No abstract

Klebsiella pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes.

Mobile genetic elements (MGEs) that frequently transfer within and between bacterial species play a critical role in bacterial evolution, and often carry key accessory genes that associate with a bacteria’s ability to cause disease. MGEs carrying antimicrobial resistance (AMR) and/or virulence determinants are common in the opportunistic pathogen Klebsiella pneumoniae, which is a leading cause of highly drug-resistant infections in hospitals. Well-characterised virulence determinants in K. pneumoniae include the polyketide synthesis loci ybt and clb (also known as pks), encoding the iron-scavenging siderophore yersiniabactin and genotoxin colibactin, respectively. These loci are located within an MGE called ICEKp, which is the most common virulence-associated MGE of K. pneumoniae, providing a mechanism for these virulence factors to spread within the population. Here we apply population genomics to investigate the prevalence, evolution and mobility of ybt and clb in K. pneumoniae populations through comparative analysis of 2498 whole-genome sequences. The ybt locus was detected in 40 % of K. pneumoniae genomes, particularly amongst those associated with invasive infections. We identified 17 distinct ybt lineages and 3 clb lineages, each associated with one of 14 different structural variants of ICEKp. Comparison with the wider population of the family Enterobacteriaceae revealed occasional ICEKp acquisition by other members. The clb locus was present in 14 % of all K. pneumoniae and 38.4 % of ybt+ genomes. Hundreds of independent ICEKp integration events were detected affecting hundreds of phylogenetically distinct K. pneumoniae lineages, including at least 19 in the globally-disseminated carbapenem-resistant clone CG258. A novel plasmid-encoded form of ybt was also identified, representing a new mechanism for ybt dispersal in K. pneumoniae populations. These data indicate that MGEs carrying ybt and clb circulate freely in the K. pneumoniae population, including among multidrug-resistant strains, and should be considered a target for genomic surveillance along with AMR determinants.

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K . pneumoniae lineages or ‘clones’ that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated (‘hypervirulent’) clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K . pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K . pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

BackgroundKlebsiella pneumoniae is a recognised agent of multidrug-resistant (MDR) healthcare-associated infections; however, individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulent K. pneumoniae clones that cause severe community-associated infections such as liver abscess and pneumonia. Concerningly, iuc has also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in the K. pneumoniae population.MethodsHere, we examined the genetic diversity, distribution and mobilisation of iuc and iro loci amongst 2503 K. pneumoniae genomes using comparative genomics approaches and developed tools for tracking them via genomic surveillance.ResultsIro and iuc were detected at low prevalence (< 10%). Considerable genetic diversity was observed, resolving into five iro and six iuc lineages that show distinct patterns of mobilisation and dissemination in the K. pneumoniae population. The major burden of iuc and iro amongst the genomes analysed was due to two linked lineages (iuc1/iro1 74% and iuc2/iro2 14%), each carried by a distinct non-self-transmissible IncFIBK virulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids of K. pneumoniae. The other iuc and iro lineages were associated with diverse plasmids, including some carrying IncFII conjugative transfer regions and some imported from Escherichia coli; the exceptions were iro3 (mobilised by ICEKp1) and iuc4 (fixed in the chromosome of K. pneumoniae subspecies rhinoscleromatis). Iro/iuc mobile genetic elements (MGEs) appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc.) but were also detected at low prevalence in others such as MDR strain ST258.ConclusionsIuc and iro are mobilised in K. pneumoniae via a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDR K. pneumoniae strains.Electronic supplementary materialThe online version of this article (10.1186/s13073-018-0587-5) contains supplementary material, which is available to authorized users.