Biology subjects

Wyres, K. L.

Publications and source records attributed to Wyres, K. L..

36 records · Page 2Linked to original sources

bioRxiv2021

Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae

Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening more than ten thousand genomes, 500 thousand metagenomes, and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.

microbiology↗

bioRxiv2021

Recovery of small plasmid sequences via Oxford Nanopore sequencing

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of [~]4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols. Impact statementResearchers who use Oxford Nanopore Technologies (ONT) platforms to sequence bacterial genomes can currently choose from two library preparation methods. The first is a ligation-based approach, which uses ligase to attach sequencing adapters to the ends of DNA molecules. The second is a rapid approach, which uses a transposase enzyme to cleave DNA and attach adapters in a single step. There are advantages to each preparation, for example ligation can produce better yields but rapid is a simpler procedure. Our study reveals another advantage of rapid preparations: they are more effective at sequencing small plasmids. We show that sequencing of ligation-based libraries yields fewer reads derived from small plasmids, making such plasmids harder to detect in bacterial genomes. Since small plasmids can contain clinically relevant genes, including antimicrobial resistance (AMR) or virulence determinants, their exclusion could lead to unreliable conclusions that have serious consequences for AMR surveillance and prediction. We therefore recommend that researchers performing ONT-only sequencing of bacterial genomes should consider using rapid preparations whenever small plasmid recovery is important. Data summarySupplementary figures, tables, data and code can be found at: github.com/rrwick/Small-plasmid-Nanopore

bioinformatics↗

bioRxiv2020

Genomic surveillance framework and global population structure for Klebsiella pneumoniae

K. 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.

genomics↗

bioRxiv2020

Detection of plasmid contigs in draft genome assemblies using customized Kraken databases

Plasmids play an important role in bacterial evolution and mediate horizontal transfer of genes including virulence and antimicrobial resistance genes. Although short-read sequencing technologies have enabled large-scale bacterial genomics, the resulting draft genome assemblies are often fragmented into hundreds of discrete contigs. Several tools and approaches have been developed to identify plasmid sequences in such assemblies, but require trade-off between sensitivity and specificity. Here we propose using the Kraken classifier, together with a custom Kraken database comprising known chromosomal and plasmid sequences of Klebsiella pneumoniae species complex (KpSC), to identify plasmid-derived contigs in draft assemblies. We assessed performance using Illumina-based draft genome assemblies for 82 KpSC isolates, for which complete genomes were available to supply ground truth. When benchmarked against five other classifiers (Centrifuge, RFPlasmid, mlplasmids, PlaScope, and Platon), Kraken showed balanced performance in terms of overall sensitivity and specificity (90.8% and 99.4%, respectively for contig count; 96.5% and >99.9%, respectively for cumulative contig length), and the highest accuracy (96.8% vs 91.8%-96.6% for contig count; 99.8% vs 99.0%-99.7% for cumulative contig length), and F1-score (94.5% vs 84.5%-94.1%, for contig count; 98.0% vs 88.9%-96.7% for cumulative contig length). Kraken also achieved consistent performance across our genome collection. Furthermore, we demonstrate that expanding the Kraken database with additional known chromosomal and plasmid sequences can further improve classification performance. Although we have focused here on the KpSC, this methodology could easily be applied to other species with a sufficient number of completed genomes. IMPACT STATEMENTThe assembly of bacterial genomes using short-read data often results in hundreds of discrete contigs due to the presence of repeat sequences in those genomes. Separating plasmid contigs from chromosomal contigs in such assemblies is required, e.g., to assess the mobility of antimicrobial resistance genes. Although several tools have been developed for that purpose, they often suffer from low sensitivity or specificity. Here, we propose that the Kraken classifier coupled with a custom Kraken database comprising plasmid-free chromosomal sequences and complete plasmid sequences can be used for detection of plasmid contigs in draft genome assemblies. We showed that Kraken achieved balanced and higher performance compared with other methods (Centrifuge, RFPlasmid, mlplasmids, PlaScope, and Platon). We therefore consider that the Kraken classifier can be the best option for predicting the origin of contigs for species with a suitable number of completed chromosomal and plasmid sequences. DATA SUMMARYTable S1: Complete chromosomes used for creating the base Kraken database. Plasmid-free chromosomal sequences and complete plasmid sequences used for creating the base Kraken database are also available via Figshare at https://doi.org/10.6084/m9.figshare.13289564. Table S2: Sequence data used for benchmarking. Draft assemblies of these 82 KpSC strains are available via Figshare at https://doi.org/10.6084/m9.figshare.13553432. The corresponding sequence read files and complete genomes were deposited in the NCBI SRA and GenBank under BioProjects PRJEB6891, PRJNA351909, PRJNA486877, and PRJNA646837 (individual BioSample IDs listed in Table S2). Kraken output files are available via Figshare at https://doi.org/10.6084/m9.figshare.13553789.

microbiology↗

bioRxiv2020

Properties of genes encoding transfer RNAs as integration sites for genomic islands and prophages in Klebsiella pneumoniae

The evolution of traits including antibiotic resistance, virulence, and increased fitness in Klebsiella pneumoniae and related species has been linked to the acquisition of mobile genetic elements through horizontal transfer. Among them, genomic islands (GIs) preferentially integrating at genes encoding tRNAs and the tmRNA (t(m)DNAs) would be significant in promoting chromosomal diversity. Here, we studied the whole set of t(m)DNAs present in 66 Klebsiella chromosomes, investigating their usage as integration sites and the properties of the integrated GIs. A total of 5,624 t(m)DNAs were classified based on their sequence conservation, genomic context, and prevalence. 161 different GIs and prophages were found at these sites, hosting 3,540 gene families including various related to virulence and drug resistance. Phylogenetic analyses supported the acquisition of several of these elements through horizontal gene transfer, likely mediated by a highly diverse set of encoded integrases targeting specific t(m)DNAs and sublocations inside them. Only a subset of the t(m)DNAs had integrated GIs and even identical tDNA copies showed dissimilar usage frequencies, suggesting that the genomic context would influence the integration site selection. This usage bias, likely towards avoiding disruption of polycistronic transcriptional units, would be conserved across Gammaproteobacteria. The systematic comparison of the t(m)DNAs across different strains allowed us to discover an unprecedented number of K. pneumoniae GIs and prophages and to raise important questions and clues regarding the fundamental properties of t(m)DNAs as targets for the integration of mobile genetic elements and drivers of bacterial genome evolution and pathogen emergence.

genomics↗

bioRxiv2019

Identification of Acinetobacter baumannii loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with Kaptive

Multiply antibiotic resistant Acinetobacter baumannii infections are a global public health concern and accurate tracking of the spread of specific lineages is needed. Variation in the composition and structure of capsular polysaccharide (CPS), a critical determinant of virulence and phage susceptibility, makes it an attractive epidemiological marker. The outer core (OC) of lipooligosaccharide also exhibits variation. To take better advantage of the untapped information available in whole genome sequences, we have created a curated reference database of the 92 publicly available gene clusters at the locus encoding proteins responsible for biosynthesis and export of CPS (K locus), and a second database for the 12 gene clusters at the locus for outer core biosynthesis (OC locus). Each entry has been assigned a unique KL or OCL number, and is fully annotated using a simple, transparent and standardised nomenclature. These databases are compatible with Kaptive, a tool for in silico typing of bacterial surface polysaccharide loci, and their utility was validated using a) >630 assembled A. baumannii draft genomes for which the KL and OCL regions had been previously typed manually, and b) 3386 A. baumannii genome assemblies downloaded from NCBI. Among the previously typed genomes, Kaptive was able to confidently assign KL and OCL types with 100% accuracy. Among the genomes retrieved from NCBI, Kaptive detected known KL and OCL in 87% and 90% of genomes, respectively indicating that the majority of common KL and OCL types are captured within the databases; 13 KL were not detected in any public genome assembly. The failure to assign a KL or OCL type may indicate incomplete or poor-quality genomes. However, further novel variants may remain to be documented. Combining outputs with multi-locus sequence typing (Institut Pasteur scheme) revealed multiple KL and OCL types in collections of a single sequence type (ST) representing each of the two predominant globally-distributed clones, ST1 of GC1 and ST2 of GC2, and in collections of other clones comprising >20 isolates each (ST10, ST25, and ST140), indicating extensive within-clone replacement of these loci. The databases are available at https://github.com/katholt/Kaptive and will be updated as further locus types become available. Data Summary1. Databases including fully annotated gene cluster sequences for A. baumannii K loci and OC loci are available for download at https://github.com/katholt/Kaptive 2. The Kaptive software, which can be used to screen new genomes against the K and O locus database is available at https://github.com/katholt/Kaptive (command-line code) and http://kaptive.holtlab.net/ (interactive web service). 3. Details of the Kaptive search results validating in silico serotyping of K and O loci using our approach are provided as supplementary files, Dataset 1 (92 KL reference sequences and 12 OCL reference sequences), Dataset 2 (642 genomes assembled from reads available in NCBI SRA) and Dataset 3 (3415 genome assemblies downloaded from NCBI GenBank). Impact statementThe ability to identify and track closely related isolates is key to understanding, and ultimately controlling, the spread of multiply antibiotic resistant A. baumannii causing difficult to treat infections, which are an urgent public health threat. Extensive variation in the KL and OCL gene clusters responsible for biosynthesis of capsule and the outer core of lipooligosaccharide, respectively, are potentially highly informative epidemiological markers. However, clear, well-documented identification of each variant and simple-to-use tools and procedures are needed to reliably identify them in genome sequence data. Here, we present curated databases compatible with the available web-based and command-line Kaptive tool to make KL and OCL typing readily accessible to assist epidemiological surveillance of this species. As many bacteriophage recognise specific properties of the capsule and attach to it, capsule typing is also important in assessing the potential of specific phage for therapy on a case by case basis.

microbiology↗

bioRxiv2019

Diversity and evolution of surface polysaccharide synthesis loci in Enterobacteriales

Bacterial capsules and lipopolysaccharides are diverse surface polysaccharides (SPs) that serve as the frontline for interactions with the outside world. While SPs can evolve rapidly, their diversity and evolutionary dynamics across different taxonomic scales has not been investigated in detail. Here, we focused on the bacterial order Enterobacteriales (including the medically-relevant Enterobacteriaceae), to carry out comparative genomics of two SP locus synthesis regions, cps and kps, using 27,334 genomes from 45 genera. We identified high-quality cps loci in 22 genera and kps in 11 genera, around 4% of which were detected in multiple species. We found SP loci to be highly dynamic genetic entities: their evolution was driven by high rates of horizontal gene transfer (HGT), both of whole loci and component genes, and relaxed purifying selection, yielding large repertoires of SP diversity. In spite of that, we found the presence of (near-)identical locus structures in distant taxonomic backgrounds that could not be explained by recent exchange, pointing to long-term selective preservation of locus structures in some populations. Our results reveal differences in evolutionary dynamics driving SP diversity within different bacterial species, with lineages of Escherichia coli, Enterobacter hormachei and Klebsiella aerogenes most likely to share SP loci via recent exchange; and lineages of Salmonella enterica, Citrobacter sakazakii and Serratia marcescens most likely to share SP loci via other mechanisms such as long-term preservation. Overall, the evolution of SP loci in Enterobacteriales is driven by a range of evolutionary forces and their dynamics and relative importance varies between different species.

evolutionary biology↗

bioRxiv2019

Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from south and southeast Asia.

BackgroundK. pneumoniae is a leading cause of blood stream infection (BSI). Strains producing extended spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial resistant (AMR) K. pneumoniae, and also for the characteristically antimicrobial sensitive, community-acquired hypervirulent strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. MethodsWe conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate. FindingsK. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common amongst South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes amongst K. pneumoniae. InterpretationSouth and Southeast Asia are high-risk regions for the emergence of AMR and convergent AMR-hypervirulent K. pneumoniae. Enhanced surveillance efforts, reporting STs, AMR and virulence information are urgently required to monitor this public health threat. FundingThis work was supported by the Wellcome Trust (grant #206194 to Wellcome Sanger Institute) and the Bill and Melinda Gates Foundation, Seattle (grant OPP1175797 to KEH). KEH is supported by a Senior Medical Research Fellowship from the Viertel Foundation of Australia. DAB and PNN are supported by the Wellcome Trust.

microbiology↗

bioRxiv2018

Convergence of virulence and multidrug resistance in a single plasmid vector in multidrug-resistant Klebsiella pneumoniae ST15

SYNOPSISO_ST_ABSBackgroundC_ST_ABSMultidrug resistance (MDR) and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options.\n\nObjectivesTo characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15.\n\nMethodsThe complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing.\n\nResultsBoth isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and eight other AMR genes (blaTEM, aac3-IIa, aph3-Ia, dfrA1, satA2, blaSHV, sul1, aadA1). The other carried remnants of these elements encoding blaTEM and aac3-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania.\n\nConclusionsThe presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.

genomics↗

bioRxiv2018

Yersiniabactin, Colibactin and Wider Resistome Contribute to Enhanced Virulence and Persistence of KPC-2-Producing Klebsiella pneumoniae CG258 in South America

The emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and outcome of infections caused by this pathogen. We performed a detailed virulome and resistome analysis of representative KPC- and/or CTX-M-producing K. pneumoniae belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. pneumoniae strains recovered from the human-animal-environment interface revealed a wide resistome characterized by the presence of genes and mutations conferring resistance to human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar way to hypervirulent K1/ST23 strain (ybt- and clb-negative) carrying the pLVPK-like plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and antimicrobial resistance, but also on the virulence factors ybt and clb.

microbiology↗

bioRxiv2018

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) has emerged as an important cause of two distinct public health threats: multidrug resistant (MDR) healthcare-associated infections1 and community-acquired invasive infections, particularly pyogenic liver abscess2. The majority of MDR hospital outbreaks are caused by a subset of Kp clones with a high prevalence of acquired antimicrobial resistance (AMR) genes, while the majority of community-acquired invasive infections are caused by hypervirulent clones that rarely harbour acquired AMR genes but have high prevalence of key virulence loci3-5. Worryingly, the last few years have seen increasing reports of convergence of MDR and the key virulence genes within individual Kp strains6, but it is not yet clear whether these represent a transient phenomenon or a significant ongoing threat. Here we perform comparative genomic analyses for 28 distinct Kp clones, including 6 hypervirulent and 8 MDR, to better understand their evolutionary histories and the risks of convergence. We show that MDR clones are highly diverse with frequent chromosomal recombination and gene content variability that far exceeds that of the hypervirulent clones. Consequently, we predict a much greater risk of virulence gene acquisition by MDR Kp clones than of resistance gene acquisition by hypervirulent clones.

microbiology↗

bioRxiv2018

Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae

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.\n\nMethodsHere we examined the genetic diversity, distribution and mobilisation of iuc and iro loci among 2503 K. pneumoniae genomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance.\n\nResultsIro 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 FII conjugative transfer regions and some imported from E. coli; the exceptions were iro3(mobilised by ICEKp1), and iuc4 (fixed in the chromosome of K. pneumoniae subspecies rhinoscleromatis). Iro/iuc 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.\n\nConclusionsIuc 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.

genomics↗

bioRxiv2018

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307

Recent reports indicate the emergence of a new carbapenemase producing Klebsiella pneumoniae clone, ST307. Here we show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 extended-spectrum beta-lactamase (ESBL) gene plus other antimicrobial resistance determinants. Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance is now emerging.

microbiology↗

bioRxiv2018

Kaptive Web: user-friendly capsule and lipopolysaccharide serotype prediction for Klebsiella genomes

As whole genome sequencing becomes an established component of the microbiologists toolbox, it is imperative that researchers, clinical microbiologists and public health professionals have access to genomic analysis tools for rapid extraction of epidemiologically and clinically relevant information. For the gram-negative hospital pathogens such as Klebsiella pneumoniae, initial efforts have focused on detection and surveillance of antimicrobial resistance genes and clones. However, with the resurgence of interest in alternative infection control strategies targeting Klebsiella surface polysaccharides, the ability to extract information about these antigens is increasingly important.\n\nHere we present Kaptive Web, an online tool for rapid typing of Klebsiella K and O loci, which encode the polysaccharide capsule and lipopolysaccharide O antigen, respectively. Kaptive Web enables users to upload and analyse genome assemblies in a web browser. Results can be downloaded in tabular format or explored in detail via the graphical interface, making it accessible for users at all levels of computational expertise.\n\nWe demonstrate Kaptive Webs utility by analysis of >500 K. pneumoniae genomes. We identify extensive K and O locus diversity among 201 genomes belonging to the carbapenemase- associated clonal group 258 (25 K and six O loci). Characterisation of a further 309 genomes indicates that such diversity is common among the multi-drug resistant clones and that these loci represent useful epidemiological markers for strain subtyping. These findings reinforce the need for rapid, reliable and accessible typing methods such as Kaptive Web.\n\nKaptive Web is available for use at kaptive.holtlab.net and source code is available at github.com/kelwyres/Kaptive-Web.

microbiology↗

bioRxiv2017

Population genomics of hypervirulent Klebsiella pneumoniae clonal group 23 reveals early emergence and rapid global dissemination

Since the mid-1980s there have been increasing reports of severe community-acquired pyogenic liver abscess, meningitis and bloodstream infections caused by hypervirulent Klebsiella pneumoniae, predominantly encompassing clonal group (CG) 23 serotype K1 strains. Common features of CG23 include a virulence plasmid associated with iron scavenging and hypermucoidy, and a chromosomal integrative and conjugative element (ICE) encoding the siderophore yersiniabactin and the genotoxin colibactin. Here we investigate the evolutionary history and genomic diversity of CG23 based on comparative analysis of 98 genomes. Contrary to previous reports with more limited samples, we show that CG23 comprises several deep branching sublineages dating back to the 1870s, many of which are associated with distinct chromosomal insertions of ICEs encoding yersiniabactin. We find that most liver abscess isolates (>80%) belong to a dominant sublineage, CG23-I, which emerged in the 1920s following acquisition of ICEKp10 (encoding colibactin in addition to yersiniabactin) and has undergone clonal expansion and global dissemination within the human population. The unique genomic feature of CG23-I is the production of colibactin, which has been reported previously as a promoter of gut colonisation and dissemination to the liver and brain in a mouse model of CG23 K. pneumoniae infection, and has been linked to colorectal cancer. We also identify an antibiotic-resistant subclade of CG23-I associated with sexually-transmitted infections in horses dating back to the 1980s. These data show that hypervirulent CG23 K. pneumoniae was circulating in humans for decades before the liver abscess epidemic was first recognised, and has the capacity to acquire and maintain AMR plasmids. These data provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To further support such studies we present an open access and completely sequenced human liver abscess isolate, SGH10, which is typical of the globally disseminated CG23-I sublineage.

genomics↗

bioRxiv2017

Antimicrobial resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital

BackgroundKlebsiella pneumoniae is a leading cause of extended-spectrum beta-lactamase (ESBL) producing hospital-associated infections, for which elderly patients are at increased risk.\n\nMethodsWe conducted a 1-year prospective cohort study, in which a third of patients admitted to two geriatric wards in a specialized hospital were recruited and screened for carriage of K. pneumoniae by microbiological culture. Clinical isolates were monitored via the hospital laboratory. Colonizing and clinical isolates were subjected to whole genome sequencing and antimicrobial susceptibility testing.\n\nResultsK. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8% and ESBL carriage 1.7%. K. pneumoniae infection incidence was 1.2%. The isolates were diverse, and most patients were colonized or infected with a unique phylogenetic lineage, with no evidence of transmission in the wards. ESBL strains carried blaCTX-M-15 and belonged to clones associated with hospital-acquired ESBL infections in other countries (ST29, ST323, ST340).\n\nOne also carried the carbapenemase blaIMP-26. Genomic and epidemiological data provided evidence that ESBL strains were acquired in the referring hospital. Nanopore sequencing also identified strain-to-strain transmission of a blaCTX-M-15 FIBK/FIIK plasmid in the referring hospital.\n\nConclusionsThe data suggest the major source of K. pneumoniae was the patients own gut microbiome, but ESBL strains were acquired in the referring hospital. This highlights the importance of the wider hospital network to understanding K. pneumoniae risk and infection control. Rectal screening for ESBL organisms upon admission to geriatric wards could help inform patient management and infection control in such facilities.\n\nSummaryPatients own gut microbiota were the major source of K. pneumoniae, but extended-spectrum beta-lactamase strains were acquired in the referring hospital. This highlights the potential for rectal screening, and the importance of the wider hospital network, for local risk management.

microbiology↗

bioRxiv2017

Best Practice Data Life Cycle Approaches for the Life Sciences

Throughout history, the life sciences have been revolutionised by technological advances; in our era this is manifested by advances in instrumentation for data generation, and consequently researchers now routinely handle large amounts of heterogeneous data in digital formats. The simultaneous transitions towards biology as a data science and towards a life cycle view of research data pose new challenges. Researchers face a bewildering landscape of data management requirements, recommendations and regulations, without necessarily being able to access data management training or possessing a clear understanding of practical approaches that can assist in data management in their particular research domain.\n\nHere we provide an overview of best practice data life cycle approaches for researchers in the life sciences/bioinformatics space with a particular focus on omics datasets and computer-based data processing and analysis. We discuss the different stages of the data life cycle and provide practical suggestions for useful tools and resources to improve data management practices.

bioinformatics↗

bioRxiv2017

Frequent emergence of pathogenic lineages of Klebsiella pneumoniae viamobilisation of yersiniabactin and colibactin

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 bacterias ability to cause disease. MGEs carrying antimicrobial resistance (AMR) and/or virulence determinants are common in opportunistic pathogen Klebsiella pneumoniae, which are 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.\n\nHere we apply population genomics to investigate the prevalence, evolution and mobility of ybt and clb in K. pneumoniae populations through comparative analysis of 2,498 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 Enterobacteriaceae population showed 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 [≥]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 show 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.\n\nAUTHOR SUMMARYKlebsiella pneumoniae infections are becoming increasingly difficult to treat with antibiotics. Some K. pneumoniae strains also carry extra genes that allow them to synthesise yersiniabactin, an iron-scavenging molecule, which enhances their ability to cause disease. These genes are located on a genetic element that can easily transfer between strains. Here, we screened 2498 K. pneumoniae genome sequences and found substantial diversity in the yersiniabactin genes and the associated genetic elements, including a novel mechanism of transfer, and detected hundreds of distinct yersiniabactin acquisition events between K. pneumoniae strains. We show that these yersiniabactin mobile genetic elements are specifically adapted to the K. pneumoniae population but also occasionally acquired by other bacterial members belonging to the Enterobacteriaceae family such as E. coli. These insights into the movement and genetics of yersiniabactin genes allow tracking of the evolution and spread of yersiniabactin in global K. pneumoniae populations and monitoring for acquisition of yersiniabactin in antibiotic-resistant strains.

microbiology↗