Fine-Scale Reconstruction of the Evolution of FII-33 Multidrug Resistance Plasmids Enables High-Resolution Genomic Surveillance

We examined 185 complete, publicly available FII-33 plasmid sequences, characterizing their backbone and various insertions. The variable characteristic insertions facilitated evolutionary reconstruction for this plasmid group, beginning with the acquisition of a primary resistance region (PRR) over 10 years ago. FII-33 plasmids have evolved by acquiring additional resistance genes in the PRR via translocatable elements and by forming cointegrates with plasmids of other types. In all cases, IS26 is suspected to have mediated cointegration. Plasmid cointegration has contributed to the accumulation of resistance genes and may have increased the transmissibility, stability, and host range of the original FII-33 lineage. A particularly important sublineage was formed by a replicative IS26 cointegration event that fused an FII-33 plasmid with a bla(KPC-2)-containing R-type plasmid, interrupting the FII-33 traI gene encoding the conjugative relaxase. The FII-33:R cointegrate arose in the Klebsiella pneumoniae ST11 clone and remains largely confined there due to the abolition of transfer ability by the FII-33:R cointegration event. However, in some cases FII-33:R cointegrates have fused with additional plasmids and acquired complete transfer regions or oriT sequences that might restore their ability to transfer horizontally. Cointegration events across FII-33 plasmid sublineages have involved plasmids of at least 15 different types. This suggests that plasmid cointegration occurs readily and is more common than previously appreciated, raising questions about the effects of cointegrate formation on plasmid host range, stability, and capacity for horizontal transfer. Resources are provided for detecting and characterizing FII-33 plasmid sublineages from complete or draft genome sequences. IMPORTANCE Effective genomic surveillance of antibiotic-resistant bacterial pathogens must consider plasmids, which are frequently implicated in the accumulation and transfer of resistance genes between bacterial strains or species. However, the evolution of plasmids is complex, and simple typing or comparison tools cannot accurately determine whether plasmids belong to the same sublineages. This precludes precise tracking of plasmid movement in bacterial populations. We have examined the FII-33 group, which has been associated with multidrug resistance and particularly carbapenem resistance in clinically significant members of the Enterobacterales in China. Our analysis has provided insight into the evolution of this important plasmid group, allowing us to develop resources for rapidly typing them to the sublineage level in complete or draft genome sequences. Our approach will improve detection and characterization of FII-33 plasmids and facilitate surveillance within and outside China. The approach can serve as a model for similar studies of other plasmid types.