Acquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan

Abstract

This study focused on the detection of the plasmid-mediated mcr colistin resistance gene in Escherichia coli isolates from wastewater treatment plants (WWTPs). Seven influent samples were collected from three WWTPs in Nagano Prefecture, Japan, during August and December 2018. Colistin-resistant E. coli isolates were selected on colistin-supplemented CHROMagar ECC plates. mcr-1-positive isolates were subjected to whole-genome sequencing (WGS) analysis. From six influent samples, seven mcr-1-positive but extended-spectrum β-lactamase (ESBL)-negative isolates belonging to different genetic lineages, namely, B2-O25:H4-ST131-fimH22, B2-O2:H1-ST135-fimH2, B1-O8:H9-ST764-fimH32, B1-O23:H16-ST453-fimH31, A-O81:H27-ST10-fimH54, A-O16:H5-ST871-fimH25, and F-O11:H6-ST457-fimH145, were detected. The MICs of colistin for these isolates ranged from 4 to 16 mg/liter. The mcr-1 genes were located on plasmids belonging to IncX4 and IncI2 in five and two isolates, respectively. Four IncX4 plasmids with the same size (33,309 bp) showed high sequence similarity (4 single-nucleotide variations). The remaining one IncX4 plasmid, with a size of 33,858 bp, carried the mcr-1 gene with the single synonymous nucleic substitution T27C. Two IncI2 plasmids with sizes of 60,710 bp and 60,733 bp had high sequence similarity (99.9% identity; 100% query coverage). Two of five isolates carrying IncX4 plasmids and both of the isolates carrying IncI2 plasmids harbored ColV plasmids carrying virulence-associated genes of avian pathogenic E. coli (APEC). In addition, another isolate of the B2-O25:H4-ST131-fimH22 lineage had those APEC-associated virulence genes on its chromosome. In conclusion, mcr-1-positive E. coli environmental isolates were mostly characterized as positive for APEC-associated virulence genes. The copresence of those genes may suggest the existence of a common source in animals and/or their associated environments.IMPORTANCE Colistin is considered a last-line therapeutic option in severe infections due to multidrug-resistant Gram-negative bacteria, in particular carbapenemase-producing Enterobacteriaceae and multidrug-resistant Acinetobacter baumannii An increasing prevalence of mcr genes in diverse Enterobacteriaceae species, mainly Escherichia coli and Klebsiella pneumoniae from humans and food animals, has become a significant concern to public health all over the world. In Japan, mcr genes have so far been detected in food animals, raw meat, wastewater, and human clinical samples. This study reports the copresence of mcr-1 and avian pathogenic E. coli (APEC)-associated virulence genes in five of seven E. coli isolates recovered from aquatic environments in Japan. Our study highlights the importance and urgency of action to reduce environmental contamination by mcr genes that may likely occur due to exposure to untreated wastewater through combined sewer overflow by recent unusual weather.

Keywords: APEC; ColV; WWTPs; mcr-1.

FIG 1

Linear comparison of complete plasmid sequences of mcr-1-carrying plasmids. (a) Comparison of IncX4 plasmids pA1, pA2, pB1, pB2, and pC3 in this study with plasmids pRYU3223C-1 (GenBank accession number AP018411), pICBEC72Hmcr (GenBank accession number CP015977), pCSZ4 (GenBank accession number KX711706), pGZ49269 (GenBank accession number MG210939), and pMR0617mcr1 (GenBank accession number CP024462). (b) Comparison of IncI2 plasmids pC1 and pC2 in this study with plasmids pMRY15-131_2 (GenBank accession number AP017622), pRYU2912C-1 (GenBank accession number AP018412), p6383 (GenBank accession number MG594798), and pAF23 (GenBank accession number KX032519). The arrows show the translation orientation of the coding genes. The mcr-1 genes are indicated by black arrows.

FIG 2

Circular genetic maps of plasmids carrying APEC-associated virulence genes. pColV-C1 (GenBank accession number LC484363) harbored by E. coli C1 (A). The APEC-associated virulence genes cva/cvi, iroBCDEN, sitABCD, hlyF, ompT, and iss are shown in red, green, blue, yellow, pink, and orange, respectively. The tra/trb conjugation genes are marked in white. pColBM-B1 (GenBank accession number LC485173) harbored by E. coli B1 (B). The colicin B and M operon, including cba and cma genes, is shown in red. The tra/trb and pil conjugation genes are marked in white.