The Plasmid-Mediated Kluyvera-Like Arnbcadtef Operon Confers Colistin (Hetero)Resistance To Escherichia Coli

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 system, or still unknown mechanisms. This work analyzed 74 Escherichia coli isolates from healthy swine, turkey, or bovine, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found, among which 45% carried this gene on highly mobile plasmids, followed by 4 strains lacking previously known resistance determinants or 2 with mcr-4 (1 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 strains with mutations that confer colistin resistance. The expression of mcr genes negatively controls the transcription of the arnBCADTEF operon itself, a downregulation that was also observed in the four isolates lacking known resistance determinants, with three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed an IncFII plasmid of 62.1 kb carrying an extra copy of the arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, called pArnT1, was cured by ethidium bromide, and the cells lost resistance to colistin in parallel. Furthermore, a susceptible E. coli strain acquired heteroresistance after transformation with pArnT1 or pBAD24 carrying the Kluyvera-like arnBCADTEF operon, revealing it as a new colistin resistance determinant.