Characterisation of plasmids harbouring qnrA1, qnrS1, and qnrB4 in E. coli isolated in the Philippines from food-producing animals and their products.

OBJECTIVES: Determinants showing plasmid-mediated quinolone resistance, which usually leads to antimicrobial ineffectiveness, have become an emerging clinical problem. In our previous study in the Philippines, a high prevalence of qnr determinants was found in clinical samples and food-producing animals and their food products. However, no qnr-carrying plasmids have been investigated in animals or animal-derived foods. Hence, in the present, we aimed to characterise qnr-carrying plasmids in Escherichia coli isolated from the food supply chain. METHODS: Plasmids from 44 qnr-positive isolates were assigned to incompatibility groups by Polymerase chain reaction (PCR)-based replicon typing, and the presence of ß-lactamase-encoding genes were investigated by PCR. Localisation of qnr in plasmids was determined by S1-PFGE and Southern blot hybridisation. The transferability of qnr-carrying plasmids was examined by conjugation analysis. RESULTS: Overall, 77.3% (95% confidence interval [CI]: 62.2-88.5) of the isolates harbouring qnr determinants were positive for seven plasmid types, and 56.8% concurrently harboured blaTEM-1. Plasmid IncFrepB was prevalent (65.9% [95% CI: 50.1-79.5]) among qnr determinants. Localisation of qnr determinants in IncFrepB and transferability of plasmids was further confirmed. CONCLUSION: The current study proved that qnr in E. coli isolated from food-producing animals and their food products could spread via plasmid IncFrepB upon selective pressure with quinolones or other antimicrobials. Therefore, to curb the emergence and spread of qnr-harbouring bacteria in the Philippines, prudent use of antimicrobials in animal production and stricter hygiene and food handling are recommended.

Genetic characterization of coliform bacterial isolates from environmental water in Thailand.

INTRODUCTION: In contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. METHODS: Coliform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. RESULTS: In 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with ß-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms' wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. CONCLUSIONS: Results obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

Quinolone Resistance Determinants of Clinical Salmonella Enteritidis in Thailand.

Salmonella Enteritidis has emerged as a global concern regarding quinolone resistance and invasive potential. Although quinolone-resistant S. Enteritidis has been observed with high frequency in Thailand, information on the mechanism of resistance acquisition is limited. To elucidate the mechanism, a total of 158 clinical isolates of nalidixic acid (NAL)-resistant S. Enteritidis were collected throughout Thailand, and the quinolone resistance determinants were investigated in the context of resistance levels to NAL, norfloxacin (NOR), and ciprofloxacin (CIP). The analysis of point mutations in type II topoisomerase genes and the detection of plasmid-mediated quinolone resistance genes showed that all but two harbored a gyrA mutation, the qnrS1 gene, or both. The most commonly affected codon in mutant gyrA was 87, followed by 83. Double codon mutation in gyrA was found in an isolate with high-level resistance to NAL, NOR, and CIP. A new mutation causing serine to isoleucine substitution at codon 83 was identified in eight isolates. In addition to eighteen qnrS1-carrying isolates showing nontypical quinolone resistance, one carrying both the qnrS1 gene and a gyrA mutation also showed a high level of resistance. Genotyping by multilocus variable number of tandem repeat analysis suggested a possible clonal expansion of NAL-resistant strains nationwide. Our data suggested that NAL-resistant isolates with single quinolone resistance determinant may potentially become fluoroquinolone resistant by acquiring secondary determinants. Restricted therapeutic and farming usage of quinolones is strongly recommended to prevent the emergence of fluoroquinolone-resistant isolates.