Biology subjects

van Vliet, A. H. M.

Publications and source records attributed to van Vliet, A. H. M..

12 recordsLinked to original sources

bioRxiv2026

Rapid whole genome- and annotation-based multilocus sequence typing of the fungal pathogen Histoplasma capsulatum

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

genomics↗

bioRxiv2025

Scalable Assembly of Ascaris Mitogenomes from Whole-Genome Data Reveals a Novel Clade

The genus Ascaris is an important group of giant parasitic roundworms, infecting over 700 million people globally, with substantial economic losses in domestic pigs. Whilst species of Ascaris are morphologically indistinguishable, analysis of mitochondrial and selected nuclear DNA loci has revealed three clades (A, B, C), which may inflate with the addition of genomic data. Here, we present a bioinformatic pipeline for de novo assembly of complete mitochondrial genomes (mtDNA) from low-coverage whole-genome data through host-read depletion or mtDNA read enrichment, followed by mtDNA-specific assembly. Our approach yielded 149 high-quality Ascaris mtDNA assemblies, enabling the study of population-level diversity, including the identification of a novel clade (Clade D, designated here) associated with human samples from Ethiopia. Our analysis further revealed Clade C to comprise of pig-derived samples from Europe based on characterisation of worms isolated in Germany. Furthermore, genetic diversity across all mitochondrial loci was high (concatenated dataset: S = 1261; h = 94; {pi} = 0.0224), indicating substantial intraspecies variation. Our methods described here provide a scalable framework for mtDNA genome reconstruction with insights into roundworm population-genomic and phylogenetic studies.

genomics↗

bioRxiv2025

Risk factors for fluoroquinolone- and macrolide-resistance among swine Campylobacter coli using multi-layered chain graphs

Campylobacter spp. resistant to fluoroquinolones and macrolides are serious public health threats. Studies aiming to identify risk factors for drug-resistant Campylobacter have narrowly focused on antimicrobial use at the farm level. Using chain graphs, we quantified risk factors for fluoroquinolones- and macrolide-resistance in Campylobacter coli isolated from two distinctive swine production systems, conventional and antibiotic-free (ABF). The chain graphs were learned using genotypic and phenotypic resistance data from 1082 isolates and host exposures obtained through surveys for 18 cohorts of pigs. The gyrA T86I point mutation alone explained at least 58 % of the variance in ciprofloxacin minimum inhibitory concentration (MIC) for ABF and 79 % in conventional farms. For macrolides, genotype and host exposures explained similar variance in azithromycin and erythromycin MIC. Among host exposures, heavy metal exposures were identified as risk factors in both conventional and ABF. Chain graph models can generate insights into the complex epidemiology of antimicrobial resistance by characterizing context-specific risk factors and facilitating causal discovery. Author summaryAntimicrobial resistance is influenced by multiple factors, including exposures to selecting agents, such as antibiotics, antiseptics, or heavy metals, and factors affecting the transmission of resistant pathogens, such as biosecurity and hygiene measures. Understanding what specific factors are associated with resistance in a given context is challenging. We developed an approach based on probabilistic graphical models to investigate context-specific antimicrobial resistance risk factors. We applied the approach to Campylobacter coli isolated from pigs in antibiotic-free and conventional farms. We demonstrated how for fluoroquinolones, risk factors were similar across both types of farms, but risk factors for macrolides were different across settings.

microbiology↗

bioRxiv2024

Use of Genome Scale Metabolic Reconstructions of Avian Pathogenic Escherichia coli (APEC) phylogroups for the identification of lineage-specific metabolic pathways

Avian Pathogenic Escherichia coli (APEC) are a genetically diverse pathotype primarily associated with extra-intestinal infections in birds. APEC lineages are predicted to have unique metabolic capabilities contributing to virulence and survival in the host environment. Here we present a genome-scale metabolic model for the APEC pathotype based on 114 APEC genome sequences, and lineage-specific models for the phylogroups B2, C and G based on a representative isolate for each phylogroup. A total of 1,848 metabolic reactions were predicted in the 114 APEC isolates before gap filling and manual correction. Of these, 89% represented core reactions, whilst the 11% accessory reactions were mostly associated with carbon and nitrogen metabolism. Predictions of auxotrophy were confirmed by inactivation of the conditionally essential lysA and the non-essential potE genes. The APEC metabolic model outperformed the E. coli K-12 iJO1366 model in the Biolog Phenotypic Array platform. Sub-models specific for phylogroups B2, C and G predicted differences in the metabolism of 3-hydroxyphenylacetate (3-HPAA), a phenolic acid derived from the flavonoid quercetin, which is commonly added to poultry feed. Two 3-HPAA associated reactions/genes distinguished APEC phylogroup C from APEC phylogroups B2 and G, and 3-HPAA supported the growth of APEC phylogroup C in minimal media, but not phylogroups B2 and G. In conclusion, we have constructed genome-scale metabolic models for the three major APEC phylogroups B2, C and G, and have identified a metabolic pathway distinguishing phylogroup C APEC. This demonstrates the importance of lineage- and pathotype-specific metabolic models when investigating genetically diverse microbial pathogens. IMPACT STATEMENTAvian Pathogenic Escherichia coli (APEC) are the cause of colibacillosis in poultry, which results in a significant economic burden to the poultry industry, and strongly affects the health and welfare of flocks. APEC isolates show a high level of genetic diversity, which complicates diagnostics, epidemiology and the design of prevention and treatment strategies. In this study we have used genome sequences derived from 114 APEC isolates to investigate their metabolic capabilities, and define the metabolic diversity of APEC within a generalised APEC metabolic model, and lineage-specific metabolic models. These models have been interrogated to find unique pathways that can be targeted for the development of anti-APEC treatments, and one such metabolic pathway was identified as a proof of principle. This approach shows great promise for the design of future strategies to prevent and deal with APEC infections, and can be adapted to other genetically diverse microbial pathogens.

microbiology↗

bioRxiv2024

Global phylogenomic analysis of Staphylococcus pseudintermedius reveals genomic and prophage diversity in multi-drug resistant lineages

Staphylococcus pseudintermedius is the foremost cause of opportunistic canine skin and mucosal infections worldwide. Multidrug resistant (MDR) and methicillin-resistant S. pseudintermedius (MRSP) lineages have disseminated globally in the last decade and present significant treatment challenges. However, little is known regarding the factors that contribute to the success of MDR lineages. In this study, we compared the genome sequence of 110 UK isolates of S. pseudintermedius to 2,166 genomes of S. pseudintermedius populations from different continents. A novel core genome multi-locus typing scheme was generated to allow large scale, rapid and detailed analysis of S. pseudintermedius phylogenies, and was used to show that the S. pseudintermedius population structure is broadly segregated into an MDR population and a non-MDR population. MRSP lineages are predicted to either encode certain resistance genes chromosomally, or on plasmids, and this is associated with their MLST sequence type. Comparison of lineages most frequently implicated in disease, ST-45 and ST-71, with the phylogenetically related ST-496 lineage that has a comparatively low disease rate, revealed that ST-45 and ST-71 genomes encode distinct combinations of phage-defence systems and concurrently encode a high number of intact prophages. In contrast, ST-496 genomes encode a wider array of phage defence systems and lack intact, complete prophages. Additionally, we show that distinct prophages are widespread in S. pseudintermedius and appear to account for the vast majority of genomic diversity. These findings indicate that MRSP lineages have significant structural genomic differences, and that prophage integration, and differential antiviral systems correlate with the emergence of successful genotypes. Impact StatementStaphylococcus pseudintermedius is a major cause of soft tissue infections in dogs but may occasionally infect other companion animals and humans, most often veterinary professionals in primary care. Methicillin-resistant Staphylococcus pseudintermedius (MRSP) have emerged worldwide and are often linked to resistance to multiple antimicrobials, resulting in a significant health burden. Here, we analysed a large collection of S. pseudintermedius genome sequences, which has allowed a detailed characterisation of the molecular epidemiology and diversity of the species using a novel typing scheme. Here we show that closely related MRSP lineages differ in whether specific antibiotic resistance genes are encoded on potentially mobilisable plasmids, or more stably on the chromosome, indicating differing evolutionary trajectories of MRSP lineages. Comparison of the S. pseudintermedius types most frequently implicated in clinical cases to those associated with commensal carriage, showed that known genes thought to contribute to disease are universal, and therefore, not associated with the high incidence rates of disease of particular lineages. We demonstrate that MRSP sequence types linked to high disease rates lack specific phage-defence systems and are associated with a high burden of prophages. Data statementAll supporting data, code and protocols have been provided either within the article, through supplementary data files, or via Figshare and Zenodo. Six supplementary figures and one supplementary file are available with the online version of this article. Data summaryAll genome sequencing data (sequencing reads and genome assemblies) are available at the Sequence Read Archive (SRA) and Genomes repositories at NCBI, in Bioproject number PRJNA1153484. The information for the individual S. pseudintermedius genomes has been included in Supplementary File 1. Genome assemblies used in this study are available from https://zenodo.org/records/13692319, DOI 10.5281/zenodo.13692318. The S. pseudintermedius cgMLST scheme developed for this study has been made available via Zenodo (https://zenodo.org/records/13633136, DOI: 10.5281/zenodo.13633135) and Figshare (https://doi.org/10.6084/m9.figshare.26911654.v1, DOI: 10.6084/m9.figshare.26911654).

microbiology↗

bioRxiv2023

A novel variant of the Listeria monocytogenes type VII secretion system EssC component is associated with an Rhs toxin

The Type VIIb protein secretion system (T7SSb) is found in Bacillota (firmicute) bacteria and has been shown to mediate interbacterial competition. EssC is a membrane-bound ATPase that is a critical component of the T7SSb and plays a key role in substrate recognition. Prior analysis of available genome sequences of the foodborne bacterial pathogen Listeria monocytogenes has shown that although the T7SSb was encoded as part of the core genome, EssC could be found as one of seven different sequence variants. While each sequence variant was associated with a specific suite of candidate substrate proteins encoded immediately downstream of essC, many LXG-domain proteins were encoded across multiple essC sequence variants. Here we have extended this analysis using a diverse collection of 37,930 L. monocytogenes genomes. We have identified a rare eighth variant of EssC present in ten L. monocytogenes Lineage III genomes. These genomes also encode a large toxin of the rearrangement hotspot (Rhs) repeat family adjacent to essC8, along with a probable immunity protein and three small accessory proteins. We have further identified nine novel LXG-domain proteins, and four additional chromosomal hotspots across L. monocytogenes genomes where LXG proteins can be encoded. The eight L. monocytogenes EssC variants were also found in other Listeria species, with additional novel EssC types also identified. Across the genus, species frequently encoded multiple EssC types, indicating that T7SSb diversity is a primary feature of the genus Listeria. DATA SUMMARYAll genome sequences used in this study are available via Genbank, and the assembly accession numbers are provided in Table S1. This file also lists relevant metadata (name, source category, country, year and clonal complex). IMPACT STATEMENTListeria monocytogenes is a soil-borne saprophytic bacterium and a food-borne pathogen of humans. Decomposing plant matter and the human GI tract are rich in diverse microbial species and to colonise these niches L. monocytogenes must be able to compete with other bacteria. The type VII secretion system (T7SS) of Bacillota has been shown to secrete protein toxins that target other bacteria. In this study we have analysed a diverse collection of L. monocytogenes genome sequences to study the diversity of the Listeria T7SS and its putative effector proteins. We show that the EssC component of the L. monocytogenes T7SS is highly diverse, clustering into one of eight sequence variants. Each EssC variant is associated with a specific toxin candidate, and the EssC8 variant T7SS likely secretes a novel rearrangement hotspot (Rhs) repeat toxin. We also identify multiple new LXG-families of T7SS toxins and describe genomic hotspots where they are encoded. We find no link between EssC variants and clinical outcome. In agreement with this, analysis of EssC variability in available genomes of other Listeria species showed that all eight L. monocytogenes EssC variants are present in non-monocytogenes Listeria species.

microbiology↗

bioRxiv2022

The immune response to lumpy skin disease virus in cattle is influenced by inoculation route

Lumpy skin disease virus (LSDV) causes severe disease in cattle and water buffalo and is transmitted by hematophagous arthropod vectors. Detailed information of the adaptive and innate immune response to LSDV is limited, hampering the development of tools to control the disease. This study provides an in-depth analysis of the immune responses of calves experimentally inoculated with LSDV via either needle-inoculation or arthropod-inoculation using virus-positive Stomoxys calcitrans and Aedes aegypti vectors. Seven out of seventeen needle-inoculated calves (41%) developed clinical disease characterised by multifocal necrotic cutaneous nodules. In comparison 8/10 (80%) of the arthropod-inoculated calves developed clinical disease. A variable LSDV-specific IFN-{gamma} immune response was detected in the needle-inoculated calves from 5 days post inoculation (dpi) onwards, with no difference between clinical calves (developed cutaneous lesions) and nonclinical calves (did not develop cutaneous lesions). In contrast a robust and uniform cell-mediated immune response was detected in all eight clinical arthropod-inoculated calves, with little response detected in the two nonclinical arthropod-inoculated calves. Neutralising antibodies against LSDV were detected in all inoculated cattle from 5-7 dpi. Comparison of the production of anti-LSDV IgM and IgG antibodies revealed no difference between clinical and nonclinical needle-inoculated calves, however a strong IgM response was evident in the nonclinical arthropod-inoculated calves but absent in the clinical arthropod-inoculated calves. This suggests that early IgM production is a correlate of protection in LSD. This study presents the first evidence of differences in the immune response between clinical and nonclinical cattle and highlights the importance of using a relevant transmission model when studying LSD.

immunology↗

bioRxiv2022

A reverse vaccinology approach identifies putative vaccination targets in the zoonotic nematode Ascaris.

Ascariasis is the most prevalent helminthic disease affecting both humans and pigs and is caused by the roundworms Ascaris lumbricoides and Ascaris suum. While preventive chemotherapy continues to be the most common control method, recent reports of anthelminthic resistance highlight the need for development of a vaccine against ascariasis. The aim of this study was to use a reverse vaccinology approach to identify potential vaccine candidates for Ascaris. Three Ascaris proteomes predicted from whole-genome sequences were analysed. Candidate proteins were identified using open-access bioinformatic tools (e.g. Vacceed, VaxiJen, Bepipred 2.0) which test for different characteristics such as sub-cellular location, T-cell and B-cell molecular binding, antigenicity, allergenicity and phylogenetic relationship with other nematode proteins. From over 100,000 protein sequences analysed, four transmembrane proteins were predicted to be non-allergen antigens and potential vaccine candidates. The four proteins are a Piezo protein, two voltage-dependent calcium channels and a protocadherin-like protein, are all expressed in either the muscle or ovaries of both Ascaris species, and all contained high affinity epitopes for T-cells and B-cells. The use of a reverse vaccinology approach allowed the prediction of four new potential vaccination targets against ascariasis in humans and pigs. These targets can now be further tested in in vitro and in vivo assays to prove efficacy in both pigs and humans.

bioinformatics↗

bioRxiv2021

Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to {beta}-tubulin proteins and destabilising microtubules. Three mutations in the {beta}-tubulin protein family are associated with BZ resistance. Seven shared {beta}-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all {beta}-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two {beta}-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris {beta}-tubulin isotype A clusters with helminth {beta}-tubulins previously shown to interact with BZ. Molecular dynamics simulations using {beta}-tubulin isotype A highlighted the key role of amino acid E198 in BZ-{beta}-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with {beta}-tubulins have been identified and show how mutations can lead to resistance in nematodes.

molecular biology↗

bioRxiv2021

Genomic screening of antimicrobial resistance markers in UK and US Campylobacter isolates highlights stability of resistance over an 18 year period

Campylobacter jejuni and Campylobacter coli are important bacterial causes of human foodborne illness. Despite several years of reduced antibiotics usage in livestock production in the UK and US, high prevalence of antimicrobial resistance (AMR) persists in Campylobacter. Both countries have instigated genome sequencing-based surveillance programs for Campylobacter, and here we have identified AMR genes in 32,256 C. jejuni and 8,776 C. coli publicly available genome sequences to compare the prevalence and trends of AMR in Campylobacter isolated in the UK and US between 2001-2018. AMR markers were detected in 68% of C. coli and 53% of C. jejuni, with 15% of C. coli being multi-drug resistant (MDR) compared to only 2% of C. jejuni. The prevalence of aminoglycoside, macrolide, quinolone and tetracycline resistance remained fairly stable from 2001-2018 in both C. jejuni and C. coli, but statistically significant differences were observed between the UK and US. There was a statistically significant higher prevalence of aminoglycoside and tetracycline resistance for US C. coli and C. jejuni, and macrolide resistance for US C. coli. In contrast, UK C. coli and C. jejuni showed a significantly higher prevalence of quinolone resistance. Specific MLST clonal complexes (e.g. ST-353/464) showed >95% quinolone resistance. This large-scale comparison of AMR prevalence has shown that the prevalence of AMR remains stable for Campylobacter in the UK and the US. This suggests that antimicrobial stewardship and restricted antibiotic usage may help contain further expansion of AMR prevalence in Campylobacter, but are unlikely to reduce it in the short term.

microbiology↗

bioRxiv2021

A Campylobacter integrative and conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare?

Microbial genomes are highly adaptable, with mobile genetic elements (MGEs) such as integrative conjugative elements (ICE) mediating the dissemination of new genetic information throughout bacterial populations. This is countered by defence mechanism such as CRISPR-Cas systems, which limit invading MGEs by sequence-specific targeting. Here we report the distribution of the pVir, pTet and PCC42 plasmids and a new 70-129 kb ICE (CampyICE1) in the foodborne bacterial pathogens Campylobacter jejuni and Campylobacter coli. CampyICE1 contains a degenerated Type II-C CRISPR system consisting of a sole Cas9 protein, which is distinct from the previously described Cas9 proteins from C. jejuni and C. coli. CampyICE1 is conserved in structure and gene order, containing blocks of genes predicted to be involved in recombination, regulation, and conjugation. CampyICE1 was detected in 134/5,829 (2.3%) C. jejuni genomes and 92/1,347 (6.8%) C. coli genomes. Similar ICE were detected in a number of non-jejuni/coli Campylobacter species, although these lacked a CRISPR-Cas system. CampyICE1 carries 3 separate short CRISPR spacer arrays containing a combination of 108 unique spacers and 16 spacer variant families. A total of 69 spacers and 10 spacer variant families (63.7%) were predicted to target Campylobacter plasmids. The presence of a functional CampyICE1 Cas9 protein and matching anti-plasmid spacers was associated with the absence of the pVir, pTet and pCC42 plasmids (188/214 genomes, 87.9%), implicating that the CampyICE1-encoded CRISPR-Cas has contributed to the exclusion of competing plasmids. In conclusion, the characteristics of the CRISPR-Cas9 system on CampyICE1 suggests a history of plasmid warfare in Campylobacter. IMPACT STATEMENTUnderstanding pathogen evolution is paramount for enhancing food safety and limiting pathogenic disease in humans and animals. Campylobacter species comprise a group of human and animal pathogens with a remarkable success rate, being the most frequent cause of bacterial food-borne disease in high-income countries. A common theme among Campylobacter evolution is genomic plasticity, and a significant proportion of this plasticity is driven by horizontal gene transfer (HGT) that results in acquisition of complex traits in one evolutionary event. Understanding the mechanisms of transfer of MGEs and how MGEs such as integrative conjugative elements (ICE) exclude other MGEs is fundamental to understanding Campylobacter evolution. CRISPR-Cas9 proteins play a role in bacterial immune systems, mediating the defence against bacteriophage, plasmids, and integrative elements. The use of CRISPR-Cas by a mobile element to fight off competing elements, possibly to the advantage or detriment to their host, also increases our understanding of how important selfish genomic islands undergo co-evolution with bacterial pathogens, and generates insight into the complex warfare between MGEs. DATA STATEMENTAll genome sequences used in this study are available on the National Center for Biotechnology Information (NCBI) Genome database or in the Campylobacter PubMLST website; the assembly accession numbers (NCBI Genome) or genome ID numbers (Campylobacter PubMLST) are listed in Table S1 (available in the online version of this article). Genome assemblies were quality checked based on N50, L50, genome size and number of contigs. CRISPR Spacer sequences and predicted targets, Cas9 alignments, presence of mobile elements and plasmids are all included in the Supplementary Information.

microbiology↗

bioRxiv2018

Chemotactic motility and biofilm formation in Campylobacter jejuni are coordinated by the CheAYWVX system

Flagellar motility plays a central role in the bacterial foodborne pathogen Campylobacter jejuni, as flagellar motility is required for reaching the intestinal epithelium and subsequent colonisation or disease. Flagellar proteins also contribute strongly to biofilm formation during transmission. Chemotaxis is the process directing flagellar motility in response to attractant and repellent stimuli, but its role in biofilm formation of C. jejuni is not well understood. Here we show that inactivation of the core chemotaxis genes cheVAWY in C. jejuni strain NCTC 11168 affects both chemotactic motility and biofilm formation. Inactivation of any of the core chemotaxis genes (cheA, cheY, cheV or cheW) impaired chemotactic motility but did not affect flagellar assembly or growth. The {Delta}cheY mutant swam in clockwise loops, while complementation restored normal motility. Inactivation of the core chemotaxis genes interfered with the ability to form a discrete biofilm at the air-media interface, and the {Delta}cheY mutant displayed reduced dispersal/shedding of bacteria into the planktonic fraction. This suggests that while the chemotaxis system is not required for biofilm formation per se, it is necessary for organized biofilm formation. Hence interference with the Campylobacter chemotaxis system at any level disrupts optimal chemotactic motility and transmission modes such as biofilm formation.

microbiology↗