Biology subjects

Thomson, N. R.

Publications and source records attributed to Thomson, N. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Refining the Serine Protease Autotransporters of Enterobacteriaceae (SPATE) gene detection in Enteroaggregative Escherichia coli genomes uncovers differential SPATE distribution by phylogeny

BackgroundEnteroaggregative Escherichia coli (EAEC) are a heterogenous pathotype, implicated in acute and persistent diarrhoea especially in developing countries. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) are Type V Secretory System trypsin-like proteases repeatedly reported from EAEC. This study aimed to determine SPATE encoding-gene prevalence among EAEC and their association with diarrhoea. We screened 881 EAEC genomes from four recent epidemiological studies in Nigeria for 23 SPATE-encoding genes, initially using ARIBA and the Virulencefinder database. ResultsInitial screening inflated SPATE gene content, particularly in genomes with multiple SPATEs, due to cross detection of highly similar sequences and other artefacts. We developed and validated refined methodology, which detected 478 of 1,156 original SPATE calls and also identified SPATE miscalls from previous datasets in the literature. The most prevalent SPATE-encoding gene in our EAEC collection was sepA 297(33.71%), closely followed by sat 360 (29.74%). pic, encoding a SPATE with mucinase activity, was found in 65 (7.4%) genomes and associated with diarrhoea (p=0.00004). EAEC strains belonging to E. coli phylogroups A, B1 or C carried, on average, one SPATE gene per genome while >1 was typically detected in phylogroup B2 EAEC. Other EAEC carried few or no SPATE genes. ConclusionsOur study shows that multifunctional genome analysis tools may have to be refined for certain gene families to avoid overestimation. SPATEs are not as prevalent as previously thought but they remain common among EAEC, particularly among phylogroup A, B1, B2 and C, pointing to the possibility that they make lineage-specific contributions to disease.

microbiology↗

bioRxiv2026

Slow-growing human cell lines are a suitable alternative to rabbit Sf1Ep cells for in vitro cultivation of Treponema pallidum

Treponema pallidum subsp. pallidum, the causative agent of syphilis, remains difficult to study owing to long-standing limitations in in vitro cultivation. Although a rabbit epithelial cell co-culture system (Sf1Ep) enabled major advances, the absence of human cell-based models limits clinical relevance and mechanistic understanding of host-pathogen interactions. Here, we established a co-culture system using human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six epithelial or epithelial-like cell lines from diverse tissues were evaluated under microaerophilic conditions using standard cultivation medium. CAL-39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviors, and long-term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility were quantified over extended culture. Using live-cell imaging, we defined two distinct host-cell interaction behaviors: surface-associated crawling and stable single-polar attachment. Both behaviors were observed across all tested cell lines but persisted only in those permissive for sustained growth. Together, our findings establish clinically relevant human epithelial co-culture models for T. pallidum, provide new insights into host-cell-dependent growth and motility, and create a platform for mechanistic studies of syphilis pathogenesis and vaccine target discovery.

microbiology↗

bioRxiv2025

Aeromonas: Genomic Insights into an Environmental Pathogen and Reservoir of Antimicrobial Resistance

Aeromonads are an ecologically versatile group of bacteria that cause infection in aquatic animals and are recognised as an emerging human pathogen. Despite this, our understanding of Aeromonas diversity, especially the relationship between clinical and environmental strains, remains limited. Here, we present a complete view of the Aeromonas genus, comprising 1,853 genomes, and a detailed comparison of clinical and environmental strains from South Asia, including 996 newly sequenced genomes from Bangladesh and India. Phylogenetic analyses revealed that Aeromonas is a highly diverse genus, with no distinct clade separating clinical and environmental isolates. We identified 28 Aeromonas species and 905 novel sequence types, comprising 72.5% of the genomes. Notably, we show a high incidence of AMR genes across all isolates, including against front and last-line antibiotics. Finally, we highlight frequent misidentification of Aeromonas as Vibrio cholerae, key to cholera-endemic regions where both genera co-exist and are associated with diarrhoeal disease. Our study underscores Aeromonas as an important environmental AMR reservoir and emerging multi-species pathogen capable of spilling over into human populations.

genomics↗

bioRxiv2025

Solubilization of Membrane Proteins using designed protein WRAPS

The development of therapies and vaccines targeting integral membrane proteins has been complicated by their extensive hydrophobic surfaces, which can make production and structural characterization difficult. Here we describe a general deep learning-based design approach for solubilizing native membrane proteins while preserving their sequence, fold, and function using genetically encoded de novo protein WRAPs (Water-soluble RFdiffused Amphipathic Proteins) that surround the lipid-interacting hydrophobic surfaces, rendering them stable and water-soluble without the need for detergents. We design WRAPs for both beta-barrel outer membrane and helical multi-pass transmembrane proteins, and show that the solubilized proteins retain the binding and enzymatic functions of the native targets with enhanced stability. Syphilis vaccine development has been hindered by difficulties in characterizing and producing the outer membrane protein antigens; we generated soluble versions of four Treponema pallidum outer membrane beta barrels which are potential syphilis vaccine antigens. A 4.0 [A] cryo-EM map of WRAPed TP0698 is closely consistent with the design model. WRAPs should be broadly useful for facilitating biochemical and structural characterization of integral membrane proteins, enabling therapeutic discovery by screening against purified soluble targets, and generating antigenically intact immunogens for vaccine development.

biochemistry↗

bioRxiv2024

High-quality transcriptome profile of Treponema pallidum subsp. pallidum: confirmation of transcriptional landscape

Syphilis remains a critical global health challenge due to its potential for severe complications and the increase in its incidence rate over recent years. Until recently, the infectious agent of syphilis, Treponema pallidum subsp. pallidum (TPA), could not be cultured in vitro. Advances in co-culture techniques have finally allowed for effective long-term cultivation of TPA, providing a platform to study its biology. Limited transcriptional data from TPA have been reported so far and many genes in treponemal genomes are annotated based on in silico prediction of putative coding sequences without functional validation. To inform future syphilis vaccine development, experimental validation of in silico predicted genes coupled with functional annotation is necessary. In this study, strand-specific RNA-sequencing was used to reconstruct a high-quality transcriptome profile of TPA, confirming the active transcription of genes previously annotated as hypothetical, paving the way for more accurate identification of vaccine target candidates. Our transcriptomic data also revealed, for the first time, the organization of genes into transcription units, an abundance of anti-sense RNAs, and transcripts from intergenic regions, providing crucial insights for future functional genomics studies of TPA. Author SummaryIn our study, we explored the genetic activity of the bacteria responsible for syphilis, Treponema pallidum subsp. pallidum (TPA). Although syphilis has been a known disease for centuries, the bacterium causing it has remained difficult to study because it couldnt be easily grown in the lab. Recently, new techniques have allowed us to cultivate TPA successfully, enabling deeper investigation into its genetics. By employing directional RNA sequencing, we have mapped out which genes are actively transcribed, including those previously labeled as hypothetical. Our study has also revealed new insights into the gene organization and uncovered the presence of antisense RNA, which may regulate gene expression. These findings offer critical information that could inform future research and vaccine development efforts for syphilis.

microbiology↗

bioRxiv2023

Genetic diversity, determinants, and dissemination of Burkholderia pseudomallei lineages implicated in melioidosis in northeast Thailand

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conducted a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identified three dominant lineages with unique gene sets enhancing bacterial fitness, indicating lineage-specific adaptation strategies. Crucially, recombination was found to drive lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage revealed heightened expression of lineage-specific genes in environmental versus infection conditions, notably under nutrient depletion, highlighting environmental persistence as a key factor in the success of dominant lineages. The study also revealed the role of environmental factors - slope of terrain, altitude, direction of rivers, and the northeast monsoons - in shaping B. pseudomallei geographical dispersal. Collectively, our findings highlight persistence in the environment as a pivotal element facilitating B. pseudomallei spread, and as a prelude to exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.

microbiology↗

bioRxiv2023

Multidrug resistance plasmids underlie clonal expansions and international spread of Salmonella enterica serotype 4,,12,i:- ST34 in Southeast Asia

Salmonella enterica serotype 4,[5],12,i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogens epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a hotspot for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors. Our work enriches the understanding on epidemiology and evolution of this variant in Southeast Asia, and determines that multidrug resistance plasmids have driven its local and potentially global success.

genomics↗

bioRxiv2022

Genomic epidemiology of the cholera outbreak in Yemen reveals the spread of a multi-drug resistance plasmid between diverse lineages of Vibrio cholerae

The humanitarian crisis in Yemen led in 2016 to the biggest cholera outbreak documented in modern history, with more than 2.5 million suspected cases to date. In late 2018, epidemiological surveillance showed that V. cholerae isolated from cholera patients had turned multi-drug resistant (MDR). We generated genomes from 260 isolates sampled in Yemen between 2018 and 2019 to identify a possible shift in circulating genotypes. 84% of V. cholerae isolates were serogroup O1 belonging to the seventh pandemic El Tor (7PET) lineage, sublineage T13 - same as in 2016 and 2017 - while the remaining 16% of strains were non-toxigenic and belonged to divergent V. cholerae lineages, likely reflecting sporadic gut colonisation by endemic strains. Phylogenomic analysis reveals a succession of T13 clones, with 2019 dominated by a clone that carried an IncC-type plasmid harbouring an MDR pseudo-compound transposon (PCT). Identical copies of these mobile elements were found independently in several unrelated lineages, suggesting exchange and recombination between endemic and epidemic strains. Treatment of severe cholera patients with macrolides in Yemen from 2016 to early 2019 coincides with the emergence of the plasmid-carrying T13 clone. The unprecedented success of this genotype where an SXT-family integrative and conjugative element (SXT/ICE) and an IncC plasmid coinhabit show the stability of this MDR plasmid in the 7PET background, which may durably reduce options for epidemic cholera case management. We advocate a heightened genomic epidemiology surveillance of cholera to help control the spread of this highly-transmissible, MDR clone.

microbiology↗

bioRxiv2021

Vibrio cholerae El Tor strains linked to global cholera are homogeneous by pulsed-field gel electrophoresis

Vibrio cholerae O1 El Tor, causative agent of the ongoing seventh cholera pandemic, is native to the aquatic environment of the Ganges Delta, Bay of Bengal (GDBB). Recent studies traced pandemic strains to the GDBB and proposed global spread of cholera had occurred via intercontinental transmission. In the research presented here, NotI-digested genomic DNA extracted from V. cholerae O1 clinical and environmental strains isolated in Bangladesh during 2004 - 2014 was analyzed by pulsed-field gel electrophoresis (PFGE). Results of cluster analysis showed 94.67% of the V. cholerae isolates belonged to clade A and included the majority of clinical isolates of spatio-temporal origin and representing different cholera endemic foci. The rest of the strains were estuarine, all environmental isolates from Mathbaria, Bangladesh, and occurred as singletons, clustered in clades B and C, or in the small clades D and E. Cluster analysis of the Bangladeshi strains and including 157 El Tor strains from thirteen countries in Asia, Africa, and the Americas revealed 85% of the total set of isolates belonged to clade A, indicating all were related, yet did not form an homogeneous cluster. Overall, 15% of the global strains comprised multiple small clades or segregated as singletons. Three sub-clades could be discerned within the major clade A, reflecting distinct lineages of V. cholerae El Tor associated with cholera in Asia, Africa, and the Americas. The presence in Asia and the Americas of non-pandemic V. cholerae El Tor populations differing by PFGE and from strains associated with cholera globally suggests different ecotypes are resident in distant geographies. Author SummaryCholera is a major health threat, especially in the Ganges Delta, Bay of Bengal (GDBB). Vibrio cholerae, causative agent of cholera, is native to the GDBB aquatic environment. Recent genomic studies suggest GDBB is the cholera hotspot where the disease spreads globally via human activity. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA from V. cholerae El Tor endemic cholera strains was done, including Bangladesh aquatic environment and clinical strains from distant geographical regions representing three cholera-prone continents. Results showed the majority of pandemic strains belonged to a major cluster, suggesting clonal relatedness. Ecotypes were detected, indicating geographically specific lineages. It is concluded that epidemic strains in Bangladesh and thirteen countries of Asia, Africa, and the Americas are geographically adapted, with independent evolution of the bacterium in respective geographical regions.

molecular biology↗

bioRxiv2021

Genomic and antigenic diversity of carried Klebsiella pneumoniae isolates mirrors that of invasive isolates in Blantyre, Malawi

Klebsiella pneumoniae is an antimicrobial resistance (AMR) associated pathogen of global importance, and polyvalent vaccines targeting K. pneumoniae O-antigens are in development. Genomes from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae complex isolates colonising adults in Blantyre, Malawi, placed these isolates in a global genomic context, and compared colonising to invasive isolates from the main public hospital in Blantyre. 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicountry collection of 484 K. pneumoniae genomes sampled to cover maximum diversity of the species, 150 previously sequenced Malawian and 66 Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule (K-) and LPS O-antigen (O-types). We find that the diversity of Malawian Klebsiella isolates is representative of the species population structure, but with local success and expansion of sequence types (STs) ST14, ST15, ST340 and ST307. Siderophore and hypermucoidy genes were more frequent in invasive versus carriage isolates (present in 13% vs 1%, p < 0.001) but still generally lacking in most invasive isolates. The population structure and distribution of O-antigen types was similar in Malawian invasive and carriage isolates, with O4 being more common in Malawian isolates (14%) than in previously published studies (2-5%). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlights the need for geographically aware sampling to robustly define secular trends in Klebsiella diversity. Colonising and invasive isolates in Blantyre are similar and hence O-typing of colonising Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development. Data SummaryAll data and code to replicate this analysis is available as the blantyreESBL v1.0.0 R package (https://doi.org/10.5281/zenodo.5554082) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, and accession numbers (as well as accession numbers of publicly available genomes used in this analysis) are provided in the R package.

microbiology↗

bioRxiv2021

Genomic analysis of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli colonising adults in Blantyre, Malawi reveals previously undescribed diversity.

Escherichia coli is a ubiquitous bacterium and one of the most prevalent Gram-negative species associated with drug resistant infections. The large number of sequenced genomes available have provided us with a consistently growing knowledge base to further understand pathogenesis and epidemiology of this organism. However, data from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts and E. coli genetic diversity from this region is poorly described. To reduce this gap, we investigated extended-spectrum beta-lactamase (ESBL)-producing E. coli colonising adults in Blantyre, Malawi to assess the bacterial diversity and AMR determinants and to place these isolates in the context of the wider population structure. We performed short-read whole-genome sequencing of 473 colonising ESBL E. coli isolated from human stool and contextualised the genomes with a previously curated multi-country species wide collection of 10,146 genomes. The most frequently identified sequence types (STs) in our collection were the globally successful ST131, ST410 and ST167, and the dominant ESBL genes were blaCTX-M, mirroring global trends. However, 37% of Malawian isolates did not cluster with any isolates in the curated multicountry collection and a core gene phylogeny was consistent with locally spreading subclades within globally dominant clones, including in ST410 and ST167. We also found Carbapenemase genes in our collection at low frequency; we used long read sequencing to characterise selected ESBL and carbapenemase-associated plasmids, demonstrating the presence of globally distributed carbapenemase carrying plasmids. Increased genomic surveillance of E. coli from Malawi and sSA is necessary to understand local, regional and global transmission of both E. coli and the AMR genes they commonly carry. Impact StatementDrug-resistant Escherichia coli producing extended-spectrum beta lactamase (ESBL) or carbapenemase enzymes have been identified by the World Health Organisation as priority pathogens of global concern, and whole genome sequencing has provided insight into mechanisms of virulence, antimicrobial resistance, and the spread of high-risk clones. However, studies analysing large numbers of E. coli using whole-genome data often focus on opportunistic use of hospital diagnostic collections in high-income settings. Understanding how the genomic epidemiology of E. coli in low- and middle-income countries (including many of the nations of sub-Saharan Africa) differs is essential to provide insight into local, and global drivers of transmission. We therefore sequenced 473 ESBL-producing E. coli genomes colonising adults in Blantyre, Malawi. We analyse determinants of antimicrobial resistance and virulence and place the isolates in wider context using a previously published global E. coli collection that was generated to represent the whole species diversity of sequences publicly available at the time of generation. We find that there is diversity in Malawian isolates not reflected in the curated global collection: widely successful antimicrobial-resistance associated E. coli sequence types are represented in Blantyre, but locally circulating subclades are apparent. Furthermore, given the high number of ESBL producing pathogens causing infections there is an unmet need for carbapenem antimicrobials which are still active against ESBL-producers but are not yet widely available in our setting. We find that carbapenemases (enzymes that can render bacteria resistant to carbapenems) in our collection are unusual but present and carried on globally disseminated plasmids. So too are globally successful, stably carbapenemase-associated E. coli lineages. Although the Malawian isolates analysed typically lacked carbapenemases, carbapenem use is increasing in Malawi and their unstewarded use will accelerate selection for carbapememases in E. coli in the future. Our study highlights the need for robust stewardship protocols and ongoing genomic surveillance as these agents are introduced. Data SummaryAll data and code to replicate this analysis are available as the blantyreESBL v1.3 R package (https://doi.org/10.5281/zenodo.5554081) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, under PRJEB26677, PRJEB28522 and PRJEB36486 (short reads) and PRJNA869071 (Nanopore reads and hybrid assemblies). Accession numbers (as well as accession numbers of publicly available genomes used in this analysis) linked to sample metadata are provided in the R package and as supplementary data to this manuscript.

microbiology↗

bioRxiv2021

Population structure analysis and laboratory monitoring of Shigella with a standardised core-genome multilocus sequence typing scheme: a validation study

The laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. We studied over 4,000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes were grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognised the natural groupings for Shigella. By contrast, the serotyping scheme was affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will enhance the laboratory surveillance of Shigella infections.

genomics↗

bioRxiv2021

Exploring bacterial diversity via a curated and searchable snapshot of archived DNA sequences

The open sharing of genomic data provides an incredibly rich resource for the study of bacterial evolution and function, and even anthropogenic activities such as the widespread use of antimicrobials. Whilst these archives are rich in data, considerable processing is required before biological questions can be addressed. Here, we assembled and characterised 661,405 bacterial genomes using a uniform standardised approach, retrieved from the European Nucleotide Archive (ENA) in November of 2018. A searchable COBS index has been produced, facilitating the easy interrogation of the entire dataset for a specific gene or mutation. Additional MinHash and pp-sketch indices support genome-wide comparisons and estimations of genomic distance. An analysis on this scale revealed the uneven species composition in the ENA/public databases, with just 20 of the total 2,336 species making up 90% of the genomes. The over-represented species tend to be acute/common human pathogens. This aligns with research priorities at different levels from individuals with targeted but focused research questions, areas of focus for the funding bodies or national public health agencies, to those identified globally as priority pathogens by the WHO for their resistance to front and last line antimicrobials. Understanding the actual and potential biases in bacterial diversity depicted in this snapshot, and hence within the data being submitted to the public sequencing archives, is essential if we are to target and fill gaps in our understanding of the bacterial kingdom.

microbiology↗

bioRxiv2021

Panakeia - A universal tool for bacterial pangenome analysis

AO_SCPLOWBSTRACTC_SCPLOWWe present Panakeia, a tool which strives to be easy to use and providing a detailed view of the pangenome structure which can efficiently be utilised for discovery, or further in-depth analysis, of features of interest. It analyses synteny and multiple structural patterns of the pangenome, giving insights into the biological diversity and evolution of the studied taxon. Panakeia hence provides both broad and detailed information on the structure of a pangenome, for diverse and highly clonal populations of bacteria. Development of new pan-genome analysis tools is important, as the pangenome of a microbial species has become an important method to define the diversity of a selected taxon, most commonly a species, in the last years. This enables comparison of strains from different ecological niches and can be used to define the functional potential in a bacterial population. It gives us a much better view of microbial genomics than can be gained from singular genomes which after all are just single representatives of a much more varied population. Previously published pangenome tools often reduce the information to a presence/absence matrix of unconnected genes or generate massive hard to interpret output graphs. However, Panakeia includes synteny and structural information and presents it in a way that can readily be used for further analysis. Panakeia can be downloaded at https://github.com/BioSina/Panakeia.

bioinformatics↗

bioRxiv2021

Different evolutionary trends form the twilight zone of the bacterial pan-genome

The pan-genome is defined as the combined set of all genes in the gene pool of a species. Pan-genome analyses have been very useful in helping to understand different evolutionary dynamics of bacterial species: an open pan-genome often indicates a free-living lifestyle with metabolic versatility, while closed pan-genomes are linked to host-restricted, ecologically specialised bacteria. A detailed understanding of the species pan-genome has also been instrumental in tracking the phylodynamics of emerging drug resistance mechanisms and drug resistant pathogens. However, current approaches to analyse a species pan-genome do not take the species population structure into account, nor do they account for the uneven sampling of different lineages, as is commonplace due to over-sampling of clinically relevant representatives. Here we present the application of a population structure-aware approach for classifying genes in a pan-genome based on within-species distribution. We demonstrate our approach on a collection of 7,500 E. coli genomes, one of the most-studied bacterial species used as a model for an open pan-genome. We reveal clearly distinct groups of genes, clustered by different underlying evolutionary dynamics, and provide a more biologically informed and accurate description of the species pan-genome.

genomics↗

bioRxiv2021

gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

Members of the bacterial genus Vibrio utilise chitin both as a metabolic substrate and a signal to activate natural competence. Vibrio cholerae is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in V. cholerae has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse V. cholerae. We show that the gene encoding GbpA, known to be an important colonisation and virulence factor in pandemic isolates, is not ubiquitous amongst V. cholerae. We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within the V. cholerae species is more complex than previously thought, and emphasise the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains. Impact statementIt is thought that the ability to metabolise chitin is ubiquitous amongst Vibrio spp., and that this enables these species to survive in aqueous and estuarine environmental contexts. Although chitin metabolism pathways have been detailed in several members of this genus, little is known about how these processes vary within a single Vibrio species. Here, we present the distribution of genes encoding key chitinase and chitin-binding proteins across diverse Vibrio cholerae, and show that our canonical understanding of this pathway in this species is challenged when isolates from non-pandemic V. cholerae lineages are considered alongside those linked to pandemics. Furthermore, we show that genes previously thought to be species core genes are not in fact ubiquitous, and we identify novel components of the chitin metabolic cascade in this species, and present functional validation for these observations. Data summaryThe authors confirm that all supporting data, code, and protocols have been provided within the article or through supplementary data files. O_LINo whole-genome sequencing data were generated in this study. Accession numbers for the publicly-available sequences used for these analyses are listed in Supplementary Table 1, Table 2, and the Methods. C_LIO_LIAll other data which underpin the figures in this manuscript, including pangenome data matrices, modified and unmodified sequence alignments and phylogenetic trees, original images of gels and immunoblots, raw fluorescence data, amplicon sequencing reads, and the R code used to generate Figure 7, are available in Figshare: https://dx.doi.org/10.6084/m9.figshare.13169189 (Note for peer-review: Figshare DOI is inactive but will be activated upon publication, please use temporary URL https://figshare.com/s/7795a2d80c13f694f8fa for review). C_LI O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/430729v1_fig7.gif" ALT="Figure 7"> View larger version (38K): org.highwire.dtl.DTLVardef@100c246org.highwire.dtl.DTLVardef@d295aborg.highwire.dtl.DTLVardef@1601f69org.highwire.dtl.DTLVardef@1ae3ecb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO ChiA-3-6xHis displays chitobiosidase and endochitinase activities, but not -N-acetylglucosaminidase activity. Lysates and supernatants included in Figures 6b and 6c were assayed for chitinase enzyme activity using a fluorometric chitinase assay kit (see Methods for details). Lysed cells from E. coli cultures harbouring pMJD157 and cultured in the presence of arabinose were the only samples which produced detectable and statistically significant signals on triacetylchitotriose and chitobiose substrates (a, b). No signal was detected in the presence of glucosaminide substrate (c). All plots are scaled equivalently. P = pellet; S = supernatant; EV = empty vector (pBAD33). Parametric t-tests performed where indicated: ns = not significant; *** = p<0.001; **** = p<0.0001. C_FIG

microbiology↗

bioRxiv2020

Global population structure and genotyping framework for genomic surveillance of the major dysentery pathogen, Shigella sonnei

Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring of the emergence and spread of resistant clones at local and global scales.

microbiology↗

bioRxiv2020

Co-evolutionary signals from Burkholderia pseudomallei population genomics highlight its survival strategy in a hostile environment

BackgroundThe soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis. It kills up to 40% of cases and contributes to human morbidity and mortality in many tropical and sub-tropical countries. As no vaccines are currently available, prevention is the key health policy and is achieved by avoiding direct contact with soil and standing water. The pathogen notoriously persists in ranges of environmental conditions which make disease prevention difficult. We aimed to scan B. pseudomallei genomes for signals of evolutionary adaptations that allow it to thrive across environmental conditions, which should ultimately inform prevention policy. MethodsWe conducted three layers of analyses: a genome-wide epistasis and co-selection study (GWES) on 2,011 B. pseudomallei genomes to detect signals of co-selection; gene expression analyses across 82 diverse physical, chemical, biological and infectious conditions to identify specific conditions in which such selection might have acted; and gene knockout assays to confirm the function of the co-selection hotspot. FindingsWe uncovered 13,061 mutation pairs in distinct genes and non-coding RNA that have been repeatedly co-selected through B. pseudomallei evolution. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions including temperature stress, osmotic stress, UV radiation, and nutrient deprivation; highlighting these conditions as the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed the role of BPSL1661 under nutrient deprivation, and explored the functional basis of the co-selection gene network surrounding BPSL1661 in facilitating bacterial survival under nutrient depletion. InterpretationOur findings suggest that B. pseudomallei has a selective advantage to survive nutrient-limited conditions. Anthropogenic activities such as shifting cultivation systems with more frequent rotations of cropping and shortened fallow periods or continuous cultivation of cash crops could directly or indirectly contribute to loss of soil nutrient; these may lead to the preferential survival of B. pseudomallei and a subsequent rise of melioidosis. Successful disease control for melioidosis needs to consider improving environmental health in addition to current preventive efforts. FundingWellcome Trust, European Research Council, UK Department of Health, Thailand Research Fund and Khon Kaen University Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with terms (co-selection AND bacteria AND population) with no date or language restrictions from database inception until April 11, 2021. We identified 44 publications of which four were conducted at a genome-wide scale. These four studies were performed on human-restricted pathogens, detected co-selection of antibiotic resistance gene networks which highlight the use of antibiotics as major selection pressures and further inform treatment options. However, none of these studies were performed on Burkholderia pseudomallei or other opportunistic pathogens that have been adapted to both natural and host environments. The selection pressures exerted on these pathogens and the genetic determinants allowed for their adaptations remain unclear, which limit our understanding on the bacterial biology and the information used for disease control. Added value of this studyBased on genomes of 2,011 B. pseudomallei collected from melioidosis endemic areas, we identified and confirmed genetic signals for co-selection. Using transcriptome profiling covering a broad spectrum of conditions and exposures, we showed that genes under co-selection displayed marked expression correlation under physical stress conditions with the gene at the co-selection hotspot conditionally expressed under nutrient starvation. Furthermore, we experimentally validated the function of the hotspot gene and demonstrated that unlike host-restricted pathogens, the B. pseudomallei co-selection network does not facilitate host infection but is focused on bacterial survival in a harsh environment, particularly under nutrient depletion. Aside from providing a data resource, the study also showcases the power of combined genetics, transcriptomics and functional analysis as a tool for biology discovery. Implications of all available evidenceOur findings provide evolutionary and biological evidence for preferential survival of B. pseudomallei under nutrient starvation. Agricultural practice that induces soil loss, which is not uncommon in melioidosis endemic areas has been linked to soil nutrient depletion and may contribute to the prevalence of B. pseudomallei and a consequent rise of melioidosis in these regions. Successful melioidosis control has to consider environmental health in addition to existing prevention policy.

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