63.33%) as compared to females 11(36.67%). The maximum number of cases were observed in the 61-80 years of age group (11 cases).

ESBL status

The analysed samples (n=93) 32.25% (30 cases) were ESBL, while 67.75% (63 cases) were non ESBL producers. Table 1

Distribution of ESBL according to organism

Escherichia coli was the most frequently isolated organism among the 30 ESBL-positive cases, accounting for 20 cases, followed by Klebsiella pneumoniae with 7 cases and Klebsiella oxytoca with 3 cases. These findings indicate that E. coli was the predominant ESBL-producing pathogen in this study. Table 2.

Antibiotic Sensitivity & Resistance in ESBL Isolates

The drug susceptibility table of ESBL isolates revealed high levels of resistance to multiple antibiotics Table 3. Ampicillin (AMP), Cefazolin (CZ), and Ceftazidime (CAZ) showed 100% resistance, indicating their ineffectiveness against ESBL-producing bacteria. Other cephalosporins, including Cefuroxime (CXM), Cefepime (CPM), Ceftriaxone (CTX), and Cefalexin (CX), also demonstrated high resistance rates ranging from 86.67% to 96.67%.

Among aminoglycosides, Gentamicin (GEN) and Amikacin (AK) showed moderate effectiveness, with 53.33% and 56.67% sensitivity, respectively. Azithromycin (AZM) and Cotrimoxazole (COT) exhibited high resistance rates of 83.33% and 86.67%, respectively.

Carbapenems such as Imipenem (IPM) and Meropenem (MRP) showed relatively better activity, with sensitivity rates of 40% and 56.67%, respectively. Piperacillin-Tazobactam (PIT) and Ticarcillin-Clavulanate (TCC) also showed intermediate sensitivity at 46.67% and 50%, respectively.

Encouragingly, Tigecycline (TGC), Polymyxin B (PB), and Colistin (CL) demonstrated 100% sensitivity, making them the most effective treatment options against ESBL isolates. Doxycycline (DO) and Chloramphenicol (C) also retained good activity, with 70% and 73.33% sensitivity, respectively.

Table 1: ESBL Status

In the present study, a total of 93 Gram-negative bacterial isolates were screened for the production of Extended-Spectrum Beta-Lactamase (ESBL). Among these, 30 isolates (32.25%) were found to be ESBL producers, while the remaining 63 isolates (67.75%) were non-ESBL producers.

Table 2: Distribution of ESBL on the basis of organism

Among the 30 ESBL-producing isolates identified in the study, Escherichia coli was the most prevalent organism, accounting for 20 isolates (66.67%), followed by Klebsiella pneumoniae with 7 isolates (23.33%), and Klebsiella oxytoca with 3 isolates (10%). This distribution indicates that E. coli is the predominant ESBL producer among the Gram-negative isolates, suggesting its significant role in antimicrobial resistance in the studied population.

Table 3: Distribution of antibiotic sensitivity and resistance in ESBL isolates

The ESBL-producing isolates showed high resistance to most beta-lactam antibiotics, including 100% resistance to cefazolin (CZ) and ceftazidime (CAZ), and 96.67% resistance to ampicillin (AMP), cefuroxime (CXM), and cefotaxime (CTX). Moderate resistance was observed against gentamicin (GEN, 46.67%), amikacin (AK, 43.33%), and imipenem (IPM, 40%). In contrast, all isolates were 100% sensitive to tigecycline (TGC), polymyxin B (PB), and colistin (CL), indicating their effectiveness against ESBL producers. Other antibiotics like chloramphenicol (C), doxycycline (DO), and meropenem (MRP) showed variable sensitivity patterns, highlighting the limited but present treatment options.

Out of 93 samples was analysed in our study, out of which 30 (32.25%) organism was identified which produces ESBL. The remaining 63 (67.75%) isolates were non-ESBL producers. A similar study conducted by Balan K (2013) [6]   reported an ESBL prevalence of 19.6% in E. coli and 41.6% in Klebsiella spp., whereas our study found E. coli (66.67%) as the most predominant ESBL producer, followed by Klebsiella pneumoniae(23.33%) and Klebsiella oxytoca (10%). These variations in ESBL prevalence may be attributed to regional differences in antimicrobial resistance patterns and infection control measures. Table 1

The common prevalent pathogens which produce ESBL in our study E. coli (66.67%), K. pneumoniae (23.33%), and K. oxytoca (10%). In comparison, Tsering et al. (2009) [7] found E. coli to be the predominant ESBL producer in bloodstream infections (3 cases), followed by K. pneumoniae (2 cases) and Pseudomonas aeruginosa (1 case). Additionally, the demographic study reported that Klebsiella pneumoniae accounted for 63.8% of bloodstream infections, which is consistent with our findings regarding its significant role in BSIs Ejaz et al., (2024).[8]   Table 2

Out of 30 ESBL-producing organisms were tested for antibiotic susceptibility. Among them, Cefazolin (CZ) and Ceftazidime (CAZ) showed 100% resistance, followed by Cefotaxime (CTX), Cefuroxime (CXM), and Levofloxacin (LE) with 96.67% resistance. High resistance was also observed for Ampicillin (AMP) (96.67%), Cefepime (CPM) (86.67%), and Cotrimoxazole (COT) (86.67%). However, Tigecycline (TGC), Polymyxin B (PB), and Colistin (CL) were 100% sensitive, making them the most effective antibiotics against these isolates. A similar study conducted by Dalela et al. (2021) [9] reported that Meropenem showed 95.2% sensitivity, whereas in our study, the sensitivity was comparatively lower at 56.67%. Additionally, Piperacillin + Tazobactam was sensitive in 87.3% of cases in their study, whereas in our findings, sensitivity was only 46.67%. Notably, Gentamicin resistance was 77.8% in Dalela et al.’s study, which was higher than our observed resistance of 46.67%. These results suggest that antibiotic resistance patterns may vary across regions and time. Table 3

According to our research, ESBL-producing Gram-negative bacteria are significantly more common in bloodstream infections, with E. coli accounting for 66.67% of cases, followed by K. pneumoniae (23.33%) and K. oxytoca (10%).

Males and older age groups had the highest incidence, and ICU settings reported the most instances. High resistance to β-lactams and aminoglycosides was found in antibiotic susceptibility testing, however Tigecycline, Polymyxin B and Colistin continued to be quite effective.

An alarming increase in carbapenem resistance is shown by comparisons with earlier research, highlighting the critical need for strict infection control protocols, ongoing surveillance of antimicrobial resistance, and prudent antibiotic usage to stop the spread of multidrug-resistant organisms.

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