Acquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan (2019)

This study focused on the detection of the plasmid-mediated <i>mcr</i> colistin resistance gene in <i>Escherichia coli</i> 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 <i>E. coli</i> isolates were selected on colistin-supplemented CHROMagar ECC plates. <i>mcr-1</i>-positive isolates were subjected to whole-genome sequencing (WGS) analysis. From six influent samples, seven <i>mcr-1</i>-positive but extended-spectrum β-lactamase (ESBL)-negative isolates belonging to different genetic lineages, namely, B2-O25:H4-ST131-<i>fimH</i>22, B2-O2:H1-ST135-<i>fimH</i>2, B1-O8:H9-ST764-<i>fimH</i>32, B1-O23:H16-ST453-<i>fimH</i>31, A-O81:H27-ST10-<i>fimH</i>54, A-O16:H5-ST871-<i>fimH</i>25, and F-O11:H6-ST457-<i>fimH</i>145, were detected. The MICs of colistin for these isolates ranged from 4 to 16 mg/liter. The <i>mcr-1</i> 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 <i>mcr-1</i> 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 <i>E. coli</i> (APEC). In addition, another isolate of the B2-O25:H4-ST131-<i>fimH</i>22 lineage had those APEC-associated virulence genes on its chromosome. In conclusion, <i>mcr-1</i>-positive <i>E. coli</i> 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.<b>IMPORTANCE</b> Colistin is considered a last-line therapeutic option in severe infections due to multidrug-resistant Gram-negative bacteria, in particular carbapenemase-producing <i>Enterobacteriaceae</i> and multidrug-resistant <i>Acinetobacter baumannii</i> An increasing prevalence of <i>mcr</i> genes in diverse <i>Enterobacteriaceae</i> species, mainly <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> from humans and food animals, has become a significant concern to public health all over the world. In Japan, <i>mcr</i> genes have so far been detected in food animals, raw meat, wastewater, and human clinical samples. This study reports the copresence of <i>mcr-1</i> and avian pathogenic <i>E. coli</i> (APEC)-associated virulence genes in five of seven <i>E. coli</i> isolates recovered from aquatic environments in Japan. Our study highlights the importance and urgency of action to reduce environmental contamination by <i>mcr</i> genes that may likely occur due to exposure to untreated wastewater through combined sewer overflow by recent unusual weather.