Identification of novel variants of the colistin resistance gene mcr-3 in Aeromonas spp. from the national resistance monitoring programme GERM-Vet and from diagnostic submissions.

Objectives: To investigate Aeromonas spp. isolates for the presence of the novel resistance gene mcr-3 or variants thereof and to characterize the positive isolates by whole genome sequence analysis.
Methods: A total of 479 unrelated Aeromonas isolates were investigated by PCR for the genes mcr-1, mcr-2 and mcr-3. Positive isolates were investigated for their colistin MICs. Species assignment was based on sequence analysis of 16s rRNA and gyrB and rpoB genes. The mcr-carrying contigs obtained by WGS were analysed for the genetic environments of the mcr genes.
Results: Four (0.84%) Aeromonas isolates were positive in the mcr-3-specific PCR assay, whereas none of the isolates harboured mcr-1 or mcr-2. Each of the four mcr-3 genes encoded a novel variant, which showed amino acid identities of 95.0%-98.0% to the original Mcr-3 protein. These variants were designated Mcr-3.6 [Aeromonas allosaccharophila from golden orfe (Leuciscus idus)], Mcr-3.7 [Aeromonas media from turkey (Meleagris gallopavo)], Mcr-3.8 [Aeromonas jandaei from koi carp (Cyprinus carpio)] and Mcr-3.9 [Aeromonas hydrophila from koi carp]. The isolate harbouring the mcr-3.9 gene carried an additional mcr-3.8 gene and showed a distinctly higher colistin MIC of ≥128 mg/L than all other isolates. The genetic environments of the mcr-3 variant genes in all four isolates differed, but in part resembled the flanking regions of mcr-3.3 from Aeromonas veronii of chicken meat. Conclusions: This study identified four novel Mcr-3 variants. The isolates carrying the respective genes dated back to 2005 suggesting that this gene has existed for more than 12 years.