Klebsiella pneumoniae is a Gram-negative bacillus that belongs to the genus Klebsiella and family Enterobacteriaceae. This organism is widely distributed in plants, soil, water and drains, and can also colonize animal hosts as well as human hosts. It is a human commensal and opportunistic pathogen that can cause severe hospital-acquired infections, especially among patients with a compromised immune system. However, over the last few decades, a number of K. pneumoniae strains have acquired additional antibiotic resistance genes or virulence factors and became either antibiotic resistant or hypervirulent; infections caused by these strains are significantly more serious than those caused by the classic strains. In K. pneumoniae, the most common mechanism of resistance is the expression of β-lactamases, including extended-spectrum β-lactamases, cephalosporinases and carbapenemases, which can hydrolyze the most frequently used β-lactam antibiotics; some of these enzymes can even act on the last sort antibiotic carbapenem. Hypervirulent K. pneumoniae can cause serious, life-threatening community-acquired infections in young and relatively healthy individuals. K. pneumoniae employs a variety of virulence factors to evade and inhibit the host immune response, colonize in various body sites of the host and obtain nutrition from host. Several virulence factors have been well characterized, including capsule polysaccharide, siderophores, lipopolysaccharide and fimbriae. Due to the fast and global dissemination of hypervirulent and multidrug resistant strains, K. pneumoniae is rapidly becoming one of the most challenging pathogens. It is urgent to identify the key hypervirulent and multidrug resistant strains and monitor their transmission trend in order to prevent further dissemination of this rapidly evolving pathogen.

In this study, we performed genomic analysis of clinical K. pneumoniae isolates collected from mainland China and Hong Kong, identified distinct sets of accessory genes associated with virulence and antimicrobial resistance, revealed the molecular mechanisms underlying the generation of the carbapenem resistant hypervirulent K. pneumoniae (CR-HvKP) strains and the dissemination of the virulence and antimicrobial resistances determinants, as well as studied the functional roles of plasmid-borne virulence factors in virulence expression. Firstly, a total of 784 blaKPC-2-bearing CRKP strains collected from three hospitals located at different geographical locales in China during the period 2014-2017 were subjected to molecular typing, screening of virulence plasmid, string test and WGS (367/784 strains). The proportion of CRKP among all clinical K. pneumoniae strains increased sharply in China during 2014-2017. A large proportion (58%) of these CRKP strains were found to harbor a virulence-encoding plasmid, yet only 13% of such strains exhibited a hypervirulence phenotype defined by string test and neutrophil assay. The lack of hypervirulence phenotype in virulent plasmid-bearing CRKP strains was found to be due to mutational changes in the rmpA and rmpA2 genes, which rendered them non-functional. However, some strains carrying the wild type rmpA gene did not exhibit a hypervirulence phenotype, suggesting that other factors might also contribute to the hypervirulence of CRKP. Phylogenetic and SNP analysis indicated that the transmission of these CRKP strains in China likely involved several major clones of ST11. Carriage of IncFII pSWU01-like, bla_KPC-2-bearing plasmid was found to be the major mechanism of carbapenem resistance in these CRKP strains.

Secondly, mechanisms underlying transmission of the virulence determinants were characterized through the study of four strains. First, a carbapenem-resistant K. variicola strain 15WZ-82 that carried a conjugative plasmid which harbored the key genes of the virulence plasmid commonly found in HvKP strains and encoded the hypervirulence phenotype has been characterized. This hypervirulence plasmid was created by integration of a 100-kbp hypervirulence-encoding region of the hypervirulence plasmid pLVPK into a conjugative IncFIB type plasmid. This resultant hypervirulence plasmid could be readily transferred to other K. pneumoniae strains via conjugation and rendered the recipient hypervirulent, posing unprecedented threat to human health. Furthermore, genetic re-arrangement and recombination events were observed during transmission of this virulence plasmid from the donor strain to the recipient strains; such events were mediated by various homologous recombination activities. Next, a carbapenem-resistant and highly virulent K. pneumoniae strain 16HN-263 has also been characterized. The virulence-encoding genes rmpA2 and iucABCDiutA were found to be located in the chromosome and flanked by IS26 elements. Detailed analysis of the genetic fragment in which these virulence genes are located showed that the fragment can readily form a circular intermediate which may promote integration of this virulence-encoding fragment into various plasmid backbones and other chromosomal regions. Next, a ST11 carbapenem-resistant K. pneumoniae strain SH12 which harbored an IncFIB/IncHI1B type virulence plasmid and an IncFII/IncR type bla_KPC-2-bearing plasmid was characterized. The virulence plasmid was found to be conjugative and harbored a 35-kbp fragment including aerobactin encoding cluster from virulence plasmid pLVPK and multiple resistance genes, resulting in formation of a mosaic multi-drug resistance and virulence plasmid. This virulence plasmid could be transferred via conjugation to Escherichia coli and K. pneumoniae strains, either alone or together with the bla_KPC-2-bearing plasmid. Co-transmission of virulence and bla_KPC-2-bearing plasmids would directly convert a classic K. pneumoniae strain into CR-HvKP strain; such event may be responsible for causing a sharp increase in the prevalence of CR-HvKP in clinical settings and poses a great threat to human health. Last, five ST11 CR-HvKP strains which harbored pLVPK-like virulence plasmids were characterized. Virulence plasmids in these CR-HvKP strains could be transferred to E. coli strain EC600 via conjugation. Transmission of the virulence plasmids was found to involve formation of fusion plasmids that involve an Incl1 type conjugative plasmid and a small ColRNAI plasmid. Upon conjugation to transconjugants, these plasmids may exist in different forms, including various formats of fusion plasmids and individual plasmids. The Incl1 plasmid was considered a helper plasmid due to its ability to undergo conjugation, whereas the functional roles of the small ColRNAI plasmid remained unknown. These fusion processes involved homologous recombination between genetically homologous regions located in the virulence plasmid and each of the other two plasmids, and also could be mediated by the insertion sequences IS26 and IS903B. The conjugative fusion event would transform different ST types of K. pneumoniae, in particular the clinically prevalent ST11 or ST258 CRKP, into CR-HvKP. These data provide new insight into mechanisms of transmission of virulence-encoding genes in K. pneumoniae and broaden our understanding of the mechanisms by which virulence plasmid evolves. Most importantly, the generation of different conjugative plasmids containing the virulence region of the virulence plasmid might dramatically increase the transmission efficiency and host spectrum, facilitating rapid transmission of hypervirulence-encoding plasmid among Gram-negative bacteria.

Contributions of the virulence profiles to the virulence level of a certain strain were evaluated. Genes encoding plasmid borne virulence factors including rmp homologues, aerobactin and salmochelin siderophores have been amplified and introduced into a ST11 CRKP strain and a K1 strain, respectively. The plasmid bore rmp homologues were able to mediate hyper-HMV and CPS over-production in both ST11 CRKP and K1 K. pneumoniae. Furthermore, introduction of these factors independently did not result in virulence expression of these strains in mice; yet introduction of the rmp locus along with the siderophore caused an increase in the virulence level of these strains in mice. Our data provide background information that allows development of methodology to screen phenotypically hypervirulent CR-HvKP strains in clinical settings.

In conclusion, this study performed molecular epidemiology of K. pneumoniae strains, revealed the mechanisms underlying dissemination of the virulence determinants and expression of the observable virulence profiles. These data will provide valuable information for future control of K. pneumoniae infections.