Genomic analysis of the diversity, virulence and antimicrobial resistance in clinical klebsiella pneumoniae from China

Abstract

Klebsiella pneumoniae is an opportunistic pathogen which could cause both community- and hospital-acquired infections. Hypervirulent K. pneumoniae (hvKp) and multidrug resistant K. pneumoniae represent the major branches of K. pneumoniae species which are commonly associated with human infections. K. pneumoniae relies on a battery of gene products to escape the innate immune mechanisms of the host; these include capsule, siderophores, lipopolysaccharide (LPS), fimbriae, outer membrane protein (OMPs), porins, efflux pump and transporters. HvKp strains normally exhibit enhanced capsules which could be semi-quantitatively defined by a "string test". They are mostly associated with serotype K1 or K2, with CC23 being the dominant K1 hvKp clone, whereas several genetically unrelated groups (ST25, ST86, etc.) constitute the K2 clone. K. pneumoniae is becoming untreatable by the last-line antibiotics since many strains exhibited resistance to multiple drugs, especially the extended-spectrum ß-lactam (ESBL) and carbapenems. Classic K. pneumoniae (cKp) frequently acquire the antimicrobial resistance phenotype through horizontal transfer of resistance genes. The majority of KPC-producing K. pneumoniae worldwide belong to the notorious CC258 clone (including ST258, ST11, ST340, ST437 and ST512). Several other clonal groups (CG) were also globally distributed and associated with multidrug resistance, including CG14/15, CG17/20, CG43 and CG147. Whole genome sequencing (WGS) is a revolutionized technology which has been widely used in both academia and clinical settings. The application of WGS in studies of K. pneumoniae mainly covers analysis of evolution trends and transmission of lineages of interest, as well as identification of genetic features of epidemiological or clinical importance. To gain better understanding of this notorious pathogen and to further control its evolution and transmission, we initiated this project. Through analyzing the genome sequences of clinical ST11 K. pneumoniae strains from China, we revealed the genetic relationship of different ST11 isolates, and identified the genetic elements associated with virulence traits and antimicrobial resistances phenotypes. We also tracked a fatal outbreak of T11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital and characterized CR-hvKp isolates that belong to different sequence types. First, we conducted comprehensive genomic analysis of 58 clinical strains of ST11 K. pneumoniae, which is the dominant KPC-producing clinical clone in China. We found that these strains, collected from geographically diverse locations in the country, were genetically diverse and could be segregated into three clades, each of which exhibited distinct capsule polysaccharide loci. Various antimicrobial resistance genes and virulence genes, such as those encoding salmochelin, aerobactin and RmpA, the hallmarks of hypervirulent K. pneumoniae, were detected in the genome of these strains. Results in this part showed that ST11 carbapenemase-resistant, hypervirulent K. pneumoniae strains have widely emerged in China, and provided insight for development of strategies for prevention, diagnosis and treatment of ST11 clinical infections. Second, we investigated a fatal ventilator-associated pneumonia outbreak of ST11 carbapenemase-resistant, hypervirulent K. pneumoniae (CR-hvKp) in patients in a Chinese hospital. ST11 CR-hvKp have disseminated across various regions of China, which accounted for around 3% of clinical ST11 carbapenem-resistant K. pneumoniae infections in China. ST11 CR-hvKp exhibits a hypervirulence phenotype which was characterized by a positive string test result, extremely high survival upon exposure to human neutrophils, and high virulence in a wax moth (Galleria mellonella) larva infection model. The emergence of ST11 CR-hvKp strains was due to acquisition of a 170 kbp virulence plasmid carrying the rmpA2 and aerobactin biosynthesis genes by classic ST11 carbapenem-resistant K. pneumoniae strains. We suggest future research to focus on the development of intervention measures for the prevention of further dissemination of CR-hvKp isolates in hospital settings. Lastly, we delineated the genetic structure of two different types of carbapenem-resistant, hypervirulent K. pneumoniae. The first belonged to ST23 CR-hvKP whose emergence was due to IS26-mediated insertion of blaKPC-2 into a pLVPK-like virulence plasmid in ST23 hypervirulent K. pneumoniae. The second was an ST11 CR-hvKP isolate which carries 5 copies of blaKPC-2, which were introduced by Tnp26-encoded activity of the translocatable units. In the two cases, the carbapenem-resistance and hypervirulence phenotypes were both mediated by the plasmid(s) each strain harbored, indicating occurrence of an alarming evolutionary event of K. pneumoniae, the trend of which should be carefully monitored in the future. The emergence of diverse CR-hvKp isolates suggest that such organisms could cause severe infections in both hospital settings and the community. This study identified genetic elements associated with virulence traits and antimicrobial resistances phenotypes in K. pneumoniae and demonstrated the emergence of different types of carbapenem-resistant, hypervirulent K. pneumoniae via plasmid-mediated gene transfer. Findings in this study provide valuable information for future control of K. pneumoniae infections in China and neighboring countries.