Biology subjects

Cole, J. A.

Publications and source records attributed to Cole, J. A..

10 recordsLinked to original sources

bioRxiv2026

Genomic epidemiology of Neisseria gonorrhoeae isolates from the G-ToG clinical trial, 2014-2016

Neisseria gonorrhoeae remains a major public health threat due to rising incidence and increasing antimicrobial resistance (AMR). To characterise the landscape of gonococci circulating in England a decade ago, we analysed whole genome sequences and clinical metadata from 354 isolates collected during the G-ToG clinical trial between 2014 and 2016. Comparison with global datasets demonstrated that the G-ToG isolates captured the breadth of UK gonococcal diversity, mapping across multiple lineages without evidence of marked bias related to participant demographic characteristics. The phylogenetic structure of gonococcal isolates within the collection was strongly shaped by sexual network, with statistically significant clustering of isolates circulating within heterosexual individuals compared with the men who have sex with men (MSM) transmission group. MSM-associated lineages exhibited higher minimum inhibitory concentration (MICs) to azithromycin and ceftriaxone and were enriched for corresponding chromosomal AMR determinants and AMR-associated plasmids. These data provide a valuable baseline preceding the implementation of several major UK public health interventions, including HIV pre-exposure prophylaxis (HIV PrEP), doxycycline post-exposure prophylaxis (Doxy-PEP) and vaccination with Bexsero. Continued integration of genomic and epidemiological surveillance will be essential to monitor whether these interventions reshape gonococcal transmission dynamics and AMR trajectories, guiding future control strategies.

genomics↗

bioRxiv2026

SeaB is a conserved Salmonella enterica extracellular matrix binding protein involved in biofilm formation and infection

Salmonella enterica is a leading cause of gastroenteritis worldwide. Exacerbating this issue is the emergence of multi-drug-resistant strains, posing a major threat to human health. Type 5 secretion system proteins play a major role in virulence and are viable vaccine targets. However, only a limited number of these proteins have been functionally characterized to date. In this study, we characterized SeaB, which belongs to the Type 5a secretion system. We demonstrated that SeaB is localized to the cell surface and is involved in binding to the extracellular matrix. Our results indicate that SeaB is involved in aggregation, biofilm formation and contributes to virulence. Furthermore, immunization with SeaB elicits antibodies and provides protection against Salmonella challenge in a mouse model of infection.

microbiology↗

bioRxiv2025

Histological techniques for the visualisation and identification of polymicrobial communities in mucosal tissue

Sequencing studies have generated masses of data linking mucosal bacteria to health and disease, particularly in organs such as the colon, mouth and stomach. However, extracting microbial DNA from mucosal sites with lower bacterial biomass can introduce significant variation given the heterogeneity of bacterial colonisation within pathological microenvironments. Therefore, a histological technique that identifies mucosal samples with high microbial mass samples prior to applying sequencing technologies, would increase reproducibility and reduce issues of low signal: noise ratios commonly observed during 16S rRNA gene sequencing. Using RNAscope technology, we have previously shown that non-H. pylori bacteria invade the gastric lamina propria in H. pylori-infected patients diagnosed with chronic gastritis and gastric intestinal metaplasia. However, RNAscope technology is costly and time consuming. Here, using gastric tissue from patients with Helicobacter pylori-associated precancer and cancer, we applied the use of a modified Gram stain technique to identify gastric tissue with high bacterial biomass. We validated the presence of bacteria using custom-designed RNAscope probes on consecutive tissue sections and then we visualised bacteria within a polymicrobial community at the Phylum, Kingdom and Genus taxonomic levels. This study validates the cost-effective modified Gram stain as an efficient way to select samples with high microbial biomass for downstream applications investigating host-microbiota relationships.

microbiology↗

bioRxiv2024

The gatekeeper to gastric cancer; gastric microbiota invade the lamina propria in Helicobacter pylori-associated gastric carcinogenesis

Stomach cancer is the fourth leading cause of cancer-related deaths worldwide. Helicobacter pylori is the main risk factor for gastric adenocarcinoma (GAC), yet the mechanism underpinning this association remains uncharacterised. Gastric intestinal metaplasia (GIM) represents the pre-cancerous stage and follows H. pylori-associated chronic gastritis (CG). Sequencing studies have revealed fewer H. pylori and more non-H. pylori bacteria in GAC. However, the spatial organisation of the gastric microbiota in health and disease is unknown. Here, we have combined RNA in situ hybridisation and immunohistochemistry to detect H. pylori, non-H. pylori bacteria and host cell markers (E-cadherin, Mucins 5AC and 2) from patients with CG (n=9), GIM (n=12), GAC and normal tissue adjacent to tumours (NATs) (n=3). Quantitative analysis of whole slide scans revealed significant correlations of H. pylori and other bacteria in CG and GIM samples. In contrast to sequencing studies, significantly fewer non-H. pylori bacteria were detected in H. pylori-negative patients. Importantly, whilst H. pylori exclusively colonised the gastric glands, non-H. pylori bacteria invaded the lamina propria in 3/4 CG and 5/6 GIM H. pylori-positive patients. Bacterial invasion was observed in 3/3 GAC samples and at higher levels than matched NATs. We propose that H. pylori holds the keys to disrupt the gastric epithelial barrier, facilitating the opportunistic invasion of non-H. pylori bacteria to the lamina propria. Bacterial invasion could be a significant driver of inflammation in H. pylori-associated carcinogenesis. This proposed mechanism would both explain the synergistic roles of H. pylori and other bacteria and redirect attempts to prevent, diagnose and treat GAC.

microbiology↗

bioRxiv2023

Inhibition of Neisseria gonorrhoeae complement-mediated killing during acute gonorrhoea is dependent upon the IgG2:IgG3 antibody ratio

Excessive binding of antibodies to the bacterial cell surface can paradoxically increase resistance of some Gram-negative pathogens to complement-mediated killing (CMK). We examined CMK of 336 Neisseria gonorrhoeae clinical isolates sampled from participants recruited to a clinical trial. Serum bactericidal assays revealed 3% (9/336) of the autologous participant sera that were tested inhibited CMK. Gonococci isolated from these participants were resistant to the autologous host serum, sensitive to a pool of healthy control sera (HCS) and protected by the host serum in a 1:1 mixture with HCS. Analysis of the clinical metadata showed that there were a significantly higher proportion of inhibitory sera found in participants with urethral infections and from men within the transmission network of men who have sex with women (MSW), when compared to the whole cohort. Following antibody purification from selected participants with inhibitory sera (5/9), IgG and IgM protected the autologous isolates from HCS-mediated killing. Only three of these isolates were protected by purified IgA. A closer examination of IgG subclasses using whole gonococcal cell ELISAs revealed a strong correlation between increased IgG2 binding and decreased IgG3 binding to the bacterial cell surface of isolates that were resistant to CMK. This suggests that IgG2 prevents bactericidal IgG3 from initiating CMK and that the IgG2:IgG3 ratio is important for determining either inhibition or killing of isolates. We therefore reveal a previously unreported mechanism by which inhibitory antibodies prevent CMK of N. gonorrhoeae.

microbiology↗

bioRxiv2023

Bam complex associated proteins in Escherichia coli are functionally linked to peptidoglycan biosynthesis, membrane fluidity and DNA replication

Biogenesis of the bacterial outer membrane is key to bacterial survival and antibiotic resistance. Central to this is the {beta}-barrel assembly machine (Bam) complex and its associated chaperones, which are responsible for transport, folding and insertion of outer membrane proteins (OMPs). The Escherichia coli Bam complex is composed of two essential subunits, BamA and BamD, and three non-essential accessory lipoproteins, BamB, BamC and BamE. Optimal Bam function is further dependent on the non-essential periplasmic chaperones DegP, Skp and SurA. Despite intensive study, the specific function of these non-essential Bam-associated proteins is not fully understood. Here, we analysed {Delta}bamB, {Delta}bamC, {Delta}bamE, {Delta}surA, {Delta}skp and {Delta}degP knockout strains by phenotypic screening, conservation analysis and high-throughput genetics. We identify hundreds of synthetic-lethal interactions and reveal that Bam complex activity is impacted by changes in outer membrane lipid composition and that enterobacterial common antigen is essential in the absence of the chaperone SurA. We also show genes responsible for synthesis of peptidoglycan are synthetically-lethal with Bam accessory lipoprotein encoding genes. Together, our data indicates potential mechanisms for coordination of OMP biogenesis with other cellular growth processes such as LPS and peptidoglycan biogenesis.

microbiology↗

bioRxiv2023

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multi-drug resistant clones both alarmingly associated with high mortality. The molecular microbial genetics underpinning these recalcitrant K. pneumoniae infections is unclear, coupled with the emergence of lineages resistant to nearly all present day clinically important antimicrobials. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani medium), human urine and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

microbiology↗

bioRxiv2022

TraDIS-validate: a method for curating ordered gene-replacement libraries

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterised genes. To address this gap in functional annotation, many high-throughput screens have been devised with the goal of uncovering novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered gene-replacement library. We applied our method (TraDIS-Validate) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates. TraDIS-validate therefore had a success rate of 99.7% for identifying the correct kanamycin cassette position. This dataset provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of an antibiotic resistance cassette in any ordered library.

genetics↗

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗

bioRxiv2018

Characterization of emetic and diarrheal Bacillus cereus strains from a 2016 foodborne outbreak using whole-genome sequencing: addressing the microbiological, epidemiological, and bioinformatic challenges

The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) has not been routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group strains were obtained from human cases (n =7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (clade III) and B. cereus sensu stricto (clade IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely-related and formed a well-supported clade relative to publicly-available emetic clade III genomes with an identical sequence type (ST 26). When compared to publicly-available emetic clade III ST 26 B. cereus group genomes, the 30 emetic clade III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly-available genomes by a mean of 301.7 to 528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. Based on both WGS and epidemiological data, we hypothesize that the outbreak was a single source outbreak caused by emetic clade III B. cereus belonging to the B. paranthracis species. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

microbiology↗