Occurrence of mcr-1 and mcr-2 colistin resistance genes in porcine Escherichia coli isolates (2010–2020) and genomic characterization of mcr-2-positive E. coli

Abstract

IntroductionThe global emergence of plasmid-mediated colistin resistance is threatening the efficacy of colistin as one of the last treatment options against multi-drug resistant Gram-negative bacteria. To date, ten mcr-genes (mcr-1 to mcr-10) were reported. While mcr-1 has disseminated globally, the occurrence of mcr-2 was reported scarcely.Methods and resultsWe determined the occurrence of mcr-1 and mcr-2 genes among Escherichia coli isolates from swine and performed detailed genomic characterization of mcr-2-positive strains. In the years 2010-2017, 7,614 porcine E. coli isolates were obtained from fecal swine samples in Europe and isolates carrying at least one of the virulence associated genes predicting Shiga toxin producing E. coli (STEC), enterotoxigenic E. coli (ETEC) or enteropathogenic E. coli (EPEC) were stored. 793 (10.4%) of these isolates carried the mcr-1 gene. Of 1,477 additional E. coli isolates obtained from sheep blood agar containing 4 mg/L colistin between 2018 and 2020, 36 (2.4%) isolates were mcr-1-positive. In contrast to mcr-1, the mcr-2 gene occurred at a very low frequency (0.13%) among the overall 9,091 isolates. Most mcr-2-positive isolates originated from Belgium (n = 9), one from Spain and two from Germany. They were obtained from six different farms and revealed multilocus sequence types ST10, ST29, ST93, ST100, ST3057 and ST5786. While the originally described mcr-2.1 was predominant, we also detected a new mcr-2 variant in two isolates from Belgium, which was termed mcr-2.8. MCR-2 isolates were mostly classified as ETEC or ETEC-like, while one isolate from Spain represented an atypical enteropathogenic E. coli (aEPEC; eae+). The ST29-aEPEC isolate carried mcr-2 on the chromosome. Another eight isolates carried their mcr-2 gene on IncX4 plasmids that resembled the pKP37-BE MCR-2 plasmid originally described in Belgium in 2015. Three ST100 E. coli isolates from a single farm in Belgium carried the mcr-2.1 gene on a 47-kb self-transmissible IncP type plasmid of a new IncP-1 clade.DiscussionThis is the first report of mcr-2 genes in E. coli isolates from Germany. The detection of a new mcr-2 allele and a novel plasmid backbone suggests the presence of so far undetected mcr-2 variants and mobilizable vehicles.

Occurrence of Mobile Colistin Resistance Genes mcr-1–mcr-10 including Novel mcr Gene Variants in Different Pathotypes of Porcine Escherichia coli Isolates Collected in Germany from 2000 to 2021

“…None of the isolates tested in our study carried mcr-6 to mcr-10, which is in accordance with previous studies in commensal isolates [7]. The majority of prior studies investigating the presence of mcr genes in E. coli isolates from diseased or healthy pigs with defined E. coli pathotypes/VAG-typed E. coli did not test for mcr-6 to mcr-10 (Table S3) [44][45][46][47][48].…”

“…Additional porcine E. coli isolates (n = 1152) were received through submissions of other veterinary diagnostic laboratories for further molecular typing in our institute. Some of these isolates have already been included in a recent study on the presence of mcr-1 and mcr-2 genes in porcine E. coli isolates [7]. Ethical review and approval were waived for this study due to the fact that the sample collection was not for research but for diagnostic purposes, and only the results obtained were used for scientific purposes.…”

“…Each study isolate that proved positive for at least one of the tested VAGs was stored in a glycerin stock at −80 • C. If isolates from the same pig showed different VAG profiles, a representative isolate of each profile was stored. Part of the pathotyping PCRs have already been conducted as part of a recent study [7].…”

“…Pathotype prediction was conducted based on the presence of VAGs, as described in Section 2.2. * mcr-2-positive isolates were previously published and will not be mentioned hereinafter [7].…”

“…In 2015, the plasmid-mediated mobile colistin resistance (mcr) gene mcr-1 was identified in a porcine E. coli isolate from China, followed by reports of nine additional mcr genes (mcr-2-mcr-10) and their variants [6]. A number of studies from across the globe, including studies from Germany, reported different frequencies of mcr genes in fecal E. coli isolates from healthy pigs, as recently summarized [7]. In contrast, many fewer studies were performed to determine the frequency of mcr genes among clinical isolates, i.e., obtained from pigs with post-weaning diarrhea or from enteric colibacillosis [8][9][10][11][12].…”