Genetic basis of β-lactam resistance in Corynebacterium auriscanis and association with otitis externa in dogs and cats

Corynebacterium (C.) auriscanis is an opportunistic pathogen regularly isolated from canine otitis externa, an important condition often hard to treat. We found a surprisingly high prevalence of β-lactam resistant isolates of C. auriscanis (47 %), even though β-lactams are not routinely used for otitis externa treatment in Switzerland. To determine the genetic base of this phenotype, a selection of isolates of C. auriscanis with high and low minimal inhibitory concentration values were subjected to whole genome sequencing. Comparative analysis revealed a gene cassette containing three genes (hdfR encoding a LysR-family transcriptional regulator, blaB encoding a β-lactamase related protein and pbp2c encoding a D,D-transpeptidase) as the likely resistance-encoding determinant in the isolates from otitis externa. This locus had previously been described in C. jeikeium as well as C. diphtheriae and was associated with mobile genetic elements. In our six C. auriscanis isolates the pbp2c locus was always associated with the same IS3 family transposase, an association also found on C. diphtheriae plasmid CP091096, indicating horizontal gene transfer between species. To elucidate the function of the three genes in the pbp2c locus, we constructed plasmids with different combinations of these genes, transformed β-lactam sensitive isolates with the plasmids and tested resistance in the mutants phenotypically. By doing so we confirmed Pbp2c to be the primary factor conferring β-lactam resistance and HdfR and BlaB being important for expression and regulation. Interestingly, resistance to all β-lactams including carbapenems was constitutive in one C. auriscanis transformant while an induction effect was visible for the other transformed C. auriscanis strain, C. glutamicum and C. rouxii as previously described for C. jeikeium. Therefore, testing of β-lactam resistance should be done in combination including induction in Corynebacterium spp.