Hypervirulence and carbapenem resistance have emerged as two distinct evolutionary directions for Klebsiella pneumoniae, which pose a great threat in clinical settings. Multiple virulence factors contribute to hypervirulence, and the mechanisms of carbapenem resistance are complicated. However, more and more K. pneumoniae strains have been identified in recent years integrating both phenotypes, resulting in devastating clinical outcomes. Hypervirulent and carbapenem-resistant K. pneumoniae (CR-hvKP) emerged in the early 2010s and thereafter have become increasingly prevalent. CR-hvKP are primarily prevalent in Asia, especially China, but are reported all over the world. Mechanisms for the emergence of CR-hvKP can be summarised by three patterns: (i) carbapenem-resistant K. pneumoniae (CRKP) acquiring a hypervirulent phenotype; (ii) hypervirulent K. pneumoniae (hvKP) acquiring a carbapenem-resistant phenotype; and (iii) K. pneumoniae acquiring both a carbapenem resistance and hypervirulence hybrid plasmid. With their global dissemination, continued surveillance of the emergence of CR-hvKP should be more highly prioritised.

An increasing prevalence of hospital acquired infections and foodborne illnesses caused by pathogenic and multidrug-resistant bacteria has stimulated a pressing need for benchtop computational techniques to rapidly and accurately classify bacteria from genomic sequence data, and based on that, to trace the source of infection. BacWGSTdb (http://bacdb.org/BacWGSTdb) is a free publicly accessible database we have developed for bacterial whole-genome sequence typing and source tracking. This database incorporates extensive resources for bacterial genome sequencing data and the corresponding metadata, combined with specialized bioinformatics tools that enable the systematic characterization of the bacterial isolates recovered from infections. Here, we present BacWGSTdb 2.0, which encompasses several major updates, including (i) the integration of the core genome multi-locus sequence typing (cgMLST) approach, which is highly scalable and appropriate for typing isolates belonging to different lineages; (ii) the addition of a multiple genome analysis module that can process dozens of user uploaded sequences in a batch mode; (iii) a new source tracking module for comparing user uploaded plasmid sequences to those deposited in the public databases; (iv) the number of species encompassed in BacWGSTdb 2.0 has increased from 9 to 20, which represents bacterial pathogens of medical importance; (v) a newly designed, user-friendly interface and a set of visualization tools for providing a convenient platform for users are also included. Overall, the updated BacWGSTdb 2.0 bears great utility in continuing to provide users, including epidemiologists, clinicians and bench scientists, with a one-stop solution to bacterial genome sequence analysis.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused increasing attention, especially ST11, the predominant CRKP clone in China. Here, we report a case of hv-CRKP-associated infection and reveal the in-host evolution of its mechanism of resistance to tigecycline and polymyxin under clinical therapy. A total of 11 K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from a male patient who suffered from continuous and multisite infections. String and antimicrobial susceptibility tests identified seven hypermucoviscous strains and three tigecycline-resistant and four colistin-resistant strains. Galleria mellonella larvae infection model confirmed the hypervirulence. Pulsed-field gel electrophoresis (PFGE) separated three PFGE clusters among all strains, and further Southern blotting detected that bla_KPC-2 was located on the same-sized plasmid. Whole-genome sequencing showed that all strains belonged to the hv-CRKP ST11-KL64 clone. Diverse hypervirulence factors and resistance genes were identified. Further sequencing with the Nanopore platform was performed on the CRKP-Urine1 strain, which contained one virulence plasmid (pVi-CRKP-Urine1) and two resistance plasmids (pKPC-CRKP-Urine1 and pqnrS1-CRKP-Urine1). The gene mutations responsible for tigecycline or colistin resistance were then amplified with PCR followed by sequencing, which indicated that mutations of ramR and lon were the potential loci for tigecycline resistance and that the pmrB, phoQ and mgrB genes for colistin resistance. A novel frameshift mutation of lon was identified in the high-level tigecycline-resistant strain (MIC, 128 mg/L). The results indicate that the hypervirulent ST11-KL64 clone is a potential threat to antiinfection treatment and is capable of rapid and diverse evolution of resistance during tigecycline and polymyxin treatment.

Multidrug-resistant bacterial infections are becoming increasingly common, with only few last-resort antibiotics such as colistin available for clinical therapy. An alternative therapeutic strategy gaining momentum is phage therapy, which has the advantage of not being affected by bacterial resistance to antibiotics. However, a major challenge in phage therapy is the rapid emergence of phage-resistant bacteria. In this work, our main aim was to understand the mechanisms of phage-resistance used by the top priority pathogen Acinetobacter baumannii. We isolated the novel phage Phab24, capable of infecting colistin-sensitive and -resistant strains of A. baumannii. After co-incubating Phab24 with its hosts, we obtained phage-resistant mutants which were characterized on both genotypic and phenotypic levels. Using whole genome sequencing, we identified phage-resistant strains that displayed mutations in genes that alter the architecture of the bacterial envelope at two levels: the capsule and the outer membrane. Using an adsorption assay, we confirmed that phage Phab24 uses the bacterial capsule as its primary receptor, with the outer membrane possibly serving as the secondary receptor. Interestingly, the phage-resistant isolates were less virulent compared to the parental strains in a Galleria mellonella infection model. Most importantly, we observed that phage-resistant bacteria that evolved in the absence of antibiotics exhibited an increased sensitivity to colistin, even though the antibiotic resistance mechanism per se remained unaltered. This increase in antibiotic sensitivity is a direct consequence of the phage-resistance mechanism, and could potentially be exploited in the clinical setting.

Graphical Overall BacAnt’s service workflow. The figure is adapted with permissions from IntegronFinder (Cury et al., 2016).