Molecular characterization and prevalence of plasmids co-harbouring mcr and ESBL genes.

Multidrug-resistant Enterobacterales isolates carrying extended-spectrum beta-lactamases (ESBLs) and mobile colistin resistance (mcr) genes pose a significant healthcare threat as they can lead to difficult-to-treat infections. This study investigates the prevalence of isolates co-harbouring ESBL and mcr genes and characterizes the plasmids co-harbouring those genes. ESBL-producing Enterobacterales (ESBL-E) isolates identified during point prevalence surveys (PPS) in a Dutch hospital were screened for mcr genes. Plasmids co-harbouring mcr and ESBL genes were identified using long- and short-read sequencing data, while detecting resistance and replicon genes using AMRFinderPlus and PlasmidFinder. The plasmid database PLSDB was searched for plasmids containing the same mcr and ESBL gene(s), and SNP and DCJ-Indel distance analyses were conducted to examine plasmid diversity. The most recent common ancestor (MRCA) was inferred through timed phylogeny analyses in BEAST, and putative composite transposons containing the mcr or ESBL genes were identified. Among 188 screened ESBL-E, 11 harboured mcr genes: 9 with mcr-9 and 2 with mcr-9 and mcr-4.3. All plasmids containing mcr and ESBL genes were IncHI plasmids harbouring mcr-9, bla _CTX-M-9 and/or bla _SHV-12. The plasmid database search resulted in 128 similar plasmids. SNP analysis showed ≤10 SNPs among the PPS study plasmids and up to 924 SNPs among all plasmids. Structural relatedness and phylogenetic analyses revealed clustering of the PPS study plasmids but no additional taxonomical or geographical clustering. The MRCA of the PPS study plasmids likely emerged between 1986 and 2008. Finally, composite transposon analysis indicated that matching complete insertion sequences rarely flanked mcr-9 genes, whereas bla _CTX-M-9 and bla _SHV-12 frequently were. The prevalence of mcr genes among ESBL-E in this study is 5.9%, with mcr-9 being the most prevalent. Limited plasmid diversity suggests a single introduction event followed by regional and global dissemination across related bacterial species. Persistent ESBL gene mobility suggests a more recent introduction of these genes compared to the mcr-9 genes.