The increasing incidence of carbapenem-resistant Enterobacteriaceae (CRE) - mediated hospital infections in China prompted a need to investigate the genetic basis of emergence of such strains. A nationwide survey was conducted in China covering a total of 1105 CRE strains collected from 25 geographical locales with results showing that acquisition of two carbapenemase genes, blaKPC-2 and blaNDM, was responsible for phenotypic resistance in 90% of the CRE strains tested (58% and 32% respectively), among which several major strain types, such as ST11 of K. pneumoniae and ST131/ST167 of E. coli, were identified, suggesting that dissemination of specific resistant clones is mainly responsible for emergence of new CRE strains. Prevalence of the fosA3 gene which mediates fosfomycin resistance, was high, while the colistin resistance determinant mcr-1 was rarely present in these isolates. Consistently, the majority of the blaNDM-bearing plasmids recoverable from the test strains belonged to IncX3, which contained a common core structure, blaNDM-blaMBL-trpF. Likewise, the core structure of ISKpn27-blaKPC-2-ISKpn2 was observed among plasmids harboring the blaKPC-2 gene, although they were genetically more divergent. In conclusion, the increasing prevalence of CRE strains in China is attributed to dissemination of conservative mobile elements carrying blaNDM or blaKPC-2 on conjugative and non-conjugative plasmids.

The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated.

Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.

Co-occurrence of hypervirulence and KPC-2 carbapenem resistant phenotypes in a highly-transmissible ST11 clone of Klebsiella pneumoniae has elicited deep concerns from public health stand point. To address this puzzle, we conducted a large-scale epidemiological, clinical and genomic study of K. pneumonia ST11 clones with both hypervirulence and carbapenem resistance in two tertiary hospitals in Zhejiang province. Most of the patients (15/23) were diagnosed with exclusively carbapenem-resistant K . pneumoniae (CRKP) infections. Ten death cases were reported, some of which are due to the failure of antibiotic therapies. As a result, we identified one new rare sequence types (ST449) to KPC-2-producing CRKP, in addition to the dominant ST11. These clinical isolates of K. pneumoniae are multi-drug resistant and possess a number of virulence factors. Experimental infections of wax moth larvae revealed the presence of hypervirulence at varied level, suggesting the complexity in bacterial virulence factors. However, plasmid curing assays further suggested that the rmpA2 -virulence plasmid is associated with, but not sufficient for neither phenotypic hypermucoviscosity nor virulence of K . pneumoniae . Intriguingly, all the rmpA2 genes were found to be inactive due to genetic deletion. In total, we reported 21 complete plasmid sequences comprising 13 rmpA2 -positive virulence plasmids and 8 bla KPC-2 -harboring resistance plasmids. In addition to the prevalent pLVKP-like virulence plasmid variants (~178kb), we found an unexpected diversity among KPC-2-producing plasmids whose dominant form is IncFII-IncR type (~120kb), rather than the previously anticipated version of ~170kb. These findings provide an updated snapshot of convergence of hypervirulence and carbapenem resistance in ST11 K. pneumoniae.