Combined Disc Method Research Articles

A Study on Extended Spectrum β-Lactamase Producing (ESBL) and Fluoroquinolone-Resistant Escherichia coli in the Japanese Quails

Antimicrobial resistance (AMR) is a growing public health threat worldwide, and members of the Enterobacteriaceae family are among the clinically important bacteria rapidly developing resistance to available antibacterial agents. The development of extended-spectrum β-lactamase (ESBL) enzymes is the primary cause of resistance to third-generation cephalosporins among Enterobacteriaceae. Quails also represent a source of animal protein deficiency in developing countries including India. This study aimed to detect the presence of extended-spectrum β-lactamases (ESBLs) and fluoroquinolone-resistant Escherichia coli from fecal samples of quails in Puducherry. A total of 36 fecal samples of quails from six different retail outlets were collected. E. coli could be isolated from 27 fecal samples and confirmed as E. coli by polymerase chain reaction by targeting the uspA gene. The isolates were preliminarily screened for ESBL production by indicator antimicrobial disc and phenotypic ESBL production was confirmed by combination disc method. The isolates were subjected to genotypic detection of ESBL (bla CTX-M, bla TEM, and bla SHV) by PCR. Out of 27 isolates, 16 isolates were positive for the presence of ESBL-producing genes, in which, 9 harbored bla CTX-M, 2 harbored bla TEM, and 5 harboured bla SHV genes. Similarly, out of 27 isolates 13 isolates were found positive for fluoroquinolone resistance in which 13 (48.18%) isolates harbored the qnrS gene, and 11 (40.74%) isolates harbored the qnrB gene. Antimicrobial susceptibility test showed that the isolates exhibited a high level of resistance against Cefpodoxime (90%), Amoxycillin/clavulanic acid (70%), Ceftriaxone (60%), Cefotaxime (55%), Ceftazidime (45%), Ciprofloxacin (40%) and Aztreonam (30%).

Phenotypic and Genotypic Profiles of Extended-Spectrum Beta-Lactamase-Producing Multidrug-Resistant Klebsiella pneumoniae in Northeastern Thailand.

The global emergence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae presents a significant public health threat and complicates antibiotic treatment for infections. This study aimed to determine the prevalence of ESBL-producing K. pneumoniae in a clinical setting, analyze their antimicrobial susceptibility profiles, and characterize both phenotypic and genetic determinants. A total of 507 non-duplicate clinical isolates of Enterobacterales were collected between 2019 and 2020, and third-generation cephalosporin resistance was screened by disk diffusion. Identification of K. pneumoniae was confirmed using biochemical tests and PCR with species-specific primers. Antimicrobial susceptibility testing was conducted using disk diffusion, and phenotypic ESBL production was confirmed using the combined disk method. Multiplex PCR detected ESBL genes (blaTEM, blaSHV, and blaCTX-M) and identified blaCTX-M groups. The genetic relatedness of ESBL-producing strains was assessed using the ERIC-PCR approach. Fitty-four isolates were confirmed as ESBL producers, all classified as multidrug-resistant (MDR). All ESBL-producing K. pneumoniae isolates exhibited resistance to ampicillin and cefotaxime, with high resistance rates for ciprofloxacin (98.2%), azithromycin (94.4%), piperacillin-tazobactam (88.9%), and trimethoprim (83.3%). Genotypic analysis revealed blaCTX-M was present in 94.4% of isolates, blaSHV in 87%, and blaTEM in 55.5%. The blaCTX-M-1 group was the most prevalent, accounting for 96.1% of isolates. Co-harboring of blaCTX-M, blaSHV, and blaTEM occurred in 42.6% of isolates, with co-carrying of blaCTX-M, and blaSHV was observed in 23/54 isolates. The ERIC-PCR analysis revealed 15 distinct types, indicating high genetic diversity. These findings highlight the urgent need for ongoing monitoring to control the spread of ESBL among K. pneumoniae and emphasize the importance of early detection and appropriate antibiotic selection for effectively treating infection caused by these pathogens.

Prevalence of plasmid-mediated quinolone resistance genes in extended-spectrum beta-lactamase producing Klebsiella pneumoniae isolates in northern Iran

Plasmid-mediated quinolone resistance (PMQR) in extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) contributes to treatment failures, extended hospital stays, and increased mortality percentages. We aimed to determine the prevalence of PMQR genes in ESBL-producing K. pneumoniae isolates from clinical samples in Babol, North of Iran region. This is the first study in this region to investigate this specific association. A total of 95 K. pneumoniae isolates were obtained from hospitalized patients with various clinical infections during March 2022 to February 2023. Disk diffusion and Combination disk method were performed to identification of antimicrobial resistance profiles and ESBL-producing strains. The presence of ESBL and PMQR genes among K. pneumoniae isolates was assessed using polymerase chain reaction (PCR) method. Of the isolates, 68 (71.57 %) were considered as ESBL-producers. The blaTEM, blaSHV and blaCTX-M genes were detected in 74.73 %, 57.89 %, and 41.05 % of K. pneumoniae isolates, respectively. Among the PMQR encoding genes, the highest and lowest frequency was associated to qepA (67.3 %) and qnrA (4.2 %), respectively. The frequency of qnrA, qnrB, qnrS, acc (6′)-Ib-cr, qepA, oqxA, and oqxB genes in 26 MDR-Kp isolates was 11.53 % (n; 3), 69.23 % (n; 18), 65.38 % (n; 17), 73.07 % (n; 19), 80.76 % (n; 21), 84.61 % (n; 22), and 76.92 % (n; 20), respectively. Our result revealed of the 68 ESBL gene-positive isolates, 60 (88.23 %) were positive for the PMQR gene. The co-occurrence of these genes within resistant isolates suggests potential linkage on mobile genetic elements such as plasmids. These findings highlight the significant burden of PMQR determinants in ESBL-producing K. pneumoniae and underscore the urgent need for effective control measures. Implementing robust antimicrobial stewardship programs and strengthening drug-resistance surveillance and control protocols are crucial to prevent the spread of resistant isolates.

Ventilator Associated Pneumonia in Tertiary Care Hospital, Maharajgunj, Kathmandu, Nepal

Introduction: Ventilator Associated Pneumonia (VAP) is the most common nosocomial infection among intensive care unit (ICU) patients and lack of much information in Nepal. So, the aim of this study was to determine prevalence and bacteriological profile of VAP with special reference to multi-drug resistant (MDR), Methicillin-resistant Staphylococcus aureus(MRSA), Metallo-β-Lactamase(MBL), Extended-Spectrum β-Lactamase(ESBL)-producing bacterial strains. Methods: A total 150 tracheal specimens were studied during June 2011 to May 2012 at Department of Microbiology, TUTH as described by American Society for Microbiology (ASM). Combination disk method was done for the detection of ESBL and MBL producing isolates. Results: Prevalence of VAP was found to be 34%. Acinetobactereal coaccticusbaumannii complex (44%) was the commonest isolate, followed by Klebsiellapneumoniae (22%), Pseudomonas aeruginosa (16%) and Staphylococcus aureus (12%). Among MDR Gram negative bacteria (GNB), 39% were MBL and 33% were ESBL-producers. All GNB (61) were sensitive to Polymyxin B and Colistinsulphate, whereas, 48% were found resistant to Carbapenems. Prevalence of MRSA was 75%, which were all sensitive to Vancomycin. Conclusion: High prevalence of VAP, MDR along with MRSA or ESBL or MBL producing strains was found in the study. Thus, suitable control measures must be adopted to cope up this alarming situation with genetic characterization.

Genotypic Investigation of Antibiotic Resistance: Presence of BlaOXA-48 Gene in Clinical Isolates of Pseudomonas aeruginosa

Background: Identifying antimicrobial resistance patterns and antibiotic resistance genes in clinical isolates of Pseudomonas aeruginosa is an important strategy for effectively combating infections. This study aimed to identify the antimicrobial resistance patterns and the presence of blaOXA-48 beta-lactamase gene in P. aeruginosa isolated from clinical samples. Methods: We collected 100 isolates of P. aeruginosa from different clinical samples, including urine, tracheal tube, blood, and burn wounds. The antimicrobial resistance patterns of the isolates were investigated using the disk diffusion method. Extended-spectrum beta-lactamase (ESBL) strains were analyzed phenotypically by the combined disk method. Furthermore, the presence of the blaOXA-48 gene in the isolates was assessed genotypically via molecular techniques. Results: The highest resistance of the isolates was detected for ceftazidime (89%) and ciprofloxacin (83%), whereas the highest sensitivity was detected for colistin (18%). The phenotypic method identified 68 (68%) ESBL-producing isolates among 100 isolates. In addition, blaOXA-48 gene was detected genotypically in 33 (48.5%) isolates among 68 ESBL-producer isolates. Moreover, a significant association was found between the presence of the blaOXA-48 gene and P. aeruginosa isolates’ resistance to cefepime, imipenem, and gentamicin (P<0.05). Conclusion: The study highlighted the significant prevalence of antimicrobial resistance in P. aeruginosa, particularly against ceftazidime and ciprofloxacin, although colistin remains largely effective. The association between the blaOXA-48 gene and resistance to key antibiotics such as cefepime, imipenem, and gentamicin underscores the need for continuous monitoring and strategic antimicrobial stewardship to mitigate the spread of resistant strains.

Detection of Carbapenem Resistance Using the Genotypic and Phenotypic Methods in Klebsiella pneumoniae

Aim: This study aimed to detect the carbapenem resistance of the Klebsiella pneumoniae strains, isolated from clinical specimens with genotypic and phenotypic methods. Material and Methods: A total of 87 Klebsiella pneumoniae strains whose carbapenem resistance was determined by disc diffusion method were included in the study. Carbapenemase was investigated using the combined disk method and polymerase chain reaction (PCR). Results: The evaluation of the PCR results demonstrated that OXA was detected in 60 (68.9%) samples, NDM was detected in 20 (22.9%), OXA + NDM in 5 (5.7%), and KPC was detected in 1 (1.1%) out of 87 clinical samples. Carbapenemase was not detected in one specimen with the PCR method. The results were found compatible with the combined disc test results for all isolates which were detected as only OXA, NDM, and KPC type carbapenemase positive. In 5 (5.7%) strains in which the co-existence of NDM and OXA type carbapenemases was detected by PCR, the combined disc method detected only OXA type carbapenemase. Conclusion: The combined disk method is inadequate in the presence of strains that have multiple carbapenemases, and also have OXA which is the most frequently detected carbapenemase in our hospital. EUCAST recommends verification by other methods in the presence of OXA-48. Genotypic methods can be used for confirmation testing. The detections of strains with NDM, multiple carbapenemases, and the first detection of KPC were striking in the study. Monitoring the spread of these strains in the hospital will be necessary for infection control.

Molecular Analysis of Antibiotic Resistance in CarbapenemResistant Pseudomonas aeruginosa Isolates Isolated from Clinical Samples

Pseudomonas aeruginosa is an opportunistic pathogen that causes increased morbidity and mortality in risky patient groups. Nowadays, carbapenem resistance has become a threat and resistance genes are spreading among species through mobile genetic elements. The dissemination of carbapenemases among P.aeruginosa is a serious public health concern due to its limited options for the treatment of bacterial infections. In this study, it was aimed to investigate the molecular epidemiology of 47 carbapenem resistant P.aeruginosa (CRPA) isolates derived from various clinical samples from the Central Laboratory Bacteriology Unit of Kocaeli University Research and Training Hospital between October 2021 and March 2023. The rates of resistance to the antibiotics, some carbapenemase and virulence genes, conjugative resistance plasmids, integron gene cassette contents and the clonal similarity of the isolates were investigated and then epidemiologically evaluated. In the study, identification of the bacterial isolates and their susceptibility to some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were determined by the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of the isolates was demonstrated by the imipenem/meropenem-EDTA (IMP/MEM-EDTA) combined disc method. Conjugation experiments were performed by the broth mating method. Alkali lysis method was used in plasmid DNA isolations. Co-transferred antibiotic resistances in transconjugants were detected by disc diffusion method. Carbapenemase genes (blaIMP , blaVIM , blaNDM , blaKPC and blaOXA-48 ), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were screened by specific polymerase chain reactions (PCRs). Clonal relationships of the CRPA isolates were investigated by evaluating the DNA f ingerprintings obtained from the ERIC (enterobacterial repetitive intergenic consensus)-PCR assay. The highest resistance rate of the isolates were to levofloxacin, while the lowest resistance rates were observed against tobramycin, gentamicin and amikacin. MBL production was detected in 25 (53.2%) isolates. In conjugation experiments, 12 (25.5%) isolates were detected to harbour conjugative resistance plasmids. In 90% of the CRPA isolates, lasR and rhlR biofilm genes (encoding for the transcriptional activator protein) were detected by PCR. The blaVIM gene was detected in six (12.8%) isolates. The blaNDM gene was detected in five (10.6%) isolates and the blaOXA-48 gene was detected in three (6.4%) isolates. The blaKPC and blaIMP genes were not detected in CRPA isolates. It was determined that two (16.6%) of the isolates that carried the blaVIM gene, one (8.3%) carried the blaNDM gene and one (8.3%) carried the blaOXA-48 gene contained conjugative plasmids.In integron-specific PCRs, intI1 gene was positive in 39 (82.9%) isolates, while class 1 integron gene cassettes were detected in 24 isolates (51%). IntI1 positive six isolates were found to harbour class 1 integron gene cassettes-bearing conjugative plasmids. Class 2 integrons were not found in the CRPA isolates. Dendrogram analysis of ERIC-PCR patterns showed that there was no clonal similarity between the CRPA isolates and the isolates did not spread by cross-contamination. As a result, it has been observed that most of the CRPA isolates which have the potential to form biofilms, are highly resistant to other antibiotic groups other than carbapenems and can co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains which have the potential to cause epidemics in the clinical arena.

Distribution of antimicrobial resistance and virulence markers in chicken originated Proteusmirabilis isolates.

Proteus mirabilis is a common enteric bacterium in livestock and humans. The increase and spread of the antimicrobial resistant P. mirabilis is considered alarming worldwide. Transmission mainly occurs through consumption of contaminated poultry products. We investigated antimicrobial resistance (AMR) and virulence markers in broiler chicken-originated P. mirabilis isolates from 380 fecal samples. Phenotypic AMR test was performed against seventeen different antimicrobials. Genotypic AMR test was performed to detect sixteen different AMR genes. The samples were also tested for the presence of eight different virulence genes and biofilm formation. P. mirabilis was isolated in 11% of the samples, with significantly high multidrug-resistant (MDR) prevalence (63%). All isolates were resistant to tetracycline (100%). The combined disc method indicated that all isolates were of extended-spectrum beta-lactamase (ESBL) producers, which was compatible with the high blaTEM prevalence (95%). This was associated with blaTEM being responsible for more than 80% of ampicillin resistance in enteric pathogens. The absence of phenotypically carbapenem-resistant isolates was compatible with the very low prevalences of blaOXA (2%) and blaNDM (0%). All isolates were positive for pmfA, atfA, hpmA, and zapA (100%) virulence genes, while biofilm formation rate (85%) indicated high adherence abilities of the isolates.

Molecular analysis of ESBL- and AmpC-producing Enterobacteriaceae in fecal samples from broiler and backyard chickens

ObjectiveTo analyse the molecular profile of antibiotic resistance in Enterobacteriaceae isolates from fecal samples of healthy broilers and backyard chickens in Tamil Nadu, India. MethodsA total of 115 and 57 bacterial strains were isolated from fecal samples of broiler and backyard chickens, respectively using conventional bacterial culture tests. Bacterial isolates were screened for antibiotic susceptibility by Kirby-Bauer’s disk diffusion method. Colistin susceptibility was tested using the broth microdilution method. Extended-spectrum β-lactamase (ESBL), AmpC β-lactamase and metallo-β-lactamase (MBL) producers were screened by combination disk method. The drug-resistant genes’ positivity was analysed by polymerase chain reaction (PCR) method. ResultsEscherichia coli was the predominant bacterium isolated from both broiler (53%) and backyard (64.9%) chickens. Bacterial isolates from broilers and backyard chickens showed 100% resistance to ampicillin, piperacillin, and piperacillin-tazobactam. Five bacterial isolates from broiler chickens were resistant to colistin. ESBL producers were 34.7% in broilers and 19.1% in backyard chickens. AmpC production was detected in 44.3% of the isolates from broilers and 26.2% of those from backyard chickens. Molecular analysis revealed that bacterial isolates from broiler chickens were positive for drug-resistant genes such as blaTEM (25%), blaSHV (5%), blaCTX-M (4%), blaAmpC (56%), integron (39%), sul1 (30%), and sul2 (23%). Bacterial isolates from backyard chickens were positive for the blaTEM (22%), blaSHV (14%), blaAmpC (54.3%), and integron (20%) genes. None of the isolates from either broiler or backyard chickens harboured New Delhi metallo-β-lactamase (blaNDM) or colistin-resistant genes (mcr-1 to −3). ConclusionsBacterial isolates from broiler chickens harboured a higher number of drug resistance genes, especially blaTEM, blaCTX-M, sul1, sul2, and integron, than those from backyard chickens.

Prevalence of drug resistant Enterobacteriaceae in a Nepalese tertiary care hospital.

Antimicrobial resistance in Enterobacteriaceae is an emerging global public health problem. Numerous studies have reported community-acquired AmpC beta-lactamase and extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in Nepal. However, there are limited data on community-acquired Metallo-beta-lactamase (MBL) producing Enterobacteriaceae. A hospital-based descriptive cross-sectional study was conducted using 294 Enterobacteriaceae isolates from a total of 2,345 different clinical specimens collected from patients attending a tertiary care hospital in Nepal. Bacteria were isolated using standard microbiological growth media and identified using biochemical tests. For antimicrobial susceptibility testing, Kirby-Bauer disc diffusion technique was used. AmpC, ESBL, and MBL productions were detected by using combined disc method. AmpC, ESBL, and MBL productions were detected in 19.4%, 29.6%, and 8.5% of total Enterobacteriaceae isolates respectively. Higher rates of beta-lactamases production were seen among the isolates from in-patients in comparison with those from out-patients. However, 11.6%, 25%, and 3.7% of the total isolates from out-patients were AmpC, ESBL, and MBL producers respectively. The co-production of the beta-lactamases was also detected, with two Klebsiella pneumoniae isolates producing all three beta-lactamases. One MBL producing Proteus vulgaris isolate that was pan-resistant with no remaining treatment options was also isolated. Prevalence of drug resistant Enterobacteriaceae in our study was very high. Detection of AmpC, ESBL, and MBL positive isolates from out-patients, who did not have recent history of hospital visit, indicated the community dissemination of the drug resistant bacteria. This is a matter of great concern and an immediate attention to formulate strategies to prevent further development and spread of antibiotic resistance is required.

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.

Extended-Spectrum β-Lactamase and carbapenemase-producing Escherichia coli O157:H7 among diarrheic patients in Shashemene, Ethiopia.

The worldwide increase in multidrug resistance is a major threat to public health. One particular concern is the presence of Escherichia coli strains that carry Extended-Spectrum β-Lactamase (ESBL) and Carbapenemase enzymes, which can make multiple antibiotics ineffective. This complicates treatment strategies and raises the risk of illness and death. The aim of this study was to isolate E. coli O157:H7, assess its susceptibility against antimicrobial agents, and determine the presence of ESBL and Carbapenemase production in stool samples collected from diarrheic patients in Shashemene, west Arsi, Ethiopia from July to November 2022. The samples were cultured McConkey Agar and E. coli were isolated and identified by standard biochemical tests using API 20E. E. coli O157:H7 was further identified using sorbitol McConkey Agar and antisera for O157 antigen test. The antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method using different antibiotics. Each identified isolate was screened and tested for phenotypical ESBL and Carbapenemase production using combined disc method and modified carbapenem inactivation method, respectively. Bivariant and multivariant analyses were employed using a logistic regression model for further analysis and were interpreted based on the odds ratio and level of statistical significance at a p-value <0.05 with 95% confidence interval. E. coli O157:H7 strain was found from 9% (38/423) study participants. The majority of the participants [61.9% (262/423)] were males; and 19.1% (81/ 423) of the participants were under five children. Living in urban areas, having domestic animals, and ≥5 family size in the household were identified as statistically significant factors associated with E. coli O157:H7. Twenty-seven (71.1%) and 12 (31.6%) of the 38 E. coli O157:H7 isolates were phenotypically confirmed to be ESBL and carbapenemase producers, respectively. All isolates were resistant against Ampicillin, but sensitive to ciprofloxacin. High resistance to Ampicillin and Amoxicillin/Clavulanic acid was observed among the ESBL and carbapenemase producing isolates also. The extent of detection of multidrug resistant E. coli O157:H7 isolates against three or more classes of antimicrobial agents tested was alarmingly very high (84%). The E. coli O157:H7 isolates in this study showed a significant resistance to certain antimicrobials that were tested. The level of ESBL and Carbapenemase production among these isolates was found to be quite high. We observed a high resistance to Ampicillin and Amoxicillin/Clavulanic acid among the ESBL and carbapenemase producing isolates. Ciprofloxacin was found to be the most effective drug against both the ESBL producers and nonproducers.

Extended Spectrum Beta Lactamase Profile of Citrobacter Species Isolated from Various Samples in the North East Coast of Tamil Nadu, India: A Cross-sectional Study

Introduction: Citrobacter species play a significant role in causing hospital-acquired infections, especially those affecting the urinary and respiratory systems, and they are commonly present in faeces. Healthcare systems are facing a major challenge due to the rise of antibiotic-resistant Gram-negative infections resistant to antibiotics. Aim: To identify the genotypic characterisation of the resistant gene blaTEM-1 in Citrobacter spp. obtained from a range of clinical samples. This investigation seeks to provide insights into the patterns of antibiotic resistance exhibited by these bacteria. Materials and Methods: This cross-sectional study was conducted between August 2021 and February 2022. Preliminary examinations included screening as well as confirmatory tests for Extended Spectrum Beta Lactamase (ESBL) production, which were confirmed by the combination disk method and double disc synergy test. Genotypic detection of the blaTEM-1 gene was done using the Polymerase Chain Reaction (PCR) technique. Demographic parameters like gender and age were assessed. Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) Software version 21.0. Results: Citrobacter koseri (66 isolates, 54.5%) was found to be the predominant species among the Citrobacter spp., followed by Citrobacter freundii (55 isolates, 45.5%). Urine samples were the primary source of Citrobacter spp. isolates, comprising 52 isolates (43%) of the total. In this study, the prevalence of ESBL producers was reported to be 49 isolates (77.8%) identified as ESBL producers. Tigecycline demonstrated a 100% effectiveness rate, followed by Piperacillin-Tazobactam (96% sensitivity) and Amikacin (95% sensitivity). Out of the 20 Citrobacter spp. isolates that tested positive for ESBL, 15 isolates (75%) were found to be positive for blaTEM-1. Conclusion: The results of this study provide insight into the changing patterns of antibiotic resistance in Citrobacter spp., with particular emphasis on ESBL profiles. Thus, it is imperative to implement intervention techniques to reduce the ongoing selection and spread of these more resistant bacteria, as well as efficient infection-control measures to manage epidemics. These results advance the comprehension of antibiotic resistance patterns and guide initiatives to combat the increasing threat of antibiotic resistance.

BlaCTX-M Mediated Extended Spectrum Beta-lactamase Production in Escherichia coli from Urine Samples

Objective: To assess the antimicrobial pattern of Escherichia coli isolates from urine samples, determine the proportion of multidrug resistant (MDR) and extended spectrum beta-lactamase (ESBL) and blaCTX-M gene of E. coli from the urine samples. Methods: A total of 2452 urine samples were collected and processed from April to October 2023 in clinically suspected urinary tract infection (UTI) patients attending B&amp;B Hospital Pvt. Ltd to identify possible bacteria using standard microbiological procedures. E coli isolates were further subjected to antimicrobial susceptibility testing using Kirby Bauer disk diffusion method. Combination disk method was used to identify the ESBL phenotypes. Molecular detection of blaCTX-M gene was performed using conventional polymerase chain reaction. Results: A total of 338 (13.7%) samples showed significant bacterial growth. Maximum bacterial growth was found in outpatients (70.4%) and in patients above 60 years of age. Gram negative bacteria (87.8%) were predominant and E. coli (54.4%) was the most frequent. Nitrofurantoin, Aminoglycosides, Carbapenems and Polymixins were effective antibiotics. Among 184 E. coli isolates, 54 (29%) were MDR, 30 (16.3%) were ESBL producer. blaCTX-M gene was detected in 90% of the phenotypically confirmed ESBL producers. Conclusion: E. coli is the major cause of UTI and blaCTX-M is a prime contributor of ESBL production. High rate of resistance to third-generation Cephalosporins, Flouroquinolones and Cotrimoxazole make these antibiotics unsuitable for the treatment of UTI.

Examining the Frequency of bla CTX-M and bla TEM Genes in Escherichia Coli Isolates with Multiple Drug Resistance Isolated from Patients Hospitalized in Ahvaz University Hospitals

Background and Objectives In the last few decades, Escherichia coli strains producing extended-spectrum beta-lactamases (ESBLs) have increased and are now considered a major problem in hospitalized patients. The aim of this study was to investigate the prevalence of blaCTX-M and blaTEM genes in Escherichia coli with multiple drug resistance. Subjects and Methods This study was conducted from October 2021 to May 2022. The research site included the microbiology laboratories of Golestan, Imam Khomeini, and Shahid Beqai hospitals where isolates suspected of Escherichia coli were isolated from urine culture samples of patients with urinary tract infection. All Escherichia coli samples were identified by conventional biochemical and molecular methods. The antibiotic sensitivity of the isolates was determined by disk diffusion method. Then, in order to finally confirm the presence of blaCTX-M and blaTE genes, PCR method was used. Results Out of the 160 bacterial isolates examined, 138 (86.3%) showed multidrug resistance (MDR). The highest and lowest percentage of resistance was related to Amoxicillin-Clavonic acid (84.1%) and Meropenem (5.8%), respectively. Based on the combined disk method, 96 isolates (69.6%) were positive for Beta-lactamase enzymes (ESBLs). Among the 96 ESBL-positive isolates, 85 (88.5%) and 38 (39.6%) carried blaCTX-M and blaTEM genes, respectively. Conclusion Given the significant prevalence of Escherichia coli strains that are resistant to multiple drugs and carrying ESBLs beta-lactamase genes in hospital, there is a need for effective surveillance measures to prevent the further spread of drug resistance in these isolates.

Inhibition and Reduction of Biofilm Production along with Their Antibiogram Pattern among Gram-Negative Clinical Isolates.

Bacterial biofilm is a significant virulence factor threatening patients, leading to chronic infections and economic burdens. Therefore, it is crucial to identify biofilm production, its inhibition, and reduction. In this study, we investigated biofilm production among Gram-negative isolates and assessed the inhibitory and reduction potential of ethylene diamine tetra acetic acid (EDTA) and dimethyl sulfoxide (DMSO) towards them. In addition, we studied the antimicrobial resistance pattern of the Gram-negative isolates. Bacterial isolation and identification was done using standard microbiological techniques, following the Clinical and Laboratory Standards Institute (CLSI) guideline, 28th edition. The Kirby-Bauer disk diffusion method was used to determine the antibiotic susceptibility pattern of the isolates, and β-lactamase production was tested via the combination disk method. Biofilm formation was detected through the tissue culture plate (TCP) method. Different concentrations of EDTA and DMSO were used to determine their inhibitory and reduction properties against the biofilm. Both inhibition and reduction by the various concentrations of EDTA and DMSO were analyzed using paired t-tests. Among the 110 clinical isolates, 61.8% (68) were found to be multidrug resistant (MDR). 30% (33/110) of the isolates were extended-spectrum β-lactamase (ESBL) producers, 14.5% (16/110) were metallo-β-lactamase (MBL), and 8% (9/110) were Klebsiella pneumoniae carbapenemase (KPC) producers. Biofilm formation was detected in 35.4% of the isolates. Biofilm-producing organisms showed the highest resistance to antibiotics such as cephalosporins, chloramphenicol, gentamicin, and carbapenem. The inhibition and reduction of biofilm were significantly lower (p < 0.05) for 1 mM of EDTA and 2% of DMSO. Isolates forming biofilm had a higher resistance rate and β-lactamase production compared to biofilm nonproducers. EDTA and DMSO were found to be potential antibiofilm agents. Hence, EDTA and DMSO might be an effective antibiofilm agent to control biofilm-associated infections.

Detection of extended-spectrum beta-lactamases and carbapenemases in clinical isolates of Stenotrophomonas maltophilia in the northeast of Iran

PurposeStenotrophomonas maltophilia has emerged as an important pathogen associated with significant mortality in pneumonia and bacteremia of severely immunocompromised, hospitalized patients. The main objectives of the present study are to describe the surveillance of antibiotic resistance and to detect the genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemase enzymes in clinical S. maltophilia isolates from blood cultures in the northeast of Iran. MethodsA total of 114 clinical human isolates of S. maltophilia were collected from blood cultures of patients admitted at three teaching hospitals in Mashhad, northeast of Iran. Antimicrobial susceptibility was determined by the Kirby-Bauer disk diffusion test. The extended-spectrum beta-lactamase (ESBL) production was confirmed by the combination disk method. The PCR was applied for molecular epidemiology of ESBLs and carbapenemase coding genes. ResultsOverall, 10 % and 8 % of isolates were resistant to co-trimoxazole and levofloxacin, respectively. The prevalence of the blaTEM, blaSHV, and blaCTX-M-15 genes in the 69 ESBL-producing isolates was 2.9 %, 5.8 %, and 10.14 %, respectively. Moreover, the prevalence of the blaKPC-1, blaIMP, blaVIM, blaNDM, and blaOXA-48 genes was 18.42 % (n = 21), 4.39 % (n = 5), 6.14 % (n = 7), 15.79 % (n = 18), and 14.91 % (n = 17), respectively. ConclusionFor the first time in Iran, ESBLs and carbapenemase coding genes have been identified in human clinical strains of S. maltophilia. Circulation of these resistant strains with transferable genes is a sign of flow of resistome genes from other bacterial genera to S. maltophilia, most probably leading to the evolution of the superbugs of this bacterium.

Evaluating the incidence of ampC-lactamase genes, biofilm formation, and antibiotic resistance among hypervirulent and classical Klebsiella pneumoniae strains.

Both immunocompetent and healthy individuals can become life-threateningly ill when exposed to the hypervirulent (hvKp) strains of Klebsiella pneumoniae (Kp). The main objectives of this study were to evaluate the presence of ampC-lactamase genes, biofilm formation, and antibiotic resistance in clinical strains of hvKp and cKp (classical K. pneumoniae). Kp strains were collected from patients referred to Shahidzadeh Hospital in Behbahan city, Khuzestan Province, Iran. Several techniques were used to identify hvKp. The hypermucoviscosity phenotype was determined using the string test. Isolates that developed dark colonies on tellurite agar were assumed to be hvKp strains. If any of the iucA, iutA, or peg-344 genes were detected, the isolates were classified as hvKp. Phenotypic and genotypic detection of AmpC β-lactamases of hvKp strains was performed by combined disk method and PCR, respectively. In addition, crystal violet staining was used to determine the biofilm formation of these isolates. For this study, 76 non-duplicative isolates of Kp were collected. Overall, 22 (28.94%) strains had positive string test results, and 31 (40.78%) isolates were grown in tellurite-containing medium. The genes iucA and iutA or peg-344 were found in 23.68% of all Kp strains and in 50% of tellurite-resistant isolates, respectively. The most effective antibiotics against hvKp isolates were tetracycline (85.52%) and chloramphenicol (63.15%). Using the cefoxitin disc diffusion method, we observed that 56.57% (43/76) of the strains were AmpC producer. A total of 30.26% (n=23/76) of the isolates tested positive for at least one ampC gene, including blaDHA (52.63%, n=40), blaCIT (40.78%, n=31), blaACC (19.76%, n=15), blaMOX (25%, n=19), and blaFOX (43.42%, n=33). Biofilm formation analysis revealed that most hvKp isolates were weak (n=6, 40%) and moderate (n=5, 33.33%) biofilm producers. Healthcare practitioners should consider the possibility of the existence and acquisition of hvKp everywhere. The exact mechanisms of bacterial acquisition are also unknown, and it is unclear whether the occurrence of infections is related to healthcare or not. Thus, there are still many questions about hvKp that need to be investigated.

Integration of phenotypic, qPCR and genome sequencing methodologies for the detection of antimicrobial resistance and virulence in clinical isolates of a tertiary hospital, India.

The emergence of antimicrobial resistance (AMR) and virulence in clinical isolates is a significant public health concern. The rapid and accurate detection of these traits in clinical isolates is essential for effective infection control and treatment. We demonstrated the integration of multiple detection methodologies, including phenotypic testing, quantitative polymerase chain reaction (qPCR), and genome sequencing, to detect AMR and virulence in clinical isolates. One hundred sixty-two gram-negative bacterial clinical isolates were selected for this study from the Shri Vinoba Bhave Civil Hospital, Silvassa, a tertiary government hospital. Antimicrobial susceptibility was detected by determining the Minimum Inhibitory Concentration (MIC) using Vitek-2, whereas the combined disk (CD) method was used for phenotypic detection of carbapenemase activity. The highest sensitivity rates were obtained for antibiotics colistin 87.93%, amikacin 67.52%, tigecycline 63.39%, nitrofurantoin 60.87%, and gentamycin 56.08%. The most resistant antibiotics were ceftazidime (71.93%), ciprofloxacin (67.95%) and trimethoprim/sulfamethoxazole (65.56%). Approximately 46.91% (76) of all the isolates were MBL isolates. The qPCR results confirmed the presence of blaNDM-1 in 29.01% of the isolates. The blaNDM-1 harbouring isolates in descending order, were Acinetobacter, Enterobacter cloacae, and Klebsiella pneumoniae. Klebsiella and Acinetobacter isolates were extensively drug-resistant. Whole genome sequencing performed on one of the Klebsiella pneumoniae isolates revealed the presence of many virulence factors, which increased the pathogenicity of the clinical isolates. The results showed that antimicrobial resistance, including carbapenem resistance, blaNDM-1, and virulence factors, was highly prevalent among isolates from tertiary clinical hospitals. The integration of multiple detection methodologies can potentially improve the detection of AMR and virulence in clinical isolates, leading to better patient outcomes and a reduced spread of these essential traits.

Genotypic Investigation of Antibiotic Resistant blaOXA-4 Gene in Clinical Isolates of Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, is responsible for 10-15% of hospital infections worldwide. The acquisition of resistance genes is one of the important mechanisms that causes the spread of resistance in this bacterium. This study aimed to conduct a phenotypic and genotypic investigation of the blaOXA-4 resistance gene in P. aeruginosa isolated from clinical samples. Methods: In this study, 110 P. aeruginosa strains were isolated from various clinical samples. The disk diffusion method was applied to reveal the resistance pattern in the isolates. Moreover, the combined disk method was used for the phenotypic analysis of extended-spectrum beta-lactamases (ESBL). Finally, the presence of the blaOXA-4 beta-lactamase gene was analyzed genotypically by polymerase chain reactions (PCR) method. Results: The highest sensitivity and resistance of the isolates were related to amikacin (65.45%) and ceftazidime (86.36%), respectively. The phenotypic analysis indicated that 72 isolates (65.45%) of P. aeruginosa are ESBL-producing. Furthermore, the presence of blaOXA-4 was approved genotypically in 33 P. aeruginosa isolates (45.83%). Conclusion: This study revealed a high prevalence of antibiotic-resistant isolates of P. aeruginosa in the East Azerbaijan population that may be associated with the presence of the blaOXA-4 gene. However, further studies are necessary to identify other resistant genes in ESBL-producing isolates and other geographical areas with larger sample size.