Whole genome sequencing analysis of antimicrobial resistant Escherichia coli : from food to human

Abstract

The main work of this study is to analyze antimicrobial resistant E. coli based on whole genome sequencing data. Three stages were included for isolates from different sources (ready-to-eat food, retail raw meats and human patients).At the first stage, a retrospective study for antimicrobial resistant E. coli in readyto-eat (RTE) food sold in retail food premises in Singapore was performed in collaboration with Environmental Health Institute under NEA. A total of 99 E. coli isolates from poultry-based dishes (n=77) and fish-based dishes (n=22) were obtained between 2009 and 2014 during the surveillance project. All the isolates were included for disk diffusion testing for antimicrobial susceptibility testing.Of the 99 isolates, 24 (24.2%) were resistant to at least one antimicrobial agent. These isolates were then subjected to broth microdilution testing against 33 antimicrobial agents, including β-lactams, aminoglycosides, tetracycline, fluoroquinolones and polymyxin E (colistin), to determine the minimum inhibitory concentration (MIC) of isolates. Whole genome sequence (WGS) was carried out on the strains in order to correlate resistant phenotypes to putative antimicrobial resistance-related genes. Of the 24 isolates, 15 (62.5%) were found to be resistant to three or more classes of antimicrobials and thus were defined as multi-drug resistant strains. Two isolates (8.3%) were confirmed as Extended-Spectrum β-lactamase (ESBL)-producing E. coli by double disk synergy test.Based on WGS data, online analysis tool ResFinder detected 7 classes of antimicrobial resistance genes and resistance-related chromosomal point mutations in 19 of the 24 E. coli isolates. By analyzing the WGS contigs using BLASTn and KmerFinder, ESBL genes and transferable colistin resistance gene mcr-1 (2/24) and mcr-5 (1/24) were determined to be located on plasmids, which could pose a greater risk of AMR transfer among bacteria. Mutations were detected in four isolates within genes previously shown to confer resistance to quinolones (gyrA and parE) and tetracycline (rrsB). Prediction of AMR using WGS data was evaluated for six antimicrobials including ampicillin, chloramphenicol, colistin, fluoroquinolones, tetracycline and trimethoprim. The evaluation indicates WGS-based genotype and phenotype showed high consistency, however, for some antimicrobial resistance whose mechanism is not totally clear yet (such as colistin), there is challenge for resistance gene detection.To have a better understanding of genetic environment and mobility of colistin resistance gene mcr-5.1, the isolates carrying mcr-5.1 were sequenced using long-read sequencing technology. The plasmid sequences were assembled into a closed circle using both long-read sequencing data and short-read sequencing data. The blasting result showed the closest plasmid sequence to pSGMCR103 in NCBI is plasmid pYD786-3 (accession number KU254580.1) with 77% query coverage and 99% identity, which was carried by one E. coli isolate from human urine in USA. They share antimicrobial resistanc...