Identification of Antimicrobial Resistant (AMR) Gene in Escherichia coli and Staphylococcus aureus isolated from bovine milk sample at Chattogram Metropolitan Area

Abstract

Antimicrobial resistance (AMR) is known as a silent pandemic; an emerging global public health issue. The problem of AMR is similarly salient and prevalent in animals. From a public health perspective, AMR in dairy cattle can also jeopardize human health by the potential dissemination of AMR pathogens to humans via consumption of infected dairy products or direct contact with infected dairy cattle. Harshly, the dairy industry is increasingly confronted with AMR Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). This study was designed to investigate the multidrug- resistant pattern along with the most frequently isolated resistant genes of S. aureus and E. coli from bovine milk in the south-eastern part of Bangladesh. A total of 175 samples were randomly collected from ten dairy farms. Isolation and identification of S. aureus and E. coli were done following the standard bacteriological method. The spa gene and 16s rRNA gene were tabbed in molecular identification ofS. aureus and E. coli, respectively. In this study 26 (14.86%; 95% CI 10% - 21%) isolates were confirmed as S. aureus whereas 20 (11.43%; 95% CI 7.12% - 17.10%) were confirmed as E. coli based on the PCR assay. Among the positive S. aureus isolates, 22 (84.6%; 95% CI 65.1%-95.6%) were characterized with the presence of AMR genes and 17 (85%; 95% CI 62.1%-96.8%) E. coli isolates were characterized with the presence of AMR genes. Afterward, the positive isolates were screened against 15 antimicrobials using the disc diffusion technique. At a fleeting glance, the highest number of S. aureus (80.7%, 95% CI 60.6-93.4) isolates were resistant to streptomycin and all the S. aureus (100%) were sensitive to meropenem. Meanwhile, all the E. coli isolates were resistant to ciprofloxacin whereas 95% (95% CI 75.1% - 99.9%) showed sensitivity to meropenem and 80% (95% CI 56.3%-94.2%) to gentamicin. Among the 26 S. aureus, 5 isolates (19.2%) were positive for the mecA gene. tetK gene was identified among the 19 (73.1%) S. aureus isolates and belonged to the highest proportion. On the other hand, the tetM gene was found within the 2 isolates showing the least percentage. In case of E. coli, the ciprofloxacin-resistant gene gyrA, was identified among the 19 (95%) E. coli isolates and it was the highest proportion. No colistin resistant gene was detected in isolated E. coli. Fifty percent of the E. coli isolates represented sulphamethoxazole-trimethoprim resistant gene sul1 and sul2. Similarly, 10 (50%) E. coli isolates were positive to ampicillin resistant gene blaTEM and 30% were blaCTX-M, resistant to ceftriaxone. xi | P a g e From the univariable logistic regression analysis, it was evident that the odds of having AMR S. aureus was 2.41 (95% CI: 0.96-6.07) times higher in the farms where the antibiotic course was not completed properly. Furthermore, the odds of having AMRE.coli in bovine raw milk was almost 23 (95% CI: 6.09-84.25; P=<0.0001) times higher in the dairy farms avoiding the practice of antibiotic course completion, 12 (95% CI: 3.31-44.12) times higher where withdrawal period was not followed, 18.38 (95% CI: 4.98-67.82) times attributed when the farms avoided proper dose maintenance and 7.62 (95% CI: 2.10-27.63; P=0.002) times greater in those dairy farms having the farm personnel without the knowledge of AMR. However, multivariable logistic regression model showed no significant association might be due to low observation number. In case of presence of S. aureus in the sample, after adjustment of the confounding effect of the factors in the multivariable logistic regression model, a potential risk factor (CMT result) was identified in this study. The odds of the presence of S. aureus in the CMT test ‘positive’ samples was 10.3 times (95% CI: 2.6-40.5) higher than the CMT test ‘negative’ samples. In the case of E. coli, the multivariable analysis showed that the presence of E. coli increased by 13.7 times if the floor had been contaminated with mud and cow dung during the milking time and exclusively a hike odds found in case of the dirty udder of cows, as well. Conclusively, the study findings will provide ample statistical evidence to develop strategies for improved antimicrobial stewardship, rejuvenate the antimicrobial drug channel and to develop efficacious and sustainable alternative approaches to tackling AMR crisis both in humans and livestock.