Biology subjects

Gladstone, R. A.

Publications and source records attributed to Gladstone, R. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Identifying candidate genetic determinants associated with invasion in pneumococcal lineages

Serotype invasiveness estimates help identify high-risk serotypes and interpret disease replacement following pneumococcal conjugate vaccination. Because serotype and lineage are strongly associated, their independent contributions must be separated to identify unexpectedly invasive lineages and investigate underlying mechanisms. Using temporally matched carriage and invasive-disease isolates from children aged <7 years in South Africa and the USA, we fitted a logistic mixed-effects model with Global Pneumococcal Sequence Cluster (GPSC) as a random effect and serotype and country as fixed effects. We conducted a species-wide genome-wide association study (GWAS) of invasion and compared the prevalence of significant unitigs in GPSC1 and GPSC21 to identify invasion-associated variants differentiating them. We additionally examined capsule-locus variation that could contribute to their contrasting invasiveness. After serotype and country adjustment, GPSC1 and GPSC21, which both express 19F and share an immediate common ancestor, ranked fifth and 149th of 150 lineages; odds multipliers were 1.51 (95% conditional interval 1.06-2.15) and 0.61 (0.41-0.93), respectively. Of 1,569 invasion-associated unitigs from the species-wide GWAS, 272 differentiated GPSC1 from GPSC21. Among these, the strongest signal mapped to hk11 (desK) and was associated with GPSC1; bgaA was the highest-ranked known antibody-binding target. Within GPSC1 CC271 had additional variants in the ATP-dependent Clp protease ATP-binding subunit ClpX compared to the CC420 subclade. Capsule-protein folds were conserved with local effects indistinguishable from prediction uncertainty. Separating serotype and lineage effects can identify unexpectedly invasive lineages and prioritize candidate serotype-independent determinants, vaccine antigens and capsule variation for experimental investigation.

microbiology↗

bioRxiv2026

The global evolutionary dynamics of natural capsule switching in a human-restricted bacterial pathogen

Exchange of genetic loci encoding extracellular capsular polysaccharides in bacteria can change capsular antigens or serotype, which, in turn, can lead to evasion of vaccines targeting the capsule. However, the evolutionary dynamics and drivers of bacterial capsule switching remain poorly understood. Here, we investigate the evolutionary dynamics and correlates of capsule switching in bacteria, using the human-restricted bacterial pathogen Streptococcus pneumoniae as an example, using 10,558 genetically and geographically diverse whole-genome sequences. Our phylogenomic and ancestral state reconstruction analysis revealed that the propensity for capsule switching varies by capsule type and genetic background. We found capsule switches only between ~4% of all possible capsule-type pairs, varying by specific pairs and switching direction. We also found associations between capsule switching patterns and genetic and ecological factors, including nasopharyngeal carriage duration, capsule biosynthesis locus size, and capsule thickness. Furthermore, the genetic similarity of the replacing and replaced capsule loci was primarily associated with the frequency of switching between specific capsule type pairs. Although a few specific non-capsule genomic loci were associated with the propensity for capsule switching across distinct lineages, the collective effect of the genetic background appears to influence capsule switching more strongly. These findings shed light on how bacterial capsule types and lineages undergo capsule switching influenced by genetic and ecological factors, informing our understanding of the emergence of novel capsule-genotype combinations that may impact the design of capsule-targeting glycoconjugate vaccines.

microbiology↗

bioRxiv2026

Global population structure and phase variation of serotype 12F Streptococcus pneumoniae following the introduction of pneumococcal conjugate vaccine

BackgroundAfter the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. MethodsA global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. FindingsThe global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. InterpretationOur work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

genomics↗

bioRxiv2026

Mapping antibiotic resistance determinants in oral streptococci

BackgroundAntibiotic resistance is a global priority in healthcare. Leveraging thousands of whole-genome sequences, here we reveal the core resistance determinants of oral streptococci, focusing on assessing pattern variability and gene exchange across commensals and pathogens. MethodsGenomic information was obtained from the National Center for Biotechnology Information. Determinants of antibiotic resistance were identified using AMRFinderPlus and CARD. ICEscreen was employed for calling of integrative and conjugative elements. Variability and recombination in penicillin-binding protein sequences were assessed with MMseqs2 and fastGEAR. ResultsA total of 2,087 genomes from 15 species were included with members from the mitis, mutans, anginosus, salivarius, and bovis groups. We observed 3,576 hits from 55 unique resistance genes conferring resistance to 11 antibiotic classes. The species with most resistance determinants per genome were identified as Streptococcus mitis (2.7), Streptococcus oralis (2.5), Streptococcus parasanguinis (1.9), Streptococcus gallolyticus (1.3), and Streptococcus anginosus (1.2). The two latter species also presented the most diverse composition of determinants. Over 1,800 integrative and conjugative elements were shown across all genomes, with nearly 18% of them carrying at least one antibiotic resistance gene. Penicillin-binding protein variation analyses showed a high diversity in the mitis group. Even though S. mitis and S. oralis composed less than 4% of the genomes included in the analyses, they were recognized as sources of DNA for over a third of recombination events in pbp1a and nearly half for both pbp2b and pbp2x in resistant isolates of Streptococcus pneumoniae. ConclusionsWe show that tetracycline and macrolide resistance were highly abundant and tightly connected to integrative and conjugative elements. Further, recent recombination data show frequent genetic exchange from oral streptococci to beta-lactam-resistant S. pneumoniae. Finally, assessing the dynamics of genetic exchange across species is central for the development of strategies to mitigate the impact of antibiotic resistance.

microbiology↗

bioRxiv2025

Mapping the evolutionary path towards multi-drug resistance in the pandemic Escherichia coli ST131 lineage

Escherichia coli sequence type (ST) 131 is the most widely studied genetic lineage of the species to date, originally identified in the early 2000s as an increasingly common cause of human urinary tract and bloodstream infections worldwide. This lineage is subdivided into four extant main subclades A, B, C1 and C2 that exhibit distinct features in terms of invasiveness, antibiotic resistance and transmissibility. However, the evolutionary pathway from the generally susceptible ST131-B to the drug-resistant ST131-C clades remains poorly mapped. To fill this knowledge gap, we analysed in detail human clinical isolates obtained in Vietnam, designated as belonging to the generally neglected minor clade ST131-B0. We sequenced them using both short- and long-read technology, and combined these data with a recently published high-resolution genomic collection to provide further insight into the evolutionary process and its timeline. Extensive genomic analyses established ST131-B0 as an intermediary progenitor in the evolutionary path leading from ST131-B to the ST131-C clades, associated with multiple type I pili switches, as well as the loss and gain of specific chromosomal genes representing diverse core functions such as metabolism, transcription, DNA binding and type II toxin-antitoxin systems. Furthermore, all Vietnamese isolates of ST131-B0 unprecedentedly harboured blaCTX-M genes encoding extended-spectrum {beta}-lactamases, a trait dominant in ST131-C clades and not previously observed in ST131-B0. Our study supports the hypothesis that the ST131-C clades have gradually evolved from ST131-B by reducing the host range with better adaptation to colonising humans under selective conditions.

evolutionary biology↗

bioRxiv2025

Co-evolution between colibactin production and resistance is linked to clonal expansions in Escherichia coli

Specific strains of Escherichia coli employ the polyketide synthase island to produce a metabolite called colibactin that is implicated in colorectal tumorigenesis via its genotoxic effect on human DNA. However, the damage to host DNA is essentially collateral from ecological competition during bacterial colonisation, where colibactin is used to displace other gut bacteria. Despite intensive recent research on colibactin and its involvement in tumorigenesis, the evolutionary dynamics related to its production remain poorly characterised. We map evolution of colibactin production in E. coli using a large collection of high-resolution genomes and show that its introduction via a mobile element has induced multiple plasmid-mediated acquisitions of previously unrecognised colibactin immunity genes in non-producing multi-drug resistant lineages. Our study suggests that in E. coli, multi-drug resistance is incompatible with colibactin production due to the unbearable combined fitness costs, supported by colibactin-producing phenotypes remaining susceptible to most classes of antibiotics and thriving in regions with low antimicrobial usage. Consequently, resistant lineages circulating in these regions are under selective pressure to acquire colibactin immunity to compete with the endemic colibactin-producing bacteria during colonisation. Conversely, high antimicrobial usage selects for a colibactin susceptible antimicrobial resistant phenotype as it drives the colibactin-producing E. coli towards extinction. Such a duality of evolutionary strategies to become endemic in a host population structured by varying ecological pressures may hold more generally across bacterial species colonizing the human gut.

microbiology↗

bioRxiv2025

Machine learning based lineage prediction from AMR phenotypes for Escherichia coli ST131 clade C surveillance across infection types

2.Rising antimicrobial resistance (AMR) in Escherichia coli bloodstream infections (BSIs) in high-income settings has typically been dominated by one clone, the sequence type (ST) 131. More specifically, ST131 clade C (ST131-C) is associated with fluoroquinolone resistance and extended-spectrum {beta}-lactamases (ESBLs). Even though urinary tract infections (UTIs) are a known common precursor to BSIs, there is currently limited knowledge on the longitudinal prevalence of ST131-C in UTIs and, therefore, the temporal link between the two infection types. Leveraging available genomic and antimicrobial susceptibility test (AST) data for ciprofloxacin, gentamicin, and ceftazidime in 2790 E. coli BSI isolates, we trained random forest and Extreme Gradient Boosting (XGBoost) classifiers to predict if an E. coli isolate belongs to ST131-C using only AST data. These models were used to predict the yearly prevalence of ST131-C in 22942 UTI and 24866 BSI isolates from Norway. The XGBoost classifier achieved a prediction F1-score of over 70% on a highly unbalanced dataset where only 4.3% of the genomic BSI isolates belonged to ST131-C. The predicted prevalence of ST131-C in UTIs exhibited a similar annual trend to that of BSIs, with a stable infection burden for eight years after its rapid expansion, confirming that the persistence of ST131-C in BSIs is largely driven by ST131-C UTIs. However, a higher prevalence of ST131-C in BSIs ([~]7%) compared to UTIs ([~]4%) suggests a subsequent enrichment of ST131-C. Our study highlights how existing epidemiological knowledge can be supplemented by utilising extensive data from AMR surveillance efforts without genomic markers. 3. Impact statementThis study proposes a potential analysis method that leverages AST data, which is already regularly collected for AMR surveillance purposes. Using such data to approximate the population-wide prevalence of MDR clones, such as ST131-C, could allow for larger-scale, retrospective studies of its prevalence in a population than genomic-based methods at a significantly lower cost. Such a method could supplement existing knowledge and epidemiology study practices. We use the proposed method to find relationships between the prevalence of the important MDR E. coli clone ST131-C in UTIs and BSIs in Norway. These results suggest that monitoring and reducing MDR in UTIs could reduce the burden of this invasive clone in hard-to-treat BSIs. 4. Data summaryAll AST data, clonal information for isolates with genomic data, and code used in this study can be found in the following repository: https://github.com/theodorross/EColi-UTI-Predictions. Only the clone and published metadata information is shown for the UTI data shared by Handal, Kaspersen et al.

bioinformatics↗

bioRxiv2025

Large-scale global molecular epidemiology of antibiotic resistance determinants in Streptococcus pneumoniae

BackgroundStreptococcus pneumoniae is a leading pathogen in terms of deaths attributable to or associated with antimicrobial resistance globally. Thus, monitoring antibiotic resistance determinants constitutes a key aspect of surveillance efforts. AimLeveraging on publicly available whole-genome sequencing data, we aimed to investigate the presence and distribution patterns of antibiotic resistance in S. pneumoniae with a focus on multi-drug resistance and serotype distribution. MethodsMetadata and genomes were obtained from the National Center for Biotechnology Information Pathogen Detection database. Curation and harmonization were performed in R and SPSS. Data on resistance patterns was defined according to AMRFinderPlus and a combination of prediction tools were employed for in silico serotyping. ResultsAnalyses involved 75,161 genomes totaling 122,673 gene/allele counts from 14 antibiotic classes. Multi-drug resistance was observed in 16.7% of isolates with the highest increasing rates in Asia and South America. Within antibiotic classes, increase in macrolide resistance genes was highlighted, particularly in the proportion of genomes presenting mefA/msrD. Over a third of isolates with serotypes 19F, 23F, 15A, 6B, and 19A showed multi-drug resistance. We further observed the highest significant increases in the presence of resistance in 33F, 22F, 10A, and 23A. Serotype 13, not included in any vaccine formulation, presented high multi-drug resistance rates with a strong increasing trend. ConclusionsThe findings of this study highlight variations in resistance determinants globally and across serotypes over time. Collectively these data underscore the added value of utilizing public whole-genome sequencing data to investigate the effectiveness and repercussions of treatment and vaccination strategies on managing antibiotic resistance.

microbiology↗

bioRxiv2024

Basic reproduction number for pandemic Escherichia coli clones is comparable to typical pandemic viruses

Extra-intestinal pathogenic Escherichia coli (ExPEC) ubiquitously colonize the human gut and represent clinically the most significant bacterial species causing urinary tract infections and bacteremia in addition to contributing to meningitis in neonates. During the last two decades, new E. coli multi-drug resistant clones such as ST131, particularly its clades C1 and C2, have spread globally, as has their generally less resistant sister clade ST131-A. Phylodynamic coalescent modeling has indicated exponential growth in the populations corresponding to these clades during the early 2000s. However, it remains unknown how their transmission dynamics compare to viral epidemics and pandemics in terms of key epidemiological quantities such as the basic reproduction number (R0). Estimation of R0 for opportunistic pathogenic bacteria poses a difficult challenge compared to viruses causing acute infections, since data on E. coli infections accumulate with a much longer delay, even in the most advanced public health reporting systems. Here, we developed a compartmental model for asymptomatic gut colonization and onward transmission coupled with a stochastic epidemiological observation model for bacteremia and fitted the model to annual Norwegian national E. coli disease surveillance and bacterial population genomics data. Approximate Bayesian Computation leveraged by the ELFI software package was used to infer R0 for the pandemic ST131 clades. The resulting R0 estimates for ST131-A, ST131-C1 and ST131-C2 were 1.47 (1.31-1.60), 1.18 (1.12-1.20) and 1.13 (1.08-1.20), respectively, indicating that the ST131-A transmission potential can be even comparable to pandemic influenza viruses, such as H1N1. The significantly lower transmissibility of ST131-C1 and ST131-C2 suggests that their global dissemination has been aided by antibiotic selection pressure and that they may be more effectively transmitted through healthcare facilities instead of primarily community-driven transmission. In summary our results provide a fundamental advance in understanding the relative transmissibility of these opportunistic pathogens and that it can vary markedly even between very closely related E. coli.

microbiology↗

bioRxiv2023

Escherichia coli plasmidome maps the game of clones

Escherichia coli is the most widely studied microbe in history, but its extrachromosomal elements known as plasmids remain poorly delineated. Here we used long-read technology to high-resolution sequence the entire plasmidome and the corresponding host chromosomes from an unbiased longitudinal survey covering two decades and over 2,000 E. coli isolates. We find that some plasmids have persisted in lineages even for centuries, demonstrating strong plasmid-lineage associations. Our analysis provides a detailed map of recent vertical and horizontal evolutionary events involving plasmids with key antibiotic resistance, competition and virulence determinants. We present genomic evidence of both chromosomal and plasmid-driven success strategies that represent convergent phenotypic evolution in distant lineages, and use in vitro experiments to verify the importance of bacteriocin-producing plasmids for clone success. Our study has general implications for understanding plasmid biology and bacterial evolutionary strategies.

microbiology↗

bioRxiv2023

Detecting co-selection through excess linkage disequilibrium in bacterial genomes

Population genomics has revolutionised our ability to study bacterial evolution by enabling data-driven discovery of the genetic architecture of trait variation. Genome-wide association studies (GWAS) have more recently become accompanied by genome-wide epistasis and co-selection (GWES) analysis, which offers a phenotype-free approach to generating hypotheses about selective processes that simultaneously impact multiple loci across the genome. However, existing GWES methods only consider associations between distant pairs of loci within the genome due to the strong impact of linkage-disequilibrium (LD) over short distances. Based on the general functional organisation of genomes it is nevertheless expected that the majority of co-selection and epistasis will act within relatively short genomic proximity, on co-variation occurring within genes and their promoter regions, and within operons. Here we introduce LDWeaver, which enables an exhaustive GWES across both short- and long-range LD, to disentangle likely neutral co-variation from selection. We demonstrate the ability of LDWeaver to efficiently generate hypotheses about co-selection using large genomic surveys of multiple major human bacterial pathogen species and validate several findings using functional annotation and phenotypic measurements. Our approach will facilitate the study of bacterial evolution in the light of rapidly expanding population genomic data.

bioinformatics↗

bioRxiv2022

Consistent typing of plasmids with the mge-cluster pipeline

Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Our method can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. We provide an intuitive visualization, classification and clustering scheme that users can explore interactively. This provides a framework that can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We illustrate the attractive features of our approach by the analysis of population-wide plasmid data from the opportunistic pathogen Escherichia coli and the distribution of the colistin resistance gene mcr-1.1 in the plasmid population.

bioinformatics↗

bioRxiv2022

Strong pathogen competition in neonatal gut colonisation

Bacterial pathogen species and their strains that colonise the human gut are generally understood to compete against both each other and the commensal species colonising this ecosystem. However, currently we are lacking a population-wide quantification of strain-level colonisation dynamics for many common bacterial pathogens and the relationship of colonisation potential to prevalence in disease is unknown. In addition, it is unclear how ecological factors might be modulating the dynamics. Here, using a combination of latest high-resolution metagenomics and strain-level genomic epidemiology methods leveraging large genomic reference libraries of key pathogens, we performed a characterisation of the competition (co-exclusion and co-presence) and colonisation dynamics for a longitudinal cohort of neonatal gut microbiomes. We found a strong inter- and intra-species competition dynamic in the gut colonisation process, but also a number of synergistic relationships among several species belonging to genus Klebsiella, which includes the prominent human pathogen Klebsiella pneumoniae. Additionally, we find no evidence of preferential colonisation by hospital-adapted pathogen lineages in either vaginal or caesarean section birth groups. Our analysis also enables the first unbiased assessment of the strain-level colonisation potential of extra-intestinal pathogenic Escherichia coli (ExPEC) in comparison with their potential to cause bloodstream infections. We determined that the established common ExPEC clones ST73 and ST95 are overall significantly more pathogenic than the more recent, globally circulating multi-drug resistant clone ST131, where only a single subclone (ST131-C2) exhibited excess pathogenic potential. Our study highlights the importance of systematic surveillance of bacterial gut pathogens, not only from disease but also from carriage state, to better inform therapies and preventive medicine in the future.

microbiology↗

bioRxiv2022

Robust analysis of prokaryotic pangenome gene gain and loss rates with Panstripe

Horizontal gene transfer (HGT) plays a critical role in the evolution and diversification of many microbial species. The resulting dynamics of gene gain and loss can have important implications for the development of antibiotic resistance and the design of vaccine and drug interventions. Methods for the analysis of gene presence/absence patterns typically do not account for errors introduced in the automated annotation and clustering of gene sequences. In particular, methods adapted from ecological studies, including the pangenome gene accumulation curve, can be misleading as they may reflect the underlying diversity in the temporal sampling of genomes rather than a difference in the dynamics of HGT. Here, we introduce Panstripe, a method based on Generalised Linear Regression that is robust to population structure, sampling bias and errors in the predicted presence/absence of genes. We demonstrate using simulations that Panstripe can effectively identify differences in the rate and number of genes involved in HGT events, and illustrate its capability by analysing several diverse bacterial genome datasets representing major human pathogens. Panstripe is freely available as an R package at https://github.com/gtonkinhill/panstripe.

evolutionary biology↗

bioRxiv2021

A high-throughput multiplexing and selection strategy to complete bacterial genomes

BackgroundBacterial whole-genome sequencing based on short-read sequencing data often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits to unambiguously bridge those contiguous sequences into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose a long-read isolate selection strategy that optimizes a representative selection of isolates from large-scale bacterial collections. ResultsDespite an uneven distribution of long-reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98) which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable: (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. ConclusionsThe proposed long-read isolate selection ensures completing bacterial genomes of isolates that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

microbiology↗

bioRxiv2021

The role of interspecies recombinations in the evolution of antibiotic resistant pneumococci

AbstractThe evolutionary histories of the antibiotic-resistant Streptococcus pneumoniae lineages PMEN3 and PMEN9 were reconstructed using global collections of genomes. In PMEN3, one resistant clade spread worldwide, and underwent 25 serotype switches, enabling evasion of vaccine-induced immunity. In PMEN9, only 9 switches were detected, and multiple resistant lineages emerged independently and circulated locally. In Germany, PMEN9s expansion correlated significantly with the macrolide:penicillin consumption ratio. These isolates were penicillin sensitive but macrolide resistant, through a homologous recombination that integrated Tn1207.1 into a competence gene, preventing further diversification via transformation. Analysis of a species-wide dataset found 183 acquisitions of macrolide resistance, and multiple gains of the tetracycline-resistant transposon Tn916, through homologous recombination, often originating in other streptococcal species. Consequently, antibiotic selection preserves atypical recom- bination events that cause sequence divergence and structural variation throughout the S. pneumoniae chromosome. These events reveal the genetic exchanges between species normally counter-selected until perturbed by clinical interventions.

genomics↗

bioRxiv2020

Producing Polished Prokaryotic Pangenomes with the Panaroo Pipeline

Population-level comparisons of prokaryotic genomes must take into account the substantial differences in gene content, resulting from frequent horizontal gene transfer, gene duplication and gene loss. However, the automated annotation of prokaryotic genomes is imperfect, and errors due to fragmented assemblies, contamination, diverse gene families and mis-assemblies accumulate over the population, leading to profound consequences when analysing the set of all genes found in a species. Here we introduce Panaroo, a graph based pangenome clustering tool that is able to account for many of the sources of error introduced during the annotation of prokaryotic genome assemblies. We verified our approach through extensive simulations of de novo assemblies using the infinitely many genes model and by analysing a number of publicly available large bacterial genome datasets. Using a highly clonal Mycobacterium tuberculosis dataset as a negative control case, we show that failing to account for annotation errors can lead to pangenome estimates that are dominated by error. We additionally demonstrate the utility of the improved graphical output provided by Panaroo by performing a pan-genome wide association study in Neisseria gonorrhoeae and by analysing gene gain and loss rates across 51 of the major global pneumococcal sequence clusters. Panaroo is freely available under an open source MIT licence at https://github.com/gtonkinhill/panaroo.

genomics↗

bioRxiv2019

A novel mosaic tetracycline resistance gene tet(S/M) detected in a multidrug-resistant pneumococcal CC230 lineage that underwent capsular switching in South Africa

SynopsisO_ST_ABSObjectiveC_ST_ABSWe reported a novel tetracycline-resistant gene in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions.\n\nMethodsWe whole genome sequenced 12,254 pneumococcal isolates from twenty-nine countries on an Illumina HiSeq Sequencer. Serotypes, sequence types and antibiotic resistance were inferred from genomes. Phylogeny was built based on single-nucleotide variants. Temporal changes of spread were reconstructed using a birth-death model.\n\nResultsWe identified tet(S/M) in 131 pneumococcal isolates, 97 (74%) caused invasive pneumococcal diseases among young children (59% HIV-positive, where HIV status was available) in South Africa. A majority of tet(S/M)-positive isolates (129/131) belong to clonal complex (CC)230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sub-lineage that exhibited multidrug-resistance. Using the genomic data and a birth-death model, we detected an unrecognised outbreak of this sub-lineage in South Africa between 2000 and 2004 with an expected secondary infections (R) of ~2.5. R declined to ~1.0 in 2005 and <1.0 in 2012. The declining epidemic coincided and could be related to the nationwide implementation of anti-retroviral treatment (ART) for HIV-infected individuals in 2004 and PCVs in late 2000s. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sub-lineage.\n\nConclusionsThe prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognised outbreak of CC230 in South Africa prior to ART and PCVs. However, capsular switching in this multidrug-resistant sub-lineage highlighted its potential to continue to cause disease in the post-PCV13 era.

microbiology↗