Kelly L. Wyres

Identification of Klebsiella capsule synthesis loci from whole genome data

Klebsiella pneumoniae is a growing cause of healthcare-associated infections for which multi-drug resistance is a concern. Its polysaccharide capsule is a major virulence determinant and epidemiological marker. However, little is known about capsule epidemiology since serological typing is not widely accessible and many isolates are serologically non-typeable. Molecular typing techniques provide useful insights, but existing methods fail to take full advantage of the information in whole genome sequences. We investigated the diversity of the capsule synthesis loci (K-loci) among 2503 K. pneumoniae genomes. We incorporated analyses of full-length K-locus nucleotide sequences and also clustered protein-encoding sequences to identify, annotate and compare K-locus structures. We propose a standardized nomenclature for K-loci and present a curated reference database. A total of 134 distinct K-loci were identified, including 31 novel types. Comparative analyses indicated 508 unique protein-encoding gene clusters that appear to reassort via homologous recombination. Extensive intra- and inter-locus nucleotide diversity was detected among the wzi and wzc genes, indicating that current molecular typing schemes based on these genes are inadequate. As a solution, we introduce Kaptive, a novel software tool that automates the process of identifying K-loci based on full locus information extracted from whole genome sequences (https://github.com/katholt/Kaptive). This work highlights the extensive diversity of Klebsiella K-loci and the proteins that they encode. The nomenclature, reference database and novel typing method presented here will become essential resources for genomic surveillance and epidemiological investigations of this pathogen.

Extensive capsule locus variation and large-scale genomic recombination within the Klebsiella pneumoniae clonal group 258

Klebsiella pneumoniae clonal group (CG) 258, comprising sequence types (STs) 258, 11, and closely related variants, is associated with dissemination of the K. pneumoniae carbapenemase (KPC). Hospital outbreaks of KPC CG258 infections have been observed globally and are very difficult to treat. As a consequence, there is renewed interest in alternative infection control measures such as vaccines and phage or depolymerase treatments targeting the K. pneumoniae polysaccharide capsule. To date, 78 immunologically distinct capsule variants have been described in K. pneumoniae. Previous investigations of ST258 and a small number of closely related strains suggested that capsular variation was limited within this clone; only two distinct ST258 capsule polysaccharide synthesis (cps) loci have been identified, both acquired through large-scale recombination events (>50 kb). In contrast to previous studies, we report a comparative genomic analysis of the broader K. pneumoniae CG258 (n = 39). We identified 11 different cps loci within CG258, indicating that capsular switching is actually common within the complex. We observed several insertion sequences (IS) within the cps loci, and show further intraclone diversification of two cps loci through IS activity. Our data also indicate that several large-scale recombination events have shaped the genomes of CG258, and that definition of the complex should be broadened to include ST395 (also reported to harbor KPC). As only the second report of extensive intraclonal cps variation among Gram-negative bacterial species, our findings alter our understanding of the evolution of these organisms and have key implications for the design of control measures targeting K. pneumoniae capsules.

Klebsiella pneumoniae Population Genomics and Antimicrobial-Resistant Clones

Antimicrobial-resistant Klebsiella pneumoniae (Kp) has emerged as a major global public health problem. While resistance can occur across a broad range of Kp clones, a small number have become globally distributed and commonly cause outbreaks in hospital settings. Here we describe recent comparative genomics investigations that have shed light on Kp population structure and the evolution of antimicrobial-resistant clones. These studies provide the basic framework within which genomic epidemiology and evolution can be understood, but have merely scratched the surface of what can and should be explored. We assert that further large-scale comparative and functional genomics studies are urgently needed to better understand the biology of this clinically important bacterium.

WGS Analysis and Interpretation in Clinical and Public Health Microbiology Laboratories: What Are the Requirements and How Do Existing Tools Compare?

Recent advances in DNA sequencing technologies have the potential to transform the field of clinical and public health microbiology, and in the last few years numerous case studies have demonstrated successful applications in this context. Among other considerations, a lack of user-friendly data analysis and interpretation tools has been frequently cited as a major barrier to routine use of these techniques. Here we consider the requirements of microbiology laboratories for the analysis, clinical interpretation and management of bacterial whole-genome sequence (WGS) data. Then we discuss relevant, existing WGS analysis tools. We highlight many essential and useful features that are represented among existing tools, but find that no single tool fulfils all of the necessary requirements. We conclude that to fully realise the potential of WGS analyses for clinical and public health microbiology laboratories of all scales, we will need to develop tools specifically with the needs of these laboratories in mind.

The diversity of Klebsiella pneumoniae surface polysaccharides

Klebsiella pneumoniae is considered an urgent health concern due to the emergence of multi-drug-resistant strains for which vaccination offers a potential remedy. Vaccines based on surface polysaccharides are highly promising but need to address the high diversity of surface-exposed polysaccharides, synthesized as O-antigens (lipopolysaccharide, LPS) and K-antigens (capsule polysaccharide, CPS), present in K. pneumoniae. We present a comprehensive and clinically relevant study of the diversity of O- and K-antigen biosynthesis gene clusters across a global collection of over 500 K. pneumoniae whole-genome sequences and the seroepidemiology of human isolates from different infection types. Our study defines the genetic diversity of O- and K-antigen biosynthesis cluster sequences across this collection, identifying sequences for known serotypes as well as identifying novel LPS and CPS gene clusters found in circulating contemporary isolates. Serotypes O1, O2 and O3 were most prevalent in our sample set, accounting for approximately 80 % of all infections. In contrast, K serotypes showed an order of magnitude higher diversity and differ among infection types. In addition we investigated a potential association of O or K serotypes with phylogenetic lineage, infection type and the presence of known virulence genes. K1 and K2 serotypes, which are associated with hypervirulent K. pneumoniae, were associated with a higher abundance of virulence genes and more diverse O serotypes compared to other common K serotypes.

Differential host susceptibility and bacterial virulence factors driving Klebsiella liver abscess in an ethnically diverse population

Hypervirulent Klebsiella pneumoniae is an emerging cause of community-acquired pyogenic liver abscess. First described in Asia, it is now increasingly recognized in Western countries, commonly afflicting those with Asian descent. This raises the question of genetic predisposition versus geospecific strain acquisition. We leveraged on the Antibiotics for Klebsiella Liver Abscess Syndrome Study (A-KLASS) clinical trial ongoing in ethnically diverse Singapore, to prospectively examine the profiles of 70 patients together with their isolates’ genotypic and phenotypic characteristics. The majority of isolates belonged to capsule type K1, a genetically homogenous group corresponding to sequence-type 23. The remaining K2, K5, K16, K28, K57 and K63 isolates as well as two novel cps isolates were genetically heterogeneous. K1 isolates carried higher frequencies of virulence-associated genes including rmpA (regulator of mucoid phenotype A), kfu (Klebsiella ferric uptake transporter), iuc (aerobactin), iro (salmochelin) and irp (yersiniabactin) than non-K1 isolates. The Chinese in our patient cohort, mostly non-diabetic, had higher prevalence of K1 infection than the predominantly diabetic non-Chinese (Malays, Indian and Caucasian). This differential susceptibility to different capsule types among the various ethnic groups suggests patterns of transmission (e.g. environmental source, familial transmission) and/or genetic predisposition unique to each race despite being in the same geographical location.

Frequent emergence of pathogenic lineages of Klebsiella pneumoniae via mobilisation 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 bacteria9s 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. Here 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.Klebsiella pneumoniae (Kp) is a commensal bacterium that causes opportunistic infections. Evidence is mounting that Kp strains carrying acquired siderophores (yersiniabactin, salmochelin and aerobactin) and/or the genotoxin colibactin are highly pathogenic and can cause invasive disease. Here we explored the diversity of the Kp integrative conjugative element (ICEKp), which mobilises the yersiniabactin locus ybt, by comparing 2499 diverse Kp genomes. We identified 17 distinct ybt lineages and 14 ICEKp structural variants (some of which carry colibactin (clb) or salmochelin synthesis loci). Hundreds of ICEKp transmission events were detected affecting hundreds of Kp lineages, including nearly >20 transfers into the globally-disseminated, carbapenem-resistant clonal group CG258. Additionally, we identify a plasmid-encoded lineage of ybt, representing a new mechanism for ybt dispersal in Kp populations. We introduce a novel sequence-based typing approach for identifying ybt and clb variants, to aid the identification of emerging pathogenic lineages and the convergence of antibiotic resistance and hypervirulence. SIGNIFICANCE Klebsiella pneumoniae infections are increasingly difficult to treat with antibiotics. Some K. pneumoniae 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 2499 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 also developed tools to identify and type yersiniabactin genes, to help track the evolution and spread of yersiniabactin in global K. pneumoniae populations and to monitor for acquisition of yersiniabactin in antibiotic-resistant strains.

Paradoxical Hypersusceptibility of Drug-resistant Mycobacteriumtuberculosis to β-lactam Antibiotics

Mycobacterium tuberculosis (M. tuberculosis) is considered innately resistant to β-lactam antibiotics. However, there is evidence that susceptibility to β-lactam antibiotics in combination with β–lactamase inhibitors is variable among clinical isolates, and these may present therapeutic options for drug-resistant cases. Here we report our investigation of susceptibility to β-lactam/β–lactamase inhibitor combinations among clinical isolates of M. tuberculosis, and the use of comparative genomics to understand the observed heterogeneity in susceptibility. Eighty-nine South African clinical isolates of varying first and second-line drug susceptibility patterns and two reference strains of M. tuberculosis underwent minimum inhibitory concentration (MIC) determination to two β-lactams: amoxicillin and meropenem, both alone and in combination with clavulanate, a β–lactamase inhibitor. 41/91 (45%) of tested isolates were found to be hypersusceptible to amoxicillin/clavulanate relative to reference strains, including 14/24 (58%) of multiple drug-resistant (MDR) and 22/38 (58%) of extensively drug-resistant (XDR) isolates. Genome-wide polymorphisms identified using whole-genome sequencing were used in a phylogenetically-aware linear mixed model to identify polymorphisms associated with amoxicillin/clavulanate susceptibility. Susceptibility to amoxicillin/clavulanate was over-represented among isolates within a specific clade (LAM4), in particular among XDR strains. Twelve sets of polymorphisms were identified as putative markers of amoxicillin/clavulanate susceptibility, five of which were confined solely to LAM4. Within the LAM4 clade, ‘paradoxical hypersusceptibility’ to amoxicillin/clavulanate has evolved in parallel to first and second-line drug resistance. Given the high prevalence of LAM4 among XDR TB in South Africa, our data support an expanded role for β-lactam/β-lactamase inhibitor combinations for treatment of drug-resistant M. tuberculosis.

Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations.

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.

Antimicrobial-Resistant Klebsiella pneumoniae Carriage and Infection in Specialized Geriatric Care Wards Linked to Acquisition in the Referring Hospital

Patients’ own gut microbiota were the major source of Klebsiella pneumoniae, but extended-spectrum β-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.