Mobilization of a Kluyvera-like arnBCADTEF operon, besides mcr genes, confers colistin resistance to Escherichia coli isolated from healthy animals

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB two-component system and also by some other(s) still unknown mechanism(s). This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine animals, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to control strains carrying mutations that confer colistin resistance. The expression of mcr genes negatively controls arnBCADTEF expression, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 60 Kb encoding an arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, named pArnT1, was cured by ethidum bromide and lost in parallel to colistin resistance. This work reveals that, besides mcr genes and chromosomal mutations, mobilization of arnBCADTEF operon represents a colistin resistance mechanism whose spread and relevance for public health should be carefully surveyed. Abstract ImportanceColistin is an old antibiotic that has returned to first-line fighting against (Gram negative) microorganisms after pandemic rising of antimicrobial resistance. However, low susceptibility to colistin is also becoming spread, mainly by plasmid mobilization of one of the enzymes (encoded by mcr genes) that modify covalently the external layer (the lipid A component of the lipopolysaccharide) of bacterial envelope, interfering antibiotic effectiveness. The second enzymatic system that performs envelope modification and confers colistin resistance when overexpressed is encoded by arnBCADTEF operon, a set of seven genes with location restricted (up to now) to the chromosome of Gram negative bacteria. This work describes plasmid mobilization of this operon between enterobacteria, from Kluyvera to Escherichia coli, where a Kluyvera-like arnBCADTEF operon carried by pArnT1 might represent, besides mcr genes, a potential risk for antimicrobial therapy and might require careful surveillance.