Phenotypic and genotypic study of hypervirulent Klebsiella pneumoniae (hvKp) and molecular characterization of its resistance and virulence determinants

Abstract

Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a significant pathogen due to its increasing incidence. Originally considered an opportunistic bacterium responsible for nosocomial infections in immunocompromised individuals, it now poses a substantial threat by causing severe infections, such as pneumonia, liver abscesses, or bacteremia, in previously healthy individuals. To comprehend the genetic and phenotypic basis of this virulence, we performed both phenotypic and genotypic characterization in hvKp identified during daily routine screening of suspected hvKp isolates in a university hospital in Spain to i) identify genes related to resistance and virulence, ii) quantify biofilm formation, iii) measure motility, iv) conduct conjugation assays, and v) assess the virulence potential in infected Galleria mellonella larvae. Whole-genome sequencing results revealed a high prevalence of key genes in both virulence (magA, wzy_K2, fim) and resistance (fosA and blaSHV). Additionally, biofilm production results varied greatly, likely due to the numerous factors involved in its formation. Surprisingly, mobile strains were found for all three types of motility studied (swimming, swarming, and twitching), despite being considered K. pneumoniae a non-motile bacteria. The conjugation of an OXA-48- encoding plasmid was demonstrated in a MDR-hvKp isolate. Infections in Galleria mellonella larvae confirmed the high virulence of selected strains, showing the virulence capability of these isolates. These findings suggest that K. pneumoniae hypervirulence is multifactorial, involving a complex interplay of genetic and phenotypic factors, which complicates the identification of specific virulence determinants and therefore treatment strategies.