Resolution of dynamic MDR structures among the plasmidome of Salmonella using MinION single-molecule, long-read sequencing.

Abstract

ISCR1 is an important mobile genetic element mediating the transfer of antibiotic resistance genes. Genetic diversity regarding distribution and copy numbers of ISCR1 within a bacterial population derived from an ancestral strain, which may reflect the degree of genetic plasticity conferred by such an element, has not been studied. To investigate the plasmid heterogeneity in Salmonella conferred by ISCR1. Nanopore long-read and other sequencing technologies were used to resolve the structures harbouring different copies of ISCR1-qnrB6 from the perspective of single molecules. Salmonella London Sa128 was positive for ISCR1-qnrB6 and harboured an MDR-encoding conjugative IncF plasmid, pSa128, containing a complex class 1 integron. The plasmid pSa128T from the transconjugant was larger compared with the original plasmid pSa128, presumably due to amplification of ISCR1-qnrB6. Single-molecule, long-read analysis indicated that both plasmids in the donor and transconjugant strains were in a heterogeneous state that contains variable numbers of ISCR1-qnrB6, with four and eight copies in single plasmids being the dominant types. This type of plasmid heterogeneity in populations of one strain can be regarded as an atypical plasmidome. This study highlights the importance of investigation of a single plasmid structure based on long-read sequencing technologies, with a focus on analysing the complex structures of the MDR region, which is expected to exhibit genetic polymorphism or plasmid heterogeneity in various MDR-encoding elements even among members of the same strain. The availability of a single-molecule sequencing technique represents a paradigm shift in the capability of performing population genetic analysis of antibiotic-resistant organisms.