Novel integrative and conjugative elements carrying cfr(B) and cfr(C) linezolid resistance genes in Clostridioides difficile isolates from calves, Italy

Objectives: To clarify the genetic basis of high-level linezolid resistance in three Clostridioides difficile strains from calves. Methods: A WGS approach was used to comprehensively characterize C. difficile A501, A505 and A516 strains exhibiting high linezolid MICs, and to clarify their phylogenetic relationships. Linezolid resistance gene transferability was assessed by filter mating experiments. Results: WGS analysis revealed the presence of cfr(B) in C. difficile A501 and A516, both exhibiting the ST11, and cfr(C) in C. difficile A505 belonging to a non-toxigenic ST15 clone. The cfr(B) gene was on a novel 25 791 bp integrative conjugative element (ICE), named ICECd-cfr(B), similar to an uncharacterized region of Clostridium sp. C1, but significantly different from the Tn6218 transposon typically associated with this gene. The cfr(C) gene was found on a novel 32 770 bp ICE, named ICECd-cfr(C), which was identical to an uncharacterized region of the C. difficile DSM 104450 chromosome. ICECd-cfr(C) exhibited high nucleotide identity, but low coverage, with a cfr(C)-carrying region previously detected in C. difficile 020482; whereas in C. difficile A505 this region was interrupted by a 16.6 kb DNA insertion. Conjugation assays failed to demonstrate the transferability of cfr(B) and cfr(C) genes. Conclusions: To the best of our knowledge, this is the first report of C. difficile isolates from calves with high linezolid MICs due to novel cfr(B)- and cfr(C)-carrying ICEs. C. difficile animal isolates, belonging to ST11 and ST15 clones with zoonotic potential, could act as reservoirs for the spread of linezolid resistance genes to human intestinal pathogens, with serious consequences for public health.