Molecular characterization and antibiotic resistance profile of ESBL-producing Escherichia coli isolated from healthy cow raw milk in smallholder dairy farms in Bangladesh

Abstract

Background and Aim: The emergence of antimicrobial-resistant bacteria, such as Escherichia coli in milk, is a serious public health concern as milk is considered a complete food and an important part of daily human diet worldwide, including in Bangladesh. However, there have been no reports on the molecular characterization and antibiotic resistance profile of extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk of healthy cows in Bangladesh. Therefore, this study aimed to detect and characterize ESBL-producing E. coli (ESBL-Ec) in milk samples from healthy cows in smallholder dairy farms in Mymensingh district, Bangladesh, and assess the potential risk of consuming this milk. Materials and Methods: A total of 100 milk samples were collected from apparently healthy cows on smallholder dairy farms. Escherichia coli was isolated from the collected samples using standard methods. The detection of ESBL-Ec was performed phenotypically using cultural methods and genotypically by ESBL genetic determinants using multiplex polymerase chain reaction. Antimicrobial susceptibility testing of the ESBL-Ec isolates was performed using the disk diffusion method with 15 common antimicrobials. Results: In this study, out of the 100 samples tested, 70 (70%) were found to be positive for E. coli. Among these, 41 (58.6%) strains were identified as ESBL-producing, both phenotypically and genotypically, with the presence of blaCTX-M, blaTEM, and blaSHV individually or combined (blaCTX-M plus blaTEM plus blaSHV). The antibiogram of these ESBL-positive isolates revealed high resistance against commonly used antibiotics, such as ampicillin, cefotaxime, and gentamicin (100%), azithromycin (88%), oxytetracycline (27%), nalidixic acid, cotrimoxazole/trimethoprim (24%), and streptomycin (22%). In addition, one isolate showed resistance to 4th generation of cephalosporin (cefepime). Most importantly, extensive multidrug resistance was found in many ESBL-Ec isolates. However, the isolates were highly sensitive to drugs such as ceftriaxone (100%) and imipenem (100%). This is the first study to detect ESBL-Ec in raw milk from healthy cows on smallholder dairy farms in Bangladesh. Conclusion: More than 58% of the E. coli isolated from raw milk of healthy cows tested positive for ESBL production and showed resistance to most commonly used antimicrobials which may be alarming for human health. A limitation of our study is that we had a small size of sample collected from one district in Bangladesh. Therefore, a larger sample size covering a wider geographic area, and using multi-locus sequence typing and whole genome sequencing could provide a more comprehensive understanding of the prevalence and characteristics of ESBL-Ec in Bangladesh. Keywords: ampicillin, antibiogram, blaCTX-M, cefotaxime, multiplex PCR, multidrug resistance.