Genotypic and phenotypic characterization of antimicrobial resistant E.coli isolated from ready-to-eat food in Singapore based on disk diffusion, broth microdilution and whole genome sequencing

This study aimed to determine the antimicrobial resistance profile of fecal indicator Escherichia coli isolated from ready-to-eat food sold in hawker centers in Singapore. A total of 99 strains were isolated between 2009 and 2014 from poultry-based dishes (n=77) and fish-based dishes (n=22). The results from disk diffusion assay showed that 24 out of 99 Escherichia coli strains were resistant to at least one antimicrobial agent. Thereafter, the 24 isolates were subjected to further phenotypic (broth microdilution) and genotypic (whole genome sequencing (WGS)) characterization. Thirty-three antimicrobial agents (including classes of ß-lactem, aminoglycosides, tetracycline, fluoroquinolones, polymyxin and others) were used for the Minimum Inhibitory Concentration (MIC) testing, which showed that 15 isolates carried multi-resistance (defined as resistance to three or more classes of antimicrobials). Two isolates which were resistant to multiple ß-lactem antimicrobial agents were confirmed as Extended-Spectrum ß-lactem (ESBL)- producing bacteria by double disk synergy method. Based on WGS data, online analysis tool ResFinder detected 7 classes of antimicrobial resistance genes and resistance-related chromosomal point mutations. For most classes of antimicrobials, the AMR genotype showed high consistency with the phenotypes. By analyzing the contigs using using blastn and KmerFinder, ESBL genes and transferable colistin resistance gene mcr were determined to be located on plasmids, increasing the risk of AMR transfer. Four known gene mutations which may cause antimicrobial resistance were detected. This study shows significant level of AMR in contaminating E.coli isolates from ready-to-eat food, and raises the concern of AMR transfer from such food to human .