Biology subjects

Holt, K. E.

Publications and source records attributed to Holt, K. E..

At least 19 recordsLinked to original sources

bioRxiv2026

A capsule polysaccharide synthesis locus database for the Klebsiella oxytoca Species Complex

The Klebsiella oxytoca Species Complex (SC) represents an emerging healthcare-associated group of opportunistic pathogens. The capsular polysaccharide is a virulence determinant and target for novel vaccines, monoclonal antibodies and phage therapy. In the absence of broadly accessible phenotyping techniques, prediction of capsule types from whole-genome sequence data is critical for understanding capsule diversity and epidemiology, and to prioritise capsule types as targets for novel anti-K. oxytoca SC interventions. Here we present the first comprehensive capsule synthesis locus (K locus) database targeted for the K. oxytoca SC, comprising 88 distinct loci defined by gene content and which is compatible with the rapid genome typing tool Kaptive. The database provides high coverage of publicly available K. oxytoca SC genomes (97.6% of 2,244 genomes, dereplicated from a total of 4,055), and the typing rate is significantly higher than that achieved with the pre-existing Klebsiella K locus database (97.6% vs 50.3%, p <0.0001), which primarily targets the Klebsiella pneumoniae SC. We demonstrate the utility of the novel K. oxytoca SC database by application to three diverse clinical K. oxytoca SC isolate collections (n=61 to 102 genomes each), suggesting a high diversity of K types. The novel K. oxytoca SC K locus database (github.com/klebgenomics/KoSC-surface-antigen-loci) will provide a key resource to support larger systematic studies and ongoing genomics surveillance efforts for the K. oxytoca SC. IMPACT STATEMENTMembers of the Klebsiella oxytoca Species Complex (SC) are an emerging cause of infections in humans and are frequently associated with antimicrobial resistance. Klebsiella species produce two key surface antigen sugars (capsular polysaccharide and lipopolysaccharide) that are immunogenic and are targets for novel control strategies such as vaccines and phage therapy. Phenotypic typing of these surface antigen sugars (serotyping) is costly and laborious, with genotyping (predicting the serotype from whole-genome sequence data) a useful alternative. Here, we present a curated capsule (K) locus reference database for the K. oxytoca SC, which represents a useful tool to assist in the epidemiological surveillance of this emerging pathogen. DATA SUMMARYAll Klebsiella oxytoca Species Complex genomes used in this work were publicly available, with accession details listed in Supplementary Tables 1, 2 and 3. The K. oxytoca Species Complex K locus reference database is available under GNU public license at github.com/klebgenomics/KoSC-surface-antigen-loci.

bioinformatics↗

bioRxiv2026

Bacterial diversity and strain dynamics in the infant respiratory microbiome during the first year of life

BackgroundMicrobial colonisation of the human body begins immediately after birth, with each body site forming a distinct ecological niche. While early gut microbial dynamics and their links to paediatric health have been considerably studied, the bacterial colonisation of the early infant respiratory system remains poorly understood, particularly at the species and strain level. ResultsHere, we generated and analysed whole genome sequencing of 925 isolates from six dominant genera in nasopharyngeal samples from 58 healthy infants enrolled in the Childhood Asthma Study (CAS) in Western Australia, collected longitudinally from birth (2, 6, and 12 months old). Our results expanded genomic reference catalogues and uncovered substantial strain-level diversity in dominant infant airway taxa. Plate-sweep metagenomics identified four microbiome profile groups (MPGs) with age-dependent membership, confirming prior observations while enabling high-resolution species and strain analyses. Community maturation was characterised by a shift from early Staphylococcus aureus dominance to increased Moraxella catarrhalis dominance by 12 months of age, alongside marked temporal changes in prevalence and cohort-level strain diversity. ConclusionsThese findings resolve infant nasopharyngeal microbiota composition at the species and strain level, revealing taxon-specific colonisation patterns. By substantially expanding publicly available reference genomes for underrepresented airway taxa, this work also provides a foundation for functional follow-up studies of the early respiratory microbiota and respiratory outcomes.

microbiology↗

bioRxiv2026

AMRgen: an R package for antimicrobial resistance genotype-phenotype analysis

Microbial whole-genome sequence data is now generated at scale, including to support antimicrobial resistance (AMR) surveillance and understand resistance mechanisms, yet analytical infrastructure for systematically linking AMR genotypes to measured phenotypes remains fragmented. Here we present AMRgen, an R package to support systematic AMR genotype-phenotype analysis. AMRgen imports and harmonises genotypic data from common bioinformatics tools, alongside phenotypic data from automated antimicrobial susceptibility testing instruments and public repositories. It supports common analyses linking data to reference distributions, modelling associations, quantifying concordance, and producing publication-ready visualisations including UpSet plots that jointly display genotypic marker combination frequencies and associated phenotypic distributions. We demonstrate AMRgens utility using publicly available surveillance data for World Health Organization priority AMR pathogens, Neisseria gonorrhoeae, Klebsiella pneumoniae, Escherichia coli and Salmonella enterica. AMRgen, available free and open-source at https://AMRgen.org, provides a reproducible end-to-end foundation for genotype-phenotype research in AMR genomics, clinical microbiology, and public health surveillance.

microbiology↗

bioRxiv2026

Verticall: A fast and robust tool for recombination detection in large-scale bacterial genomic datasets

The inference and removal of horizontally acquired genomic regions is a crucial step in phylogenomics analyses for evolutionary studies. Existing tools perform well on clonal lineage-focused datasets on the scale of hundreds of genomes, but are limited in their ability to analyse larger or more diverse datasets. Here we present Verticall, a tool to identify recombinant regions in bacterial assemblies and generate recombination-free phylogenies, which scales to thousands of genomes from clonal to genus-level diversity. Verticall uses a non-parametric approach to assign genomic regions as horizontally or vertically related based on the distribution of pairwise genetic distances between genomes. Recombination-free phylogenetic trees may be inferred by either calculating a pairwise genetic distance matrix from vertical-only regions (distance-tree approach) or by pairwise comparisons of all genomes to a reference and then masking horizontally acquired regions in a pseudo-alignment to the reference (alignment-tree approach). We demonstrate Verticalls performance using four publicly available whole-genome sequence datasets of varying sample sizes (range: 154 - 4,857 genomes) and evolutionary scales (ranging from within-lineage to genus-wide diversity). Across all four datasets, Verticall showed comparable or superior performance to the established tools Gubbins and ClonalFrameML in terms of computational efficiency, plausibility of inferred phylogenetic trees, and recovery of temporal signal for molecular dating. Our results show that Verticall is a useful tool to more efficiently and accurately detect recombination, particularly applied to datasets for which existing tools are limited, including large datasets with hundreds to thousands of genomes and those that span entire species or genera. Verticall is available free and open source at https://github.com/rrwick/Verticall. Impact StatementMany bacterial species can acquire genetic material from external sources and stably incorporate them into their own genomes through homologous recombination. During phylogenomic analyses to investigate outbreaks or for evolutionary studies, a core objective is often to reconstruct the evolutionary history of the studied organisms independent of these horizontally acquired genomic regions. This is particularly desirable when the aim is to construct dated phylogenies, as horizontally acquired variation can interfere with the molecular clock signal on which dating relies. Existing recombination detection programs perform well in certain contexts, but their algorithms are not suitable for datasets with very high diversity or thousands of genomes. We addressed this gap by developing the software package Verticall. We show this approach produces comparable results to existing software for smaller more clonal datasets, but also performs well on datasets that the existing packages cannot handle. Data SummaryVerticall is available free and open source at https://github.com/rrwick/Verticall. We used published whole-genome sequence data deposited in public databases (Pathogenwatch [https://pathogen.watch/]; European Nucleotide Archive [https://www.ebi.ac.uk/ena/], Sequence Read Archive [https://www.ncbi.nlm.nih.gov/sra/]). Accession numbers for the raw whole-genome sequences are presented in Tables S2-S6. All data, code, and analysis commands used to generate the results and figures presented in this paper are available on figshare (DOI: 10.6084/m9.figshare.31930821) and GitHub (https://github.com/erkison/verticall_paper).

bioinformatics↗

bioRxiv2026

Genomic analysis of Klebsiella pneumoniae causing community-acquired respiratory deaths among Zambian infants and children using targeted RNA-probe hybridization-capture metagenomics

Klebsiella pneumoniae (Kp) is a leading cause of neonatal and infant deaths in sub-Saharan Africa and frequently associated with antimicrobial resistance. Previously, we identified Kp as a major cause of fatal community-associated lower respiratory infections among infants and children under five years in Lusaka, Zambia, using postmortem tissue sampling and pathogen specific multiplex qPCR. In this follow-up study, we employed a novel culture-independent RNA-probe hybridization-capture metagenomic sequencing approach, targeting Kp pan-genome core and accessory genes, to perform in-depth genomic analysis of Kp from eleven post-mortem lung biopsy samples from seven of these children. Analysis detected Kp in all cases except one, which identified Klebsiella quasipneumoniae subspecies similipneumoniae. Core-genome multi-locus sequence typing (cgMLST) revealed six clonal groups (CG607, CG1123, CG10072, CG280, CG3648, and CG10344) belonging to five sublineages (SL607, SL17, SL280, SL37, and SL10072), with perfect concordance between paired samples from the same case. Two infants sampled the same month harbored SL607 lineages sharing 621 out of 629 cgMLST alleles, suggesting clonal spread. Kp capsule (K) loci were detected in all but one case and included potential vaccine targets KL25, KL23, and KL122. Antimicrobial resistance genes were widespread among samples, particularly encoding resistance toward aminoglycosides, {beta}-lactams, sulphonamides, tetracyclines, and trimethoprim. Extended spectrum {beta}-lactamases were identified in four cases, three of which were blaCTX-M-15. The acquired Kp sideophore yersiniabactin (lineage ybt14) was identified in both cases associated with SL607, and the acquired siderophore aerobactin (lineage iuc5) was identified in one of these, suggesting possible convergence of antimicrobial resistance and hypervirulence. The detection of Kp with extensive antimicrobial resistance causing fatal community acquired pneumonia signals a deeply concerning epidemiologic shift from a largely nosocomial pathogen. This calls for urgent epidemiological investigations to better understand the burden, transmission dynamics, antimicrobial resistances, and potential vaccine targets for Kp in other community settings across sub-Saharan Africa. Author SummaryKlebsiella pneumoniae is a major cause of infections and death among newborns and young children, particularly in low-income countries, where it is frequently resistant to antibiotics. While well-known as a hospital-associated pathogen, we previously showed K. pneumoniae is also a leading cause of fatal community lung infections among infants and children in Lusaka, Zambia. In this follow-on analysis, we performed deeper genetic analysis of K. pneumoniae detected from the cluster of community pneumonia deaths using lung tissue samples from seven of these children. Since traditional bacterial cultures were unavailable, we instead used a novel approach that enriched and sequenced specific regions of the K. pneumoniae genome directly from the biopsy samples without culturing bacterial isolates. We identified five different K. pneumoniae genetic subtypes, known as sublineages. Two sublineages, which came from children sampled the same month, were highly similar, suggesting clonal spread. Multiple acquired antimicrobial resistance genes were detected across all sublineages. Acquired virulence factors, which may cause more aggressive infections, were also detected in two cases. We also identified capsule types previously suggested as potential vaccine targets. This study underscores the urgent need to better understand and address the emerging burden of antibiotic-resistant K. pneumoniae pneumonia and other invasive infections among infants and children in community settings in sub-Saharan Africa.

microbiology↗

bioRxiv2025

The acquisition of rmpADC can increase virulence of classical Klebsiella pneumoniae in the absence of other hypervirulence-associated genes

Klebsiella pneumoniae is one of the most common causes of nosocomial infections, and the rise of drug-resistant K. pneumoniae strains is complicating treatment and contributing to a mounting global health crisis. K. pneumoniae has two pathotypes, classical (cKp) and hypervirulent (hvKp). CKp typically cause opportunistic infections in immunocompromised individuals in healthcare settings and often are multi-drug resistant. HvKp can be community acquired and cause high-mortality infections in immunocompetent individuals. Concerningly, antibiotic-resistant cKp strains with hypervirulence-associated genes and traits have recently emerged. Determining if and how hv-associated genes contribute to increased virulence of cKp strains is essential to addressing this growing threat. The rmpADC operon is an hv-associated locus that confers hypermucoviscosity (HMV), a key virulence phenotype, and rmp genes are often found in convergent strains. In this study, we aimed to determine if the rmp genes alone could increase the virulence of classical strains in the absence of other hv-associated genes. We introduced genetically distinct rmp loci from different lineages into a broad array of cKp isolates and found that, while many isolates became HMV positive, only a subset of these strains showed an increase in virulence in a mouse model of pneumonia. Sequence type and capsule type were not predictive of how rmp acquisition impacted the clinical isolates. Our results indicate that HMV is likely necessary but not sufficient for hypervirulence and that rmp sequence can influence virulence potential in classical strains. IMPORTANCEKlebsiella pneumoniae is a global pathogen, and gene exchange between hypervirulent (hvKp) and classical (cKp) strains is a rising threat. It is essential to understand how hvKp genes impact virulence phenotypes and identify the cKp strain backgrounds most amenable to enhanced virulence. Hypermucoviscosity (HMV) is a critical virulence factor in hypervirulent K. pneumoniae, conferred by the rmpADC locus. The rmp genes are encoded on mobile genetic elements and have been detected in convergent antibiotic-resistant K. pneumoniae strains of concern. In this study, we explored the impact of rmp acquisition in a broad set of classical clinical isolates. We observed that HMV appears necessary, but not sufficient, for increased virulence. Sequence type, capsule type, and HMV capacity could not predict which classical isolates gain an rmp-dependent colonization benefit. These insights increase our understanding of the distinctions between cKp and hvKp and further our ability to identify and treat new strains of concern.

microbiology↗

bioRxiv2025

Simple Sequence Repeats Mediate Phase Variation of the Mucoid Phenotype in Hypervirulent Klebsiella pneumoniae

Hypervirulent Klebsiella pneumoniae (HVKp) express a thick mucoid capsular polysaccharide which is essential for virulence and survival in the bloodstream but inhibits epithelial adhesion and invasion. The molecular mechanisms regulating capsule expression to rapidly adapt to these distinct niches are unknown. Here, we show that high frequency mutations in a poly-G repeat in the regulator of mucoid phenotype gene, rmpA, mediates reversible ON/OFF phase variation of the mucoid phenotype. We find that these mutations generate subpopulations of bacteria with reduced capsule expression and mucoidy. In vitro and in vivo infection models reveal that phase variation to the OFF state facilitates epithelial cell infection while ON switching enhances resistance to the bactericidal activity of human serum and systemic spread to the liver and spleen in a murine infection model. Genomic analysis reveals that the phase variation mechanism is a conserved feature of all HVKp and that additional novel DNA repeats in rmpD and rmpC drive different combinations of capsule mucoidy and hyperexpression phenotypes. These findings establish a mechanistic basis for how HVKp occupy such a diverse array of niches during infection, with important implications for the design and implementation of Kp-specific vaccines and therapies.

microbiology↗

bioRxiv2025

EnviroAmpDesigner, a tool for designing high specificity multiplex PCR primer panels for detecting and subtyping a target organism in environmental surveillance samples

BackgroundAmplicon sequencing is a popular method for understanding the diversity of bacterial communities in environmental or similar samples as exemplified by 16S rRNA sequencing. This approach has been extended into multiplex amplicon sequencing in which multiple targets are amplified in the same PCR reaction such as virus sequencing using tiled amplicons. Multiple tools exist to design PCR primers, and some support design of multiplex panels. However, despite increasing interest in the use of environmental or wastewater sampling for detecting and typing specific antimicrobial resistant (AMR) bacteria such as typhoid or cholera agents, we were unable to find a tool for designing a multiplex PCR panel for environmental samples that not only focused on detection of a specific organism, but also on amplifying lineage-specific or AMR-associated alleles of the target organism while minimising amplification of off-target genomes present in the sample. We found that existing tools either depend on the target organism being very distinct from the rest of the organisms in the sample, or focus on detection rather than genotyping of the organism of interest. We have developed EnviroAmpDesigner (v0.1.3, DOI: 10.5281/zenodo.14967337) to fill this gap, which we used to design a multiplex amplicon panel for detection, genotyping and AMR profiling of the typhoidal pathogens Salmonella enterica serovars Typhi and Paratyphi A. EnviroAmpDesigner can design amplicons for both short and long-read sequencing. The software first identifies single nucleotide polymorsphisms (SNPs) that distinguish individual genotypes of the target pathogen (in our case, S. Typhi). It then identifies SNPs that distinguish the target organism from others. Finally, the software designs primers that simultaneously target genotyping SNPs and have at least one primer with a 3 end at each SNP that distinguishes the target organism. The purpose of the latter condition is to increase the specificity of the primers, to minimise amplification of homologous sequences in non-target organisms. While our use case focuses on S. Typhi and Paratyphi A, the tool is organism agnostic and should work for any haploid organism. The tool is freely available via Bioconda and GitHub (https://github.com/AntonS-bio/EnviroAmpDesigner). Impact statementWe have developed EnviroAmpDesigner (v 0.1.3) software to design a multiplexed panel of long-read amplicons for detection and lineage typing of bacteria in environmental samples. We apply this tool to Salmonella Typhi and Paratyphi A, the bacterial agents of typhoid fever, but the tool is organism agnostic. The tool is computationally efficient and can be used on mid-range laptop. It has potential to support more widespread deployment of sequencing-based surveillance for bacterial pathogens in environmental and wastewater samples. Data SummaryFor the purposes of designing primers for S. Typhi and Paratyphi A we used 13,134 samples from the Global Typhoid Genomics Consortium data (target organisms) (1) and 1,810 publicly available Enterobacterales genomes from NCBIs RefSeq database (representing off-target related organisms) (2). To validate the primers, we applied an amplicon sequencing protocol (3) to DNA extracted from two S. Typhi isolates (NCBI accessions SRR5949979 and SRR7165748) provided by Satheesh Nair (UKHSA) (4), and a pooled DNA sample of American Type Culture Collection (ATCC) isolates S. Paratyphi A (ATCC 9150D), S. Paratyphi B (ATCC-BAA-1250D), S. Paratyphi C (ATCC-BAA-1715D), Aeromonas hydrophila (ATCC-7965D), Klebsiella pneumoniae (ATCC-BAA-1706D), and Citrobacter freundii (ATCC-8090D) chosen for their close relationship to S. Typhi. The resulting amplicon sequence data was deposited in European Nucleotide Archive (project accession: PRJEB81565).

bioinformatics↗

bioRxiv2025

Enhanced virulence and stress tolerance are signatures of epidemiologically successful Shigella sonnei

Shigellosis is a leading cause of diarrhoeal deaths worldwide, with Shigella sonnei increasingly implicated as a dominant agent. S. sonnei is divided into five monophyletic lineages all sharing a single O-antigen, yet most contemporary infections are caused by just a few clonal sub-lineages within globally dominant Lineage 3 that are quite distinct from the widely used Lineage 2 laboratory strain 53G. Factors underlying the success of these globally dominant lineages remain poorly understood in part due to a lack of complete genome sequences and available animal models. Here, we utilise a novel reference collection of representative Lineage 1, 2 and 3 isolates with complete genome sequences, and find that epidemiologically successful S. sonnei harbour fewer genes encoding putative immunogenic components whilst key virulence-associated regions (such as the type three secretion system and O-antigen) remain highly conserved. Using a zebrafish infection model, we discover that Lineage 3 isolates are most virulent, driven by significantly increased dissemination and a greater neutrophil response. We show that Lineage 3 isolates have increased tolerance to complement-mediated killing and acidic conditions alongside upregulated expression of group four capsule synthesis genes. Consistent with these observations, infection of primary human neutrophils revealed that Lineage 3 isolates are more tolerant of phagosomal killing. Together, our findings link the epidemiological success of S. sonnei to heightened virulence and stress tolerance and highlight zebrafish as a valuable platform to illuminate factors underlying establishment of Shigella epidemiological success.

microbiology↗

bioRxiv2025

A public resource of 15 genomically characterised representative strains of Shigella sonnei

Shigella sonnei is rapidly emerging as the dominant agent of shigellosis, an enteric disease responsible for a significant burden of morbidity and mortality worldwide. Whole-genome sequencing of S. sonnei isolated over the last three decades has revealed phylogenomic diversity within the population and the emergence of multiple lineages associated with distinct epidemiological patterns such as resistance to critical antimicrobials and/or transmission within different groups. However most experimental work on S. sonnei biology and pathogenicity has focused on a single laboratory strain (53G), which is phylogenetically distant from currently circulating strains. Here we introduce a set of 15 phylogenetically diverse and epidemiologically relevant S. sonnei isolates made available through publicly accessible culture collections as a resource for laboratory science. We present their complete whole-genome sequences, including the pINV invasion plasmid (missing from a large proportion of public genome data due to loss during laboratory culture). Finally, the characterisation and comparison of these complete genome sequences highlight evidence for ongoing adaptive evolution in S. sonnei, featuring the accumulation of insertion sequences, gene pseudogenisation and structural variation. Significance as a BioResource to the communityGenomic analysis of Shigella has historically been challenging due to presence of hundreds of repetitive sequence elements (which can cause fragmented assemblies) and loss of the pINV invasion plasmid (essential to virulence) during laboratory culture. Furthermore, most experimental work on S. sonnei pathogenicity uses a lab strain that is phylogenetically distant from circulating isolates. To support S. sonnei experimental and in silico research and increase its relevance to current clinical problems, we report here the complete, high-quality genome sequences of 15 S. sonnei isolates, each selected to represent distinct sub-clades of epidemiological interest. We also make the corresponding strains publicly available in national reference culture collections. Data summaryAll sequencing reads and complete assemblies have been deposited into the National Center for Biotechnology Information (NCBI) database (accessions to be determined). Genome assemblies and Bakta annotations used in the analysis can be found in Figshare (https://doi.org/10.6084/m9.figshare.28302986) together with Mauve multiple-sequence alignments for the chromosome and pINV plasmid sequences, and genome-scale metabolic models produced for each strain. Pure cultures of all strains were deposited in the publicly accessible National Collection of Type Cultures (NCTC, UK) or the "Collection de lInstitut Pasteur" (CIP, France) (accessions to be determined).

microbiology↗

bioRxiv2024

Genomic Diversity and Antimicrobial Resistance of Vibrio cholerae Isolates from Africa: A PulseNet Africa Initiative Using Nanopore Sequencing to Enhance Genomic Surveillance

ObjectivesVibrio cholerae remains a significant public health threat in Africa, with antimicrobial resistance (AMR) complicating treatment. This study leverages whole-genome sequencing (WGS) of V. cholerae isolates from Cote dIvoire, Ghana, Zambia and South Africa to assess genomic diversity, AMR profiles, and virulence, demonstrating the utility of WGS for enhanced surveillance within the PulseNet Africa network. MethodsWe analysed Vibrio isolates from clinical and environmental sources (2010-2024) using Oxford Nanopore sequencing and hybracter assembly. Phylogenetic analysis, multilocus sequence typing (MLST), virulence and AMR gene detection were performed using Terra, Pathogenwatch, and Cloud Infrastructure for Microbial Bioinformatics (CLMB) platforms, with comparisons against 88 global reference genomes for broader genomic context. ResultsOf 79 high-quality assemblies, 67 were confirmed as V. cholerae, with serogroup O1 accounting for the majority (43/67, 67%). ST69 accounted for 60% (40/67) of isolates, with eight sequence types identified overall. Thirty-seven isolates formed novel sub-clades within AFR12 and AFR15 O1 lineages, suggesting local clonal expansions. AMR gene analysis revealed high resistance to trimethoprim (96%) and quinolones (83%), while resistance to azithromycin, rifampicin, and tetracycline remained low ([≤]7%). A significant proportion of the serogroup O1 isolates (41/43, 95%) harboured resistance genes in at least three antibiotic classes. ConclusionsThis study highlights significant genetic diversity and AMR prevalence in African V. cholerae isolates, with expanding AFR12 and AFR15 clades in the region. The widespread resistance to trimethoprim and quinolones raises concerns for treatment efficacy, although azithromycin and tetracycline remain viable options. WGS enables precise identification of species and genotyping, reinforcing PulseNet Africas pivotal role in advancing genomic surveillance and enabling timely public health responses to cholera outbreaks. Data summaryAll supporting data and protocols have been provided within the article or as supplementary data files. The ONT reads have been deposited under BioProject accession PRJNA1192988, while the high-quality Vibrio spp. assemblies have been shared via figshare (Foster-Nyarko, Ebenezer (2024). Genomic Diversity and Antimicrobial Resistance of Vibrio spp. Isolates from Africa: A PulseNet Africa Initiative Using Nanopore Sequencing to Enhance Genomic Surveillance. figshare. Dataset. https://doi.org/10.6084/m9.figshare.27941376.v1). Individual accession numbers for these reads and Biosample IDs are provided in File S2, available with the online version of this article. The accession numbers for the 88 reference genome assemblies included in our analysis are also provided in File S3. Impact statementCholera remains a significant public health challenge in Africa, disproportionately affecting the region due to the ongoing transmission of Vibrio cholerae O1 and the emergence of antimicrobial resistance (AMR). This study demonstrates the utility of Oxford Nanopore Technology (ONT) sequencing in providing high-resolution insights into the genomic diversity, transmission dynamics, and AMR profiles of V. cholerae isolates across Africa. By generating and analysing whole-genome sequences, we identified novel sublineages, high prevalence rates of AMR genes, and virulence traits critical to cholera pathogenesis. These findings contribute to a deeper understanding of the epidemiology and evolution of V. cholerae in Africa, informing targeted intervention strategies. Furthermore, the study highlights the growing threat posed by AMR among V. cholerae isolates, including resistance to key therapeutic antibiotics, such as quinolones and trimethoprim, which could undermine current treatment protocols. Despite this, the absence of resistance to azithromycin and rifampicin among the O1 isolates suggests these drugs may remain viable treatment options, offering a critical avenue for preserving treatment efficacy. This research also underscores the importance of sustained genomic surveillance, capacity building, and regional collaboration to mitigate the public health impact of cholera and other foodborne pathogens. By leveraging WGS technologies and training initiatives, such as the PulseNet Africa genomics workshop, this study provides a framework for strengthening regional capacities to detect, monitor, and respond to cholera outbreaks and the spread of AMR. These efforts align with the African Union and Africa CDCs strategic priorities on health security and AMR, contributing to improved public health systems and cholera control across the continent.

genomics↗

bioRxiv2024

Wild type domestication: loss of intrinsic metabolic traits concealed by culture in rich media

BackgroundBacteria are typically isolated on rich media to maximise isolation success, removing them from their native evolutionary context. This eliminates selection pressures, enabling otherwise deleterious genomic events to accumulate. Here we present a cautionary tale of these quiet mutations which can persist unnoticed in bacterial culture lines. MethodsWe used a combination of microbiological culture (standard and minimal media conditions), whole genome sequencing and metabolic modelling to investigate putative Klebsiella pneumoniae L-histidine auxotrophs. Additionally, we used genome-scale metabolic modelling to predict auxotrophies among completed public genomes (n=2,637). ResultsTwo sub-populations were identified within a K. pneumoniae frozen stock, differing in their ability to grow in the absence of L-histidine. These sub-populations were the same strain, separated by eight single nucleotide variants and an insertion sequence-mediated deletion of the L-histidine biosynthetic operon. The His- sub-population remained undetected for >10 years despite its in inclusion in independent laboratory experiments. Genome-scale metabolic models predicted 0.8% public genomes contained [≥]1 auxotrophy, with purine/pyrimidine biosynthesis and amino acid metabolism most frequently implicated. DiscussionWe provide a definitive example of the role of standard rich media culture conditions in obscuring biologically relevant mutations i.e. nutrient auxotrophies, and estimate the prevalence of such auxotrophies using public genome collections. While the prevalence is low, it is not insignificant given the thousands of K. pneumoniae that are isolated for global surveillance and research studies each year. Our data serve as a pertinent reminder that rich-media culturing can cause unnoticed wild type domestication.

microbiology↗

bioRxiv2024

A genome-wide One Health study of Klebsiella pneumoniae in Norway reveals overlapping populations but few recent transmission events across reservoirs

Members of the Klebsiella pneumoniae species complex (KpSC) are opportunistic pathogens that cause severe and difficult-to-treat infections. KpSC are common in non-human niches, but the clinical relevance of these populations is disputed. Utilising 3,255 whole-genome sequenced isolates from human, animal and marine sources collected during 2001-2020 in Norway, we showed the KpSC populations in different niches were distinct but overlapping. Notably, human infection isolates showed greatest connectivity with each other, followed by isolates from human carriage, pigs, and bivalves. Nearly 5% of human infection isolates had close relatives ([≤]22 substitutions) amongst animal and marine isolates, despite temporally and geographically distant sampling of these sources. Infection prevention measures are essential to limit transmission within human clinical settings and reduce disease burden. However, as colonisation often precedes infection, preventing transmission that leads to colonisation, e.g. transmission between animals and humans in the community, and in the food chain, could also be beneficial.

microbiology↗

bioRxiv2024

A metabolic atlas of the Klebsiella pneumoniae species complex reveals lineage-specific metabolism that supports persistent co-existence of diverse lineages

The Klebsiella pneumoniae species complex inhabits a wide variety of hosts and environments, and is a major cause of antimicrobial resistant infections. Genomics has revealed the population comprises multiple species/subspecies and hundreds of distinct co-circulating sub-lineages that are associated with distinct gene complements. A substantial fraction of the pan-genome is predicted to be involved in metabolic functions and hence these data are consistent with metabolic differentiation as a driver of population structure. However, this has so far remained unsubstantiated because in the past it was not possible to explore metabolic variation at scale. Here we used a combination of comparative genomics and high-throughput genome-scale metabolic modelling to systematically explore metabolic diversity across the K. pneumoniae species complex (n=7,835 genomes). We simulated growth outcomes for each isolate using carbon, nitrogen, phosphorus and sulfur sources under aerobic and anaerobic conditions (n=1,278 conditions per isolate). We showed that the distributions of metabolic genes and growth capabilities are structured in the population, and confirmed that sub-lineages exhibit unique metabolic profiles. In vitro co-culture experiments demonstrated reciprocal commensalistic cross-feeding between sub-lineages, effectively extending the range of conditions supporting individual growth. We propose that these substrate specialisations promote the existence and persistence of co-circulating sub-lineages by reducing nutrient competition and facilitating commensal interactions via negative frequency-dependent selection. Our findings have implications for understanding the eco-evolutionary dynamics of K. pneumoniae and for the design of novel strategies to prevent opportunistic infections caused by this World Health Organization priority antimicrobial resistant pathogen.

microbiology↗

bioRxiv2024

Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies

Sequencing DNA directly from patient samples enables faster pathogen characterisation compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich for the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in bacterial isolates before comparing enriched and unenriched sequencing of three human faecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56x and 11.3x the number of AMR and MLST reads respectively compared to unenriched sequencing and required approximately one third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterisation outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonised with high-risk strains.

microbiology↗

bioRxiv2024

The TyphiNET data visualisation dashboard: Unlocking Salmonella Typhi genomics data to support public health

BackgroundSalmonella enterica subspecies enterica serovar Typhi (abbreviated as Typhi) is the bacterial agent of typhoid fever. Effective antimicrobial therapy reduces complications and mortality; however, antimicrobial resistance (AMR) is a major problem in many endemic countries. Prevention through vaccination is possible through recently-licensed Gavi-supported typhoid conjugate vaccines (TCVs), and national immunisation programs are currently being considered or deployed in several countries where AMR prevalence is known to be high. Pathogen whole genome sequence data are a rich source of information on Typhi variants (genotypes or lineages), AMR prevalence, and mechanisms. However, this information is currently not readily accessible to non-genomics experts, including those driving vaccine implementation or empirical therapy guidance. ResultsWe developed TyphiNET (https://www.typhi.net), an interactive online dashboard for exploring Typhi genotype and AMR distributions derived from publicly available pathogen genome sequences. TyphiNET allows users to explore country-level summaries such as the frequency of pathogen lineages, temporal trends in resistance to clinically relevant antimicrobials, and the specific variants and mechanisms underlying emergent AMR trends. User-driven plots and session reports can be downloaded for ease of sharing. Importantly, TyphiNET is populated by high-quality genome data curated by the Global Typhoid Pathogen Genomics Consortium, analysed using the Pathogenwatch platform, and identified as coming from non-targeted sampling frames that are suitable for estimating AMR prevalence amongst Typhi infections (no personal data is included in the platform). As of February 2024, data from a total of n=11,836 genomes from 101 countries are available in TyphiNET. We outline case studies illustrating how the dashboard can be used to explore these data and gain insights of relevance to both researchers and public health policy-makers. ConclusionsThe TyphiNET dashboard provides an interactive platform for accessing genome-derived data on pathogen variant frequencies to inform typhoid control and intervention strategies. The platform is extensible in terms of both data and features, and provides a model for making complex bacterial genome-derived data accessible to a wide audience.

microbiology↗

bioRxiv2024

Genomic and functional analysis of rmp locus variants in Klebsiella pneumoniae

AbstractO_ST_ABSBackgroundC_ST_ABSKlebsiella pneumoniae is an opportunistic pathogen and a leading cause of healthcare-associated infections in hospitals, which are frequently antimicrobial resistant (AMR). Exacerbating the public health threat posed by K. pneumoniae, some strains also harbor additional hypervirulence determinants typically acquired via mobile genetic elements such as the well-characterised large virulence plasmid KpVP-1. The rmpADC locus is considered a key virulence feature of K. pneumoniae and is associated with upregulated capsule expression and the hypermucoid phenotype, which can enhance virulence by contributing to serum resistance. Typically such strains have been susceptible to all antimicrobials besides ampicillin, however the recent emergence of AMR hypermucoid strains is concerning. MethodsHere, we investigate the genetic diversity, evolution, mobilisation and prevalence of rmpADC, in a dataset of 14000 genomes from isolates of the Klebsiella pneumoniae species complex, and describe the RmST virulence typing scheme for tracking rmpADC variants for the purposes of genomic surveillance. Additionally, we examine the functionality of representatives for variants of rmpADC introduced into a mutant strain lacking its native rmpADC locus. ResultsThe rmpADC locus was detected in 7% of the dataset, mostly from genomes of K. pneumoniae and a very small number of K. variicola and K. quasipneumoniae. Sequence variants of rmpADC grouped into five distinct lineages (rmp1, rmp2, rmp2A, rmp3 and rmp4) that corresponded to unique mobile elements, and were differentially distributed across different populations (i.e. clonal groups) of K. pneumoniae. All variants were demonstrated to produce enhanced capsule production and hypermucoviscosity. ConclusionThese results provide an overview of the diversity and evolution of a prominent K. pneumoniae virulence factor and support the idea that screening for rmpADC in K. pneumoniae isolates and genomes is valuable to monitor the emergence and spread of hypermucoid K. pneumoniae, including AMR strains.

genomics↗

bioRxiv2024

Bacterial strain nomenclature in the genomic era: Life Identification Numbers using a gene-by-gene approach

Unified strain taxonomies are needed for the epidemiological surveillance of bacterial pathogens and international communication in microbiological research. Core genome multilocus sequence typing (cgMLST) holds great promise for standardized high-resolution strain genotyping. However, this approach faces challenges including classification instability and disconnection of new nomenclature from widely adopted classical MLST identifiers. This essay discusses the cgMLST-based Life Identification Number (LIN) method, recently proposed as a stable multilevel strain taxonomy system applicable to most bacterial pathogens. We describe how LIN codes are implemented and used in practice for precise strain definitions and epidemiological tracking. Glossary Multilocus sequence typing (MLST)A genotyping method applied mostly to microbial strains to study population structure and epidemiology, based on comparing the nucleotide sequences of a small number (typically seven) of housekeeping protein-coding genes. In MLST, allele numbers are assigned to each sequence variant (allele) of a given gene. The MLST genotype of a bacterial strain is defined by the combination of the allele numbers observed at the genes that are included in the genotyping scheme. A sequence type (ST) is assigned to each unique combination of alleles, called an MLST profile. MLST was invented in 1998 and became a de-facto standard taxonomy of bacterial strains, albeit at low resolution. Core genome MLSTAn extension of MLST that analyzes sequence variation across hundreds to thousands of conserved (core) genes, shared by all strains of a species, providing higher resolution typing for genomic epidemiology and evolutionary studies. cgMLST schemes typically comprise 2000 to 4000 genes, depending on the genome size and genetic variation (in terms of presence/absence of genes) within bacterial species. A core genome sequence type (cgST) can be assigned to unique cgMLST profiles, i.e., a unique combination of cgMLST allelic numbers. Whole Genome Sequencing (WGS)A method that determines the complete DNA sequence of an organisms genome in a single process, providing comprehensive information for comparative genetic analyses based on cgMLST or other analytic methods. Single Nucleotide Polymorphisms (SNPs)Variations at a single base position in the DNA sequence among individuals isolates, strains or species, used as genetic markers for studying for example, evolutionary relationships or strain identity. Average nucleotide identity (ANI)A measure of genomic similarity between two organisms, calculated as the average percentage of identical nucleotides in orthologous genomic regions; commonly used to assess species-level relatedness in prokaryotes. TaxonomyHere, we apply the word taxonomy to bacterial strains as a system of classifying, naming and identifying strains based on shared genetic characteristics as defined by e.g., cgMLST.

microbiology↗