Genomic characterization of virulence factors in hypermucoviscous Klebsiella pneumoniae isolates using long-read sequencing

Abstract Background The virulence of Klebsiella pneumoniae is mediated by a diverse repertoire of genetic determinants involved in immune evasion, iron acquisition, colonization, and invasive disease. These virulence-associated traits have contributed to its recognition as an important cause of bloodstream and healthcare-associated infections. This study aimed to characterize the virulence genes, sequence types, capsule types, and O antigen types of Klebsiella pneumoniae isolates exhibiting hypermucoviscous phenotypes using whole-genome sequencing analysis. Methods Five hypermucoviscous K. pneumoniae isolates recovered from blood cultures were subjected to whole-genome sequencing using the Oxford Nanopore MinION platform. Sequence types, Capsule types, and O-antigen types were identified using the CGE, BIGSdb, and Kaptive databases, respectively. Virulence genes were detected through VFDB and BacWGSTdb, while plasmid profiles were characterized using PLSDB and PlasmidFinder. Results Among 244 K . pneumoniae bloodstream isolates screened, 10 were identified as hypermucoviscous by the string test. Of these, five representative isolates were selected for whole-genome sequencing. The DEUKp9961_18 isolate was identified as ST1322, DEUKp1212_19 as ST268, DEUKp4822_20 as ST461, DEUKp6718_20 as ST14, and DEUKp5889_19 as ST29. These isolates carried the KL139, K20, K62, K2, and K54 capsule types, respectively, and all exhibited the O2a antigen type. All isolates carried genes encoding Type 3 fimbriae, Type I fimbriae, efflux pumps, aerobactin, enterobactin, salmochelin, yersiniabactin, and T6SS-associated virulence factors. Additionally, two isolates carried plasmid-encoded virulence determinants. Capsule regulation genes ( rmpA/rmpA2 ) and aerobactin-associated genes ( iucABCD and iutA ) were located on the IncFIB(pNDM_Mar)–IncHI1(pNDM_MAR)–IncR plasmid of DEUKp6718_20, whereas these genes, together with the salmochelin-related genes ( iroBCDN ), were identified on the repB plasmid of DEUKp5889_19. Conclusions Although two isolates carried genomic determinants associated with hypervirulence, these findings should not be interpreted as evidence of a confirmed hypervirulent phenotype because no functional virulence assays were performed. Confirmation of hypervirulence requires evidence from invasive clinical disease together with functional and/or in vivo virulence studies.