Plasmid double locus sequence typing for IncHI2 plasmids, a subtyping scheme for the characterization of IncHI2 plasmids carrying extended-spectrum β-lactamase and quinolone resistance genes

IncHI2 plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance genes. These plasmids are usually > 250 kb, and technical difficulties can impair plasmid DNA purification and comparison by restriction fragment length polymorphism. We analysed the available IncHI2 whole DNA plasmid sequences to devise a rapid typing scheme to categorize the members of this plasmid family into homogeneous groups. We compared the available full IncHI2 plasmid sequences, identifying conserved and variable regions within the backbone of this plasmid family, to devise an IncHI2 typing method based on sequence typing and multiplex PCRs. A collection of IncHI2 plasmids carrying extended-spectrum beta-lactamase and quinolone resistance genes, identified in strains from different sources (animals and humans) and geographical origins, was tested by these typing systems. We devised a plasmid double locus sequence typing (pDLST) scheme and a multiplex PCR discriminating IncHI2 plasmid variants. These systems were tested on a collection of IncHI2 plasmids, demonstrating that the plasmids carrying bla(CTX-M-2) and bla(CTX-M-9) belonged to two major plasmid variants, which were highly conserved among different enterobacterial species disseminated in several European countries. The ability to recognize and subcategorize plasmids by pDLST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful to analyse their distribution in nature and to discover of their evolutionary origin.