mcr-5 and a novel mcr-5.2 variant in Escherichia coli isolates from food and food-producing animals, Germany, 2010 to 2017

Sir, Colistin is considered a last-resort antibiotic used to treat severe human infections caused by MDR Gram-negative bacteria. Thus, spread of colistin resistance among humans would be associated with major public health concerns.1,2 In 2015, the first mobile colistin resistance gene, mcr-1, encoding a phosphoethanolamine transferase enzyme, was identified on a transmissible plasmid.3 Soon after, mcr-2 and mcr-3 were detected on other conjugative plasmids in Enterobacteriaceae.4,5 Recently, Carattoli et al.6 and Borowiak et al.7 reported two novel genes, mcr-4 and mcr-5, in Salmonella enterica serovar Typhimurium and d-tartrate-fermenting S. enterica serovar Paratyphi B [Salmonella Paratyphi B (dTa+)], respectively. Both genes were located on non-conjugative ColE plasmids, either transmissible by a helper plasmid (mcr-4) or mobilizable by a Tn3-type transposon (mcr-5, registered as Tn6452 on the LSTM website, http://transposon.lstmed.ac.uk/) found on the Salmonella Paratyphi B (dTa+) chromosome and plasmids.7 Identification of different mcr genes in Enterobacteriaceae raised concerns about their distribution and genetic diversity. Thus, a molecular survey on the detection of mcr-5-harbouring Escherichia coli was initiated. In the German national monitoring programme for antimicrobial resistance in zoonotic agents the National Reference Laboratory for Antimicrobial Resistance has received 19 216 E. coli isolates from food and food-producing animals between 2010 and 2017 for antimicrobial resistance testing. Of these, 737 isolates exhibited an MIC of colistin ≥4 mg/L. During routine laboratory work, all colistin-resistant isolates were subjected to mcr-1 qPCR screening as previously described.8 Using the PCR assay of Borowiak et al.7 on mcr-1-negative E. coli (n = 135), three mcr-5-positive isolates were detected in samples recovered from the caecal contents of pigs at slaughter (10E01066) and faecal samples from pigs at farms (11E02380, 15-AB00674). These isolates exhibit a non-WT phenotype (resistant) for colistin and other antimicrobials using the microdilution method according to CLSI guidelines (M07-A9) following EUCAST epidemiological cut-off values (Table S1, available as Supplementary data at JAC Online). S1-PFGE profiling and DNA hybridization9 showed a plasmid location of mcr-5 in the isolates. Short-read paired-end MiSeq sequencing of genomic DNA and de novo assembling were performed as previously described.7 Relevant genome features of the isolates are summarized in Table S1. The complete genomes of the mcr-5-harbouring plasmids were derived from WGS data by raw-read mapping against Salmonella Paratyphi B (dTa+) plasmid pSE13-SA01718 (KY807921). In 10E01066 and 11E02380 mcr-5 was detected on 12 201 bp (pEC1066, MG587003) and 11 708 bp (pEC2380, MG587004) plasmids, respectively. Comparative analyses using BRIG10 revealed that pEC1066 and pEC2380 are ColE plasmids with a close relationship to pSE13-SA01718 of Salmonella Paratyphi B (dTa+) (Figure 1a).7 Isolate 15-AB00674 carries mcr-5 on a 6268 bp plasmid (pEC0674, MF684783) (Figure 1b) exhibiting a stronger similarity to Klebsiella pneumoniae plasmid Kp13 (CP003996.1), potentially belonging to an as yet unknown incompatibility type. Interestingly, pEC0674 lacks tnpA and tnpR of the mcr-5 transposon. Dissection of the mcr-5-harbouring plasmids of E. coli 10E01066, 11E02380 and 15-AB00674. (a) Organization of mcr-5-harbouring plasmids from E. coli. The central circle represents the GC content of the reference plasmid. (b) A gene map of the complete nucleotide sequence of plasmid pEC0674 (assemblies from raw reads of isolate 15-AB00674). Relevant features of the plasmid sequences are indicated. (c) An MCR-5 protein comparison (Accelrys DS Gene, version 2.5) of E. coli plasmids pEC1066 and pEC2380. Different domains predicted in the primary structure of MCR-5 are indicated as previously described.7 The domains of transmembrane structures and sulphatases are indicated in dark and light grey, respectively. Sequences highlighted in mid grey illustrate DUF1705 domains. Conserved amino acids that may be involved in substrate binding and colistin resistance are indicated with bold letters. The position of the amino acid deletion is indicated by an asterisk. Further DNA alignments showed that pEC1066 and pEC2380 carry identical mcr-5 transposon sequences as described for pSE13-SA01718.7 Interestingly, mcr-5 on pEC2380 exhibits a deletion of three nucleotides encoding an amino acid in the central part of the protein (Figure 1c). As this isolate exhibits the highest MIC (8 mg/L) of colistin for the isolates of this study (Table S1), this deletion may not affect the domain structure of the enzyme or functional amino acids involved in resistance development (Figure 1c). Since this mcr-5 allele is the first variant, we suggest designating it mcr-5.2 (MG384740). The increased colistin resistance (MIC = 8 mg/L) of isolate 11E02380 may be caused by a mutation in pmrB (V161G), which is known to contribute to colistin resistance in E. coli.11 In contrast to isolate 11E02380, no colistin resistance-associated pmrA or pmrB gene mutations were detected in E. coli 10E01066 and 15-AB00674. The mcr-5-carrying plasmids of this study do not carry transfer genes involved in plasmid conjugation. Further investigations were performed with pEC1066, which was therefore introduced by transformation into chemically competent E. coli cells (DH5α, Thermo Fisher) under colistin (2 mg/L) selection. The presence of pEC1066 in the transformants was confirmed by S1-PFGE and broth microdilution. An MIC of 4 mg/L was observed for DH5α transformants harbouring pEC1066, while cells without this plasmid exhibited an MIC ≤1 mg/L. Conjugation studies in E. coli with (F, RP4, RK2) and without helper plasmids indicated that pEC1066 was not transmissible by the mating systems and conditions used (room temperature, 37°C, 42°C). Our findings indicate that mcr-5-harbouring E. coli isolates sporadically occur in the intestinal tracts of pigs in Germany, which raises the concern of vertical transmission along the food chain and to consumers. The observed isolates have different characteristics and carry mcr-5 on two different plasmid types. The resistance gene is located on the Tn3-family transposon Tn6452 on the Salmonella Paratyphi B (dTa+) chromosome and plasmids.7 To determine the impact of mcr-5, further information on the stability of the mcr-5-harbouring plasmids, their transmission routes and distribution in livestock, food products and humans is needed. These data are necessary to develop efficient risk management strategies to control the spread of mobile phosphoethanolamine transferase conferring reduced susceptibility to colistin in Enterobacteriaceae. We thank Britta Lesniewsky and Gaby Carl for excellent laboratory assistance, as well as Anna-Louisa Hauffe, Cornelia Göllner and Josephine Grützke for library preparation and WGS sequencing. This work was supported by the German Federal Institute for Risk Assessment (43-001 and 1322-648). None to declare. Table S1 is available as Supplementary data at JAC Online.