A Single-Reaction Multiplex PCR Assay for Rapid Screening and Epidemiological Analysis of Hypervirulent Klebsiella pneumoniae

Hypervirulent Klebsiella pneumoniae (HvKp) causes invasive infections, and rapid clone estimation and virulence gene detection are important for treatment and infection control. We modified a previously developed PCR-based open reading frame typing (POT) method and developed HvKp-POT, a single-reaction 13-plex PCR assay for simultaneous clone estimation and detection of hypervirulence-associated genes. The assay includes a K. pneumoniae marker, six ORFs for clone estimation, and targets for K1, K2, iucA, iroB, rmpA/A2, and peg-344. We validated the assay using 106 genome-analyzed Klebsiella strains, including 44 HvKp strains. POT results were expressed as HvKp-POT-1 for clone estimation, and HvKp-POT-2 for virulence gene detection. HvKp-POT-1 values corresponded to major HvKp clones and showed one-to-one concordance with sequence types in 75 of 95 K. pneumoniae strains. Strains with the same POT-1 value sometimes included both HvKp and classical K. pneumoniae, but these were generally distinguished using HvKp-POT-2. HvKp-POT may be useful for rapid HvKp screening and epidemiological analysis.