Phenotypic and Molecular Characterization of the blaTEM Gene in Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae

Abstract Introduction There has been a notable rise in antibiotic resistance among enterobacteria. This issue is primarily attributed to the emergence of extended-spectrum beta-lactamases (ESBLs), which present a significant concern for public health worldwide. This study investigates the prevalence of ESBL production, antibiotic resistance profiles, and molecular identification of the blaTEM gene in Klebsiella pneumoniae isolates. Methods The samples were randomly collected from several medical facilities in Erbil city. The VITEK 2 system was used for bacterial identification, antibiotic susceptibility, and ESBL production testing. The Double Disc Synergy Test (DDST) confirmed ESBL production. Polymerase chain reaction was conducted on all DNA samples, and the amplified DNA was analyzed using agarose gel electrophoresis to detect the blaTEM gene. Results A total of 43 samples were collected, of which the majority were urine (56%), followed by sputum (28%), blood (9%), and wound (7%). Klebsiella pneumoniae isolates exhibited the highest prevalence of resistance against ceftazidime (72%), ceftriaxone (70%), ciprofloxacin (63%), amoxicillin-clavulanic acid (60%), amikacin (58%), cefotaxime/tazobactam (56%), and gentamicin (53%). The DDST results indicated positive ESBL production in 15 isolates (35%), as evidenced by an increase or distortion in the inhibition zone toward the amoxicillin-clavulanate disc. Of the 43 isolates, 34 (79%) carried the blaTEM gene. Conclusion The study area shows a significant level of antibiotic resistance in ESBL-producing Klebsiella pneumoniae isolates, which, if not adequately addressed, could soon lead to severe health and therapeutic consequences. Introduction Antibiotic resistance is a dynamic and ongoing challenge. Over the past three decades, there has been a notable rise in antibiotic resistance among enterobacteria, particularly concerning third-generation cephalosporins. This issue is primarily attributed to the emergence of extended-spectrum beta-lactamases (ESBLs). ESBLs are a group of beta-lactamase enzymes produced by gram-negative bacteria that can hydrolyze and inactivate many antibiotics, including aztreonam, penicillin, and cephalosporins. These enzymes, such as TEM, SHV, and CTX-M, and their variants, allow enterobacteria to resist β-lactam antibiotics. The encroachment of these multi-drug-resistant pathogens into community settings raises significant alarm. The swift spread of the genes that encode these ESBLs, primarily via plasmids, has facilitated their rapid proliferation, leading to an alarming increase in the global prevalence of ESBL-producing bacteria. This situation presents a significant concern for public health worldwide [1,2]. Klebsiella pneumoniae is an opportunistic pathogen responsible for nosocomial and community-acquired infections [3]. This bacterium is usually found as an intestinal microflora. It can cause severe diseases like ventilator-associated pneumonia, catheter-related urinary tract infections, meningitis, bacteremia, septicemia, infection of surgical and non-surgical wounds, diarrhea, prosthetic valve endocarditis, peritonitis, and osteomyelitis [1]. The first strains of Klebsiella pneumoniae with ESBL (KP-ESBL) were identified in Europe in 1982, marking the emergence of resistance to ceftazidime and aztreonam due to plasmid-mediated beta-lactamase enzymes. These resistance traits rapidly disseminated to other gram-negative bacteria, including Escherichia coli. Since the discovery of these enzymes, their prevalence has continued to rise, with over 200 distinct ESBL enzymes identified today. The impact of ESBL-producing strains is particularly pronounced in intensive care units, which pose a high risk for epidemic outbreaks. K. pneumoniae and E. coli are the two most frequently encountered bacterial species associated with ESBL production. Implementing proactive monitoring of ESBL-producing pathogens in high-risk populations is crucial through suitable antimicrobial strategies. This necessity arises from the tendency of these pathogens to display multidrug resistance, which complicates treatment options and increases the potential for severe infections [4]. This study investigates the prevalence of ESBL production, antibiotic resistance profiles, and molecular identification of the blaTEM gene in Klebsiella pneumoniae isolates in Erbil City. Methods Study design and sample collection This retrospective cross-sectional study was conducted between September 2021 and January 2022. The ethics board of the College of Health Sciences at Hewler Medical University approved the study. Patient consent was verbally obtained from the patients to collect their samples and be published in this study. The samples were randomly collected from several medical facilities in Erbil city, such as Nanakali Hospital, Hawler Teaching Hospital, and private laboratories. The collected samples included urine, sputum, or wound infection. Isolation, identification, and susceptibility test of Klebsiella pneumoniae The samples were cultured on MacConkey agar and incubated at 37°C for 24 hours. Klebsiella pneumoniae colonies were isolated based on their large, pink-to-red mucoid appearance. The VITEK 2 system (bioMérieux, France) was used for bacterial identification and antibiotic susceptibility testing. A sterile swab or stick was used to transfer an adequate number of colonies from the cultured sample into 3.0 ml of sterile saline (0.45% to 0.50% NaCl, pH 4.5 to 7) in a 12 × 75 mm polystyrene test tube. The suspension’s turbidity was then adjusted to match the 0.5 McFarland standard for antimicrobial susceptibility testing and measured using a DensiChek™ turbidimeter. Phenotypic Detection and Confirmation of ESBLs ESBL production was detected among the isolates using the VITEK 2 system. The testing panel included six wells containing 10 μg/ml ceftriaxone, 0.5 μg/ml cefotaxime, or 0.5 μg/ml ceftazidime, alone or in combination with clavulanic acid (10 μg/ml, 4 μg/ml, and 4 μg/ml, respectively). Bacterial growth in each well was quantitatively assessed using an optical scanner. A proportional reduction in growth in the wells containing cephalosporin combined with clavulanic acid, compared to those containing cephalosporin alone, was considered indicative of ESBL production. The Double Disc Synergy Test (DDST) confirmed ESBL production. Each Klebsiella pneumoniae isolate was inoculated on Müller-Hinton agar plates for susceptibility testing. Discs containing 30 μg of cefotaxime and ceftazidime were placed on either side of a disc with co-amoxiclav (20/10 μg), positioned 20 mm apart (center to center). ESBL production was indicated when the inhibition zone of either cephalosporin was expanded by clavulanic acid, often resulting in a distinctive "champagne-cork" or "keyhole" shape zone [1]. Molecular detection of blaTEM gene DNA extraction of Klebsiella pneumoniae was carried out using the simple boiling method. Isolated colonies from overnight cultures were placed in a test tube containing 1 ml of distilled water and heated in a water bath for 10 minutes. The sample was centrifuged at 1,000 rpm for 5 minutes to complete the extraction process. Polymerase chain reaction (PCR) was conducted on all DNA samples of Klebsiella pneumoniae. The concentration of extracted double-stranded DNA was combined with a PCR master mix (Promega, USA), which included Taq DNA polymerase, deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), buffers, and salts. Specific forward (5'-GAG TAT CAA CAT TTC CGT GTC-3') and reverse (5'-TAA TCA GTG GGC ACC TTC TC-3') primers were added to amplify the blaTEM target gene. The PCR mixture was then incubated in a thermocycler, undergoing 32 cycles of alternating temperatures: a denaturation step at 94°C for one minute to separate the double-stranded DNA into single strands, an annealing step at 57°C for one minute to allow the primers to bind to the target DNA, and an extension step at 72°C for 10 minutes for the Taq DNA polymerase to synthesize new DNA strands. This process generated billions of copies of the target DNA sequence within a few hours. Subsequently, the amplified DNA was separated and analyzed using agarose gel electrophoresis. Results A total of 43 samples were collected; 22 (51%) were obtained from females and 21 (49%) from males. The majority of the samples were urine (56%), followed by sputum (28%), blood (9%), and wound (7%). Klebsiella pneumoniae isolates exhibited the highest prevalence of resistance against ceftazidime (72%), ceftriaxone (70%), ciprofloxacin (63%), amoxicillin-clavulanic acid (60%), amikacin (58%), cefotaxime/tazobactam (56%), and gentamicin (53%). In contrast, the lowest prevalence of resistance was observed with imipenem (26%) and meropenem (28%) (Table 1). The DDST results indicated positive ESBL production in 15 isolates (35%), as evidenced by an increase or distortion in the inhibition zone toward the amoxicillin-clavulanate disc. The remaining samples (65%) tested negative for ESBL production (Figure 1). Of the 43 isolates, 34 (79%) carried the blaTEM gene (Figure 2). Overall, 11 isolates (26%) tested positive in the DDST and PCR, while five isolates (12%) were negative. Additionally, four isolates (9%) were positive in DDST but negative in PCR, whereas 23 isolates (53%) were negative in DDST but positive in PCR (Table 2). Table 1. Antibiotic resistance pattern of K. pneumoniae isolates. Antibiotic No. of resistant isolates Frequency (%) No. of sensitive isolates Frequency (%) Ceftazidime 31 72% 12 28% Ceftriaxone 30 70% 13 30% Ciprofloxacin 27 63% 16 37% Amoxicillin clavulanic acid 26 60% 17 40% Amikacin 25 58% 18 42% Cefotaxime 24 56% 19 44% Tazobactam 24 56% 19 44% Gentamicin 23 53% 20 47% Meropenem 12 28% 31 72% Imipenem 11 26% 32 74% Table 2. Outcomes o