The dynamic evolution and IS26-mediated interspecies transfer of a blaNDM-1-bearing fusion plasmid leading to a hypervirulent carbapenem-resistant Klebsiella pneumoniae strain harbouring blaKPC-2 in a single patient

Abstract

To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient. Minimum inhibitory concentrations were measured using broth microdilution assays. Conjugation assays, string tests, and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants, insertion sequences, and virulence factors were identified using ABRicate/ResFinder database, ISFinder, and virulence factor database. Wzi and capsular polysaccharide (KL) were typed using Kleborate and Kaptive. Multi-locus sequence typing (MLST), replicon typing, and single nucleotide polymorphism analyses were conducted using the BacWGSTdb server. The carbapenem-resistant K. pneumoniae 2111KP was characterized as ST11, wzi64, and KL64, with a positive string test result and a relatively high virulence phenotype. Analysis of the 2111KP genome revealed that blaNDM-1 was located in a 268,400-bp IncFIB/IncHI1B/IncX3 conjugative plasmid (p2111KP-1), regulated by IS26, IS5, and ISKox3. p2111KP-1 was also a rmpA2-associated virulence plasmid with an iutA-iucABCD gene cluster and a IS26-mediated multidrug-resistant fusion plasmid, which contained 8-bp (AGCTGCAC or GGCCTTTG) target site duplications. Segments flanked by IS26 of p2111KP-1 were 99.99% identical to a 49,016-bp E. coli plasmid. This study provided direct evidence of plasmid fusion via IS26 between two different bacterial species within one patient and revealed the process by which genetic elements conferring carbapenem resistance and virulence were simultaneously transferred between these species. It highlights the need for strategic antibiotic use and rigorous monitoring to prevent the plasmid-mediated fusion and transmission of drug-resistance/virulence factors.