Klebsiella pneumoniae is an important Gram-negative opportunistic pathogen that causes serious and fatal community-acquired and nosocomial bacterial infections worldwide and is regarded as a critical threat to public health. Development of carbapenem resistance and hypervirulence have been regarded as two different evolutionary directions of K. pneumoniae that led to the emergence of epidemic and high-risk strains. Among them, carbapenem-resistant K. pneumoniae (CRKP) strains have posed an imminent threat to clinical treatment of K. pneumoniae infections due to the extended phenotypic drug resistance and high transmissibility of such strains, which are known to cause infections of much higher mortality than their carbapenem-susceptible counterparts. Hypervirulent K. pneumoniae (hvKP) has also emerged as a superbug that causes severe pyogenic liver abscess and aggressive infections. For a long period of time, hypervirulence and multidrug resistance were considered as two non-overlapping phenotypes, since CRKP isolates are less virulent and hvKP isolates are generally sensitive to antimicrobial agents. These two types of strains usually belong to separate clonal lineages, as CRKP generally belong to sequence type (ST) 11 and ST258, and hvKP are mostly strains of ST23, ST86 and ST65 types. However, an increasing number of K. pneumoniae isolates that encode both phenotypes have recently been identified, suggesting that carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) strains, which can cause infections that are even more fatal and harder to treat when compared to infections caused by CRKP and hvKP, have emerged. Almost all CR-hvKP isolates reported to date could be categorized into three evolutionary patterns: i) typical hvKP lineages which have acquired extra carbapenem resistance determinants through horizontal transfer of mobile elements; ii) CRKP lineages which have acquired a pLVPK-like virulence plasmid; and iii) carriage of both carbapenem resistance and virulence genes in a single plasmid, which is the most worrying trend as such plasmids may rapidly disseminate among clinical bacterial strains, rendering them multidrug-resistant and hypervirulent. Recent evidence showed that these newly emerged CR-hvKP strains start to cause severe invasive infections of high mortality worldwide, even among healthy individuals. Understanding the genetic basis of multidrug-resistance and hypervirulence phenotypes in K. pneumoniae is essential for the development of effective strategies to prevent the further dissemination of such notorious superbugs. Therefore, it is urgent to identify key multidrug-resistant and hypervirulent K. pneumoniae strains, monitor their transmission routes and evolution trend, as well as develop valid control strategies to prevent worldwide dissemination of these clinically important pathogens.

In this study, we performed genomic and phenotypic characterization of clinical multidrug-resistant and hypervirulent K. pneumoniae strains, revealed the molecular mechanisms underlying the emergence and rapid dissemination of CR-hvKP isolates, and investigated the impact of bla_KPC-2-bearing plasmid and pLVPK-like virulence plasmid in mediating the virulence expression, phenotypic fitness and adaptation of ST11 CR-hvKP strains in clinical settings, as well as developed a potential therapeutic strategy to eradicate these clinically notorious CR-hvKP strains.

In the first section, we characterized four clinical multidrug-resistant and hypervirulent K. pneumoniae isolates and investigated the molecular mechanisms underlying the transmission of virulence plasmids. First, we discovered a conjugative plasmid that encodes carbapenem resistance and hypervirulence in a clinical ST86 K2 CR-hvKP isolate. The conjugative plasmid p17ZR-91-Vir-KPC was formed by fusion of a non-conjugative pLVPK-like plasmid and a conjugative bla_KPC-2-bearing plasmid and was present dynamically with two other non-fusion plasmids. Conjugation of plasmid p17ZR-91-Vir-KPC to other K. pneumoniae enabled them to rapidly express the carbapenem resistance and hypervirulence phenotypes. More importantly, genome analysis provided direct evidence that p17ZR-91-Vir-KPC could be directly transmitted from K2 CR-hvKP isolates to ST11 clinical K. pneumoniae strains, transforming them into ST11 CR-hvKP. This finding elucidates the evolutionary mechanisms of the recently emerged ST11 CR-hvKP strains. Second, a clinical carbapenem-resistant K. pneumoniae isolate 16ZR-187 which harbors a novel IncFIA plasmid that can be fused to a hypervirulence-encoding plasmid to form a hybrid conjugative virulence plasmid was characterized. Such fusion events involved homologous recombination between a 241bp homologous region located in each of the two plasmids. The fusion hypervirulence-encoding plasmid can be conjugated to both classic and carbapenem-resistant K. pneumoniae strains through conjugation, enabling such strains to acquire the ability to express the hypervirulence phenotype. Third, we reported the emergence of an ST11 K64 K. pneumoniae isolate which was resistant to third-generation cephalosporin and exhibited a moderate level of virulence. Whole genome sequencing revealed that this isolate harbored a plasmid pHB25-1, which carried multidrug resistance genes (bla_DHA-1, qnrB4, dfrA12, aadA2, sul1, aac(3)-lld, bla_TEM-1 and mph(E)) and virulence-encoding genes (regulator of mucoid phenotype A2 gene rmpA2 and aerobactin gene cluster iutAiucABCD). Genomic analysis indicated that plasmid pHB25-1 was formed though co-integration of structural regions located in two different plasmids, enabling it to encode both resistance and virulence phenotypes. These findings provide evidence of active plasmid evolution in clinical K. pneumoniae strains and suggest that surveillance of multidrug-resistant and hypervirulent K. pneumoniae is urgently needed. Fourth, we characterized clinical carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae isolates that belong to ST367 and K1 and contain several virulence genes, including those which encode salmochelin (iroBCDN), aerobactin (iucABCDiutA), regulator of mucoid phenotype (rmpA/A2) as well as various resistance genes, including bla_KPC-2. These carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae strains, which contain various virulence genes, exhibited a higher level of virulence and serum resistance than the classic K. pneumoniae strains, but their virulence levels were slightly lower when compared with typical ST11 CR-hvKP and ST23 K1 hvKP strains. This study reported for the first time the genetic and virulence characteristics of clinical K. quasipneumoniae subsp. similipneumoniae strains that simultaneously contained bla_KPC-2 and virulence genes, and provides better understanding of their underlying mechanisms of resistance and pathogenicity, as well as their epidemiological features. These findings also provide important insight into the prevention of worldwide dissemination of multidrug-resistant and hypervirulent K. pneumoniae, and development of new approaches for treatment of infections caused by such pathogens.

In the second section, to investigate factors that promote dissemination of CR-hvKP in clinical settings in China, we assessed the functional characteristics of four isogenic K. pneumoniae strains, namely a non-resistant and non-hypervirulent isolate, and three transconjugants which have acquired the virulence plasmid, bla_KPC-2-bearing plasmid or both (designated as HKU3, HKU3-Vir, HKU3-KPC and HKU3-KPC-Vir). Acquisition of virulence plasmid by hvKP and CR-hvKP led to reduced fitness and abolishment of colonization in rat gastrointestinal tract, which explained why hvKP are not prevalent after emergence for a long time. However, treatment with tigecycline facilitated the colonization of hvKP and CR-hvKP. Tigecycline was shown to reduce the population of Lactobacillus spp. in gut microbiome and feeding with Lactobacillus spp. could significantly reduce the colonization of CR-hvKP in rat. Our data implied that clinical use of tigecycline to treat CRKP infections facilitated the wide spread of CR-hvKP in clinical settings in China.

In the third section, we screened a collection of compounds that can dissipate bacterial proton motive force (PMF) and intermediate metabolites that can suppress antibiotic tolerance, and identified an antifungal drug, sulconazole, that act in combination with glucose or trehalose to exert strong antibacterial effect against starvation-induced CR-hvKP persisters. Investigation of the underlying mechanisms showed that sulconazole caused dissipation of transmembrane PMF, and that sulconazole act in combination with glucose or trehalose to significantly inhibit the efflux activity, reduce NADH and ATP levels, and cause intracellular accumulation of reactive oxygen species (ROS) in CR-hvKP persisters, eventually resulting in bacterial death. These findings suggested that sulconazole, when used in combination with glucose or trehalose, was highly effective in eradicating multidrug-resistant and hypervirulent K. pneumoniae persisters; this is likely a feasible strategy for treatment of chronic and recurrent K. pneumoniae infections.

In conclusion, this study characterized clinical CR-hvKP isolates and revealed the mechanisms underlying the emergence and rapid dissemination. Findings in this study provide valuable information for further control of K. pneumoniae infections. Active surveillance should be implemented to prevent the further dissemination of CR-hvKP. It is urgent to develop effective therapeutic guidelines to combat K. pneumoniae infections and establish an extensive collaborative network of scientific and medical personnel to perform this daunting task.