High frequency of chromosomal polymyxin resistance in Escherichia coli isolated from dairy farm animals and genomic analysis of mcr-1-positive strain

Abstract

Escherichia coli strains are naturally susceptible to polymyxins. The frequency of polymyxin resistance in E. coli associated with chromosomal mutations is usually low (0.2-0.6%). Here, we report polymyxin-resistant E. coli strains isolated from flies (Diptera: Muscomorpha) and animals (bovine, equine, canine, and sheep) cohabitants on two dairy farms, A and B (Botucatu, State of Sao Paulo, Brazil), in 2015. We isolated 877 E. coli from the external surface of flies and healthy animal feces. The screening of polymyxin-resistant E. coli was performed using MacConkey agar with polymyxin B (2 µg/mL). We detected a high percentage of polymyxin-resistant isolates from flies (33.33%; 66/198) and farm animals (46.09%; 313/679). Fisher's exact test revealed no associations between polymyxin resistant and multidrug resistant strains. We investigated mcr genes (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) using PCR, and five E. coli from calves (farm B) present the mcr-1 gene. Sanger sequencing and conjugation assays were performed for gene confirmation. All five mcr-1-positive E. coli showed fingerprints more than 80% similar assayed by pulsed-field gel electrophoresis. The minimum inhibitory concentrations (MICs) for polymyxin B against mcr-1-positive E. coli were 4 µg/mL (two strains), and 8 µg/mL (three strains). Whole-genome sequencing showed mcr-1.1 gene in the IncX4 plasmid and a class 1 integron unusual harboring sul3 and qacL as a non-variable structure. In Brazil, the therapeutic use of polymyxin is allowed, but stewardship in dairy cattle is uncommon; thus, our data indicate a probable selection of polymyxin-resistant strains related to environmental pressure.

Keywords: Escherichia coli; Antimicrobial resistance; Cattle; Colistin; Flies; Milking environment; Public health.