Chromosomal Locations of mcr-1 and bla CTX-M-15 in Fluoroquinolone-Resistant Escherichia coli ST410.

To the Editor: Recently, Yi-Yun Liu et al. reported on the discovery of mcr-1, a plasmidborne resistance gene mediating resistance to colistin, in isolates obtained from humans and animals (). Since the original publication, mcr-1 with or without the insertion element ISApl1 has been detected on plasmids of different incompatibility groups, including IncI2, IncHI2, and IncX4, and in many different countries (–). Because colistin is a last-resort parenteral antimicrobial drug, the transfer of mcr-1 by conjugation or through mobilizable plasmids raises concern about the emergence of pan-resistant Enterobacteriaceae.

We previously described extended-spectrum β-lactamase (ESBL)–producing and carbapenemase-producing isolates obtained from livestock and a human in Germany that harbored the mcr-1 gene (). Because the transfer of mcr-1 through the food chain is highly likely, we looked for its presence in 62 whole-genome sequenced ESBL-producing Escherichia coli isolates obtained during 2012–2013 from food products sampled in Germany (Technical Appendix). We detected 4 isolates harboring the mcr-1 gene (E. coli RL138, RL145, RL158, and RL465) that displayed a colistin MIC of 4 mg/L (Technical Appendix Table 1). The raw sequencing reads and the assembled contigs of the mcr-1–positive isolates were deposited in the European Nucleotide Archive under project accession no. PRJEB13470. We conducted conjugation experiments to analyze the transferability of mcr-1 (Technical Appendix). For all isolates except RL465, mcr-1 was transferable to E. coli J53 Azr. For isolates RL138, RL145, and RL158, the mcr-1 gene was present on IncX4 and IncHI2 plasmids (Figure, panel A; Technical Appendix Table 2). The sequence type (ST) 410 E. coli isolate RL465 was detected in a turkey hen meat sample from 2013 and harbored blaCTX-M-15 and mcr-1, a gene combination hitherto identified only in travelers from the Netherlands and children from China (). Both the blaCTX-M-15 and mcr-1 genes were not transferable, indicating that neither gene was plasmid-encoded. Examination of the genetic environment of mcr-1 in the assembled gapped genome showed a chromosomal location for the mcr-1 transposition unit that included an ISApl1 element (Figure 1, panel A; Technical Appendix Figure 1, panel A) flanked by the inverted repeats (IR-R1, IR-R2, and IR-L1). We verified the chromosomal location for the mcr-1 gene by sequencing the genome to completion, using long-read single-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA, USA; Technical Appendix Figure 2); the resulting contigs of E. coli RL465 Were deposited in the European Nucleotide Archive under accession no. PRJEB14095. One copy of the ISApl1-mcr-1 transposition unit was located in the region between a predicted 4Fe-4S ferredoxin-type protein (ydhY) and ldtE (L,D-transpeptidase) (bp 2652307–2665241), and flanked on either side by a 2-bp direct repeat (CA). We observed a similar situation for the ISEcp1-blaCTX-M-15–orf477 transposition unit (Technical Appendix Figure 1, panel B). However, this insertion mapped to a different chromosomal location in a region encoding a defective lambdoid prophage inserted between the molybdate ABC transporter operon (modABC) and the biotin biosynthesis operon (bioABCDF) (bp 1662140–1716472). It was flanked by direct repeats (TGGTT).

Figure

A) Genetic environment of mcr-1 in extended-spectrum β–lactamase (ESBL)–producing food isolates including the original environment found in pHNSHP45. Colors represent gene functions and insertion sequences: purple, transposase of ISApl1; dark blue, ISApl1 element; green, mcr-1; pink, hypothetical protein within the mcr-1 transposition unit; red, light blue, yellow, flanking genes specific for the respective isolate. B) Phylogenetic tree (unrooted) of the Escherichia coli sequence type 410 isolates harboring the blaCTX-M-15 transposition unit in the chromosome and a schematic depiction of the location of the mcr-1 and blaCTX-M-15 transposition unit. Isolates prefixed with the letter E represent consecutive isolates from 1 patient. Scale bar indicates nucleotide substitutions per site.

We reexamined our collection of 424 genome sequenced ESBL- and carbapenemase-encoding E. coli isolates, obtained during 2010–2014 (), for isolates that harbored blaCTX-M-15 at a chromosomal location identical to that found in E. coli RL465. We detected 3 such isolates from 2010–2011 from companion animals and livestock (R107, sock swab dairy cattle farm, 2011; R208, sock swab pig fattening farm, 2011; V177, sick dog, 2010), and 11 consecutive isolates from a hemato-oncologic patient (), obtained within an 11-month period during 2011–2012 (E006910, E007337, E007651, E007825, E000565, E002592, E002816, E003488, E005417, E006587, E006874) (Figure, panel B). All of these isolates were ST410 and negative for the mcr-1 gene. Phylogenetic analysis of the core genome of these isolates with E. coli RL465 using the program Harvest Suite () indicated they were highly related and separated from E. coli V177 (the oldest isolate) by 66 (E006910, E007651) to 110 (E007337) single-nucleotide polymorphisms (core genome size 94%, representing 4.58 Mbp). Thus, our results suggest that transposition of the ISApl1-mcr-1 unit to the chromosome in E. coli RL465 is a later event and probably occurred after transfer of the blaCTX-M-15 allele to the distinct chromosomal location into this E. coli ST410 subclone.

These findings highlight 2 independent points. First, our results extend data on the mobility of ISApl1-mcr-1 to a chromosomal location and reveal a new dimension in the transmissible nature of mcr-1 in colistin-resistant Enterobacteriaceae isolates and their ecology. Second, clonal isolates of ST410 have been isolated from diverse environments, livestock, companion animals, and humans and, as we demonstrate here, in turkey hen meat (,). Thus, the simultaneous spread of the mcr-1 and blaCTX-M-15 genes mediated by a single bacterial clone is real and suggests that mcr-1 is already present in the diverse reservoirs inhabited by these isolates.

Technical Appendix

Collection of isolates; antimicrobial susceptibility testing; whole-genome sequencing; in silico analyses; conjugation experiments; depiction of the minimal inhibitory concentration of the mcr-1–encoding and extended-spectrum β-lactamase (ESBL)–producing isolates from retail food; characteristics of the mcr-1–encoding ESBL-producing Escherichia coli isolates from retail meat; genetic environments of the chromosomally located antimicrobial resistance genes; schematic depiction of the chromosome, its IncFII/FIB plasmid, and the 2 phage elements in E. coli RL465.