During 2004–2010, we cultured 150 E. coli strains from fecal samples collected from cattle with suspected enteric infection or milk-aliquots collected from cattle with suspected mastitis in France and Germany. We conducted antimicrobial susceptibility testing by using disk diffusion against a panel of 17 compounds consisting of penicillin G, amoxicillin, and amoxicillin/clavulanic acid; cephalothin, cefoxitin, cefotaxime, and cefepime; ertapenem, meropenem, and imipenem; marbofloxacin, ciprofloxacin, and nalidixic acid; gentamicin; tetracycline; florfenicol; and trimethoprim/sulfamethoxazole. We interpreted results according to the criteria of the Clinical and Laboratory Standards Institute where appropriate (8,9).

A subset of these E. coli (n = 45) were classified as MDR and expressed resistance to >3 drug classes. We determined plasmid profiles and PCR-based replicon types as described previously (10,11) and detected plasmids ranging in size from 2 to 200 kbp. Our PCR-based replicon type analysis identified several incompatibility (Inc) types, including IncX4 in E. coli strain 11-1896 and the previously reported IncHI2 type in E. coli strain 29957 (Table). We then determined the MICs of these 45 MDR isolates for colistin by using broth microdilution. Three of 45 demonstrated MICs >2 mg/L, which we interpreted as being colistin resistant based on breakpoint tables of the European Committee on Antimicrobial Susceptibility Testing (12). We identified these isolates as E. coli 22134 O9:H9 U/ST10, E. coli 11-1896 O9:H12 U/ST58, and E. coli 29957 O101:H9 A or C/ST167 (Table). All were additionally resistant to >2 drug classes, including aminoglycosides, aminopenicillins, cephalosporins, fluoroquinolones, phenicols, tetracyclines, and trimethoprim and sulfonamides. One of the 3 isolates (E. coli 29957) was resistant to all of the antimicrobial compounds tested, including β-lactams, florfenicol, and fluoroquinolone compounds (Table). In addition, PCR results indicated that this isolate was positive for the presence of the mcr-1 gene (Technical Appendix Figure, panel A) (7).

We conducted whole-genome sequencing of 3 isolates with increased MICs for colistin by using the Nextera XT DNA Library Preparation Kit and the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA) to produce 300-bp paired end reads (v3 chemistry). We assembled these data de novo using SPAdes version 3.6.2 (http://bioinf.spbau.ru/spades) and then generated queries by using the PlasmidFinder 1.3 (https://cge.cbs.dtu.dk/services/plasmidfinder) and ResFinder 2.1 (http://cge.cbs.dtu.dk/services/resfinder) databases to identify plasmid replicon types and antibiotic resistance genes using BLAST+ (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Several antibiotic resistant genotypes, including some that were acquired, were identified. Four of these genotypes occurred in E. coli 22134 isolates, 8 in E. coli 11-1986 isolates, and 21 in E. coli 22957 isolates (Table). These isolates harbored genes or mutations that confirmed the phenotypes detected in most of the suspected cases of infection in the cattle in our study. We also identified plasmid replicons in all 3 isolates, including 3 types in E. coli 22134; 5 in E. coli 11-1986, and 7 in E. coli 29957. We did not detect the mcr-1 gene in E. coli 22134 or E. coli 11-1896; however, we identified several nonsynonymous amino acid substitutions in genes previously shown to be associated with colistin resistance, including pmrA and pmrB. We also identified phoP and eptB in E. coli 11-1986. Similarly, we identified the mcr-1 gene in E. coli 29957 (a feature that was previously confirmed by PCR) and 1 nonsynonymous substitution in pmrB. The mcr-1 gene was located in a 4,752-bp contig, which when used to query the current databases matched an identical region containing a transposase gene, a phosphoethanolamine transferase gene (the mcr-1 encoding gene), a hypothetical protein/phosphoesterase gene, and another transposase. The mcr-1 gene was 100% similar at the nucleotide level to that reported in China and was found to be located distal to the same insertion sequence element ISApl1 that mapped to the IncHI2 type plasmid pHNSHP45 (Technical Appendix Figure, panel B) (7).

Plasmid-mediated colistin resistance identified in MDR bacteria of animal origin represents a serious risk to public health. Our data further support recent findings demonstrating that the mcr-1 gene is not just present in Asia but can also be found in some countries in Europe (e.g., the mcr-1 gene identified in an E. coli strain cultured from a food-producing animal in France in 2007) (Table). Other arrangements of the mcr-1 gene on plasmids can occur, such as that observed in the IncX4 type (13). Liu et al. (7) reported that plasmid pHNSHP45 exhibited an in vivo transfer rate between different E. coli strains (measured at 10⁻¹ to 10⁻³ per recipient) (7), a feature that could contribute to the successful dissemination of the mcr-1 gene. Similarly, in our study, we can also confirm the transfer of the mcr-1 gene from E. coli 29957 via conjugation, albeit at a reduced frequency (data not shown). Especially concerning is the extensive resistance profile of E. coli 29957, a feature noted in other studies, which have indicated that colistin resistance might be co-selected after the use of cephalosporins and other compounds (14,15).

The mcr-1 gene has now been reported in food-producing animals and in humans located in different geographic regions. In several of these regions, the gene was linked to extended-spectrum β-lactam and florfenicol resistance in the same bacterial isolate (15). Because E. coli 29957 was identified in 2007, this finding cannot be considered a recent occurrence. Given the genetic mapping reported to date, selective pressure imposed after the administration of broad-spectrum cephalosporins and other compounds might have the potential to co-select for colistin resistance and vice versa, thereby contributing to the dissemination of mcr-1 (15). Molecular epidemiologic studies are required to discover the origin and means of transmission of this gene as a first step in attempting to limit its dissemination, particularly among pathogenic bacteria that threaten human health.