Mobile colistin resistance (mcr) gene prevalence and molecular characterization of mcr and extended-spectrum beta-lactamase (ESBL) genes containing mobile genetic elements

Abstract Introduction Multidrug-resistant (MDR) Enterobacterales isolates carrying extended-spectrum beta-lactamases (ESBLs) and mobile colistin resistance (mcr) genes pose a significant global healthcare threat as they can lead to difficult-to-treat infections. This study investigates the prevalence of isolates co-harboring ESBL and mcr genes and characterizes the plasmids co-harboring those genes. Methods ESBL-producing Enterobacterales (ESBL-E) isolates identified during point prevalence surveys (PPS) in a Dutch hospital were screened for mcr genes. Long- and short-read sequencing data of ESBL-E containing mcr genes, were used to identify plasmids co-harboring mcr- and ESBL-genes. Resistance genes and replicon genes within these plasmids were detected using the AMRFinderPlus and PlasmidFinder database in Abricate, respectively. The plasmid database PLSDB was searched for plasmids containing the same mcr gene(s) and at least one of the same ESBL genes. SNP analyses and maximum likelihood phylogeny analyses were conducted to examine plasmid diversity. The emergence of the most recent common ancestor of the plasmids was inferred through timed phylogeny analyses in BEAST, and putative composite transposons surrounding the mcr and ESBL genes were identified using plasmid annotations through Bakta, which has the ISFinder database integrated. Results Among 188 screened ESBL-E, 11 (5.9%) harbored mcr genes: nine with mcr-9 and two with both mcr-9 and mcr-4.3. All identified plasmids containing mcr and ESBL genes were IncHI plasmids harboring mcr-9 and blaCTX-M-9 with or without blaSHV-12. The plasmid database search resulted in 128 IncHI plasmids containing mcr-9, blaCTX-M-9, and/or blaSHV-12. SNP analysis showed =10 SNPs among the PPS study plasmids and up to 924 SNPs among all plasmids. Phylogenetic analysis revealed clustering of the PPS study plasmids but no additional taxonomical or geographical clustering. Time phylogeny estimated that the most recent common ancestor of the PPS study plasmids likely emerged between 2003-2007. Lastly, composite transposon analysis indicated that matching complete insertion sequences rarely flanked mcr-9 genes, whereas blaCTX-M-9 and blaSHV-12 frequently were. Conclusions The prevalence of mcr genes among ESBL-E in a Dutch hospital 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, which are mostly stabilized in the plasmids.