Plasmidome sequencing in K. pneumoniae. plasmidome

Plasmid remains an important element mediating horizontal gene transfer (HGT) and the dissemination of antibiotic resistance. To systematically examine the HGT profile of plasmids, and it participating mobile genetic elements (MGEs) and antibiotic resistance genes, a novel single-molecule real time “plasmidome” sequencing assay was developed to profile pooled conjugative plasmids from 53 clinical isolates of multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over the course of 9-months, whereby their resistance profiles could be transferred to transconjugants via plasmid conjugation. Eighty-two assembled plasmid fragments were acquired after masking the recipient chromosome sequence. Eventually, 123 antibiotic resistance genes (41 non-redundant) and 317 MGEs, such as transposons, insert sequences (ISs) and integrons (97 non-redundant) were further derived from these fragments. Most MGEs could be linked to antibiotic resistance genes, paving a foundation of the “MGE-resistance genes” network, that can be further used to reflect the dissemination of resistance by mobile elements in a confined plasmid environment. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, ISKpn19, etc. This network can be used as an early predictor of the development of plasmid-mediated antibiotic resistance. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplication.