Biology subjects

Matlock, W.

Publications and source records attributed to Matlock, W..

10 recordsLinked to original sources

bioRxiv2026

Mobility shapes plasmid GC content evolution

Plasmids are frequently AT-rich relative to their bacterial hosts. Despite this tendency towards lower GC content, plasmid and host chromosome GC content are positively correlated across diverse collections of plasmid-host pairs. However, the evolutionary processes underlying this pattern remain unclear. The classic model of amelioration predicts that horizontally acquired DNA gradually converges on host nucleotide composition, but since plasmids can repeatedly transfer between bacterial hosts, the opportunity for such host-associated evolution may depend on their transmission dynamics. Using 50,936 plasmid-host pairs from a public sequence database, we found that the apparent global correlation between plasmid and host chromosome GC content was largely driven by differences between bacterial species rather than within species. We therefore accounted for plasmid and host population structure when testing how plasmid mobility shaped host-associated compositional evolution. We compared two contrasting regimes: a population of 3,682 Enterobacterales plasmids distributed across diverse host backgrounds, and six long-term host-associated plasmids from a Rhizobium leguminosarum lineage with INSeq-determined gene essentiality data. In the Enterobacterales population, GC content variation was overwhelmingly explained by plasmid lineage rather than host phylogeny, and conjugative plasmids showed greater similarity to their host chromosomes than mobilisable or non-mobilisable plasmids. In the Rhizobium leguminosarum plasmids, synonymous-site composition was more similar to the host chromosome among genes required across multiple host life stages. Together, these results support a model in which plasmid mobility influences the opportunity for host-associated evolutionary processes to alter nucleotide composition.

genomics↗

bioRxiv2026

DNA-damaging combination treatments impose genotype-specific constraints on hypermutator evolvability

Bacterial hypermutator strains drive rapid evolution of antibiotic resistance in chronic infections. Inspired by cancer therapy approaches that exploit synthetic lethality by targeting DNA repair deficiencies in hypermutator tumours, we tested whether pairing a conventional antibiotic with a secondary DNA-damaging agent could constrain hypermutator evolution in bacteria. Using high-throughput experimental evolution of Escherichia coli repair-deficient strains, we evolved populations under carbapenem selection in combination with ciprofloxacin or mitomycin C. Strains lacking oxidative damage repair, double-strand break repair, or transcription-coupled repair showed significantly reduced evolvability, particularly under constant antibiotic pressure and increasing genotoxic stress. However, mismatch repair (MMR) hypermutators, the predominant clinical genotype, did not show reduced evolvability under these combination treatments. This is consistent with pathway orthogonality: MMR does not repair the structural DNA lesions induced by ciprofloxacin or mitomycin C, and the elevated mutation supply of MMR-deficient strains may allow rapid adaptation despite background DNA damage. Our findings demonstrate that combination strategies can constrain the evolvability of specific repair-deficient genotypes in vitro, but success requires matching DNA damage type to specific repair vulnerabilities. This work establishes proof of principle for genotype-directed antimicrobial strategies that exploit DNA repair vulnerabilities to constrain hypermutator evolution. SignificanceThis work demonstrates that combining DNA-damaging agents with antibiotics can constrain resistance evolution in specific hypermutator genotypes, but not others. These findings establish that evolution-informed antimicrobial strategies must be genotype-specific, opening new paths for precision approaches to delay or prevent the evolution of antibiotic resistance by exploiting DNA repair vulnerabilities.

microbiology↗

bioRxiv2026

Conjugation structures plasmid populations through host-lineage restriction

Conjugation mediates plasmid transfer between bacterial species, driving the horizontal spread of traits like antibiotic resistance. However, genomic and experimental evidence indicates that many conjugative plasmids are restricted to particular host lineages despite broad theoretical host ranges. Using 4,281 plasmids from an epidemiologically coherent sample of 1,739 Escherichia coli bloodstream infection isolates, we quantify the host-lineage associations of 30 plasmid backbones and assess how these associations structure plasmid co-occurrence. To achieve this, we develop a novel Bayesian modelling framework that separates genuine backbone-backbone associations from patterns arising due to shared host ancestry or abundance. First, we find that conjugative backbones exhibit stronger host-lineage restriction than mobilisable backbones, and that restriction increases with plasmid size independently of mobility class. Consistent with this pattern, host-lineage restricted backbones demonstrate fewer independent acquisitions across the host phylogeny in ancestral state reconstructions. Comparison with global plasmid diversity shows that the most host-lineage restricted backbones remain restricted beyond the studied population, whereas the least restricted backbones span a mean of seven host species. Next, after accounting for host phylogeny and abundance, we find that two thirds of backbone pairs show no strong association or avoidance; however, backbones sharing host lineages co-occur more frequently than expected. Lastly, we identify a clique of strongly associated mobilisable backbones that appear to exploit a shared set of lineage-restricted conjugative partners. A mathematical model demonstrates that hostlineage restriction of conjugative backbones, together with specificity in conjugative-mobilisable transfer, is sufficient to generate clustering among mobilisable plasmids. Collectively, our findings reframe conjugation as a mechanism that promotes within-lineage persistence and shapes plasmid community structure, with potentially important consequences for the accumulation of resistance and virulence determinants.

microbiology↗

bioRxiv2025

Plasmids link antibiotic resistance genes and phage defense systems in E. coli

Phage therapy has been proposed as an alternative to antibiotics to treat resistant infections. However, we have a limited understanding of how antibiotic resistance genes (ARGs) associate with bacterial phage defense systems (PDSs). Here, we explore the relationship between ARGs and PDSs in a sample of 2,559 plasmids originating from 1,044 E. coli isolates, representing a snapshot of clinical and non-clinical diversity in Oxfordshire, UK (2008-2020). In total, we identify 3,193 ARGs and 14,013 PDSs (180 unique types). We demonstrate that E. coli plasmids are enriched for ARGs and PDSs (both p<0.001), with a bias towards toxin-antitoxin/abortive-infection, TIR-domain and CBASS systems (all q<0.025). We proceed to show that ARGs and PDSs are physically linked by plasmids (p<0.001). Together, our results suggest that phage therapy may inadvertently select for antibiotic resistant bacteria, and that antibiotic use may similarly drive resistance to phage.

evolutionary biology↗

bioRxiv2024

E. coli phylogeny drives co-amoxiclav resistance through variable expression of blaTEM-1

Co-amoxiclav resistance in E. coli is a clinically important phenotype associated with increased mortality. The class A beta-lactamase blaTEM-1 is often carried by co- amoxiclav-resistant pathogens, but exhibits high phenotypic heterogeneity, making genotype-phenotype predictions challenging. We present a curated dataset of n=377 E. coli isolates representing all 8 known phylogroups, where the only acquired beta- lactamase is blaTEM-1. For all isolates, we generate hybrid assemblies and co-amoxiclav MICs, and for a subset (n=67/377), blaTEM-1 qPCR expression data. First, we test whether certain E. coli lineages are intrinsically better or worse at expressing blaTEM-1, for example, due to lineage differences in regulatory systems, which are challenging to directly quantify. Using genotypic features of the isolates (blaTEM-1 promoter variants and copy number), we develop a hierarchical Bayesian model for blaTEM-1 expression that controls for phylogeny. We establish that blaTEM-1 expression intrinsically varies across the phylogeny, with some lineages (e.g. phylogroups B1 and C, ST12) better at expression than others (e.g. phylogroups E and F, ST372). Next, we test whether phylogenetic variation in expression influences the resistance of the isolates. With a second model, we use genotypic features (blaTEM-1 promoter variants, copy number, duplications; ampC promoter variants; efflux pump AcrF presence) to predict isolate MIC, again controlling for phylogeny. Lastly, we use a third model to demonstrate that the phylogenetic influence on blaTEM-1 expression causally drives the variation in co- amoxiclav MIC. This underscores the importance of incorporating phylogeny into genotype-phenotype predictions, and the study of resistance more generally.

microbiology↗

bioRxiv2024

Global genomic epidemiology of blaGES-5 carbapenemase-associated integrons

Antimicrobial resistance (AMR) gene cassettes comprise an AMR gene flanked by short recombination sites (attI x attC or attC x attC). Integrons are genetic elements able to capture, excise, and shuffle these cassettes, providing adaptation on demand, and can be found on both chromosomes and plasmids. Understanding the patterns of integron diversity may help to understand the epidemiology of AMR genes. As a case study, we examined the clinical resistance gene blaGES-5, an integron-associated class A carbapenemase first reported in Greece in 2004 and since observed worldwide, which to our knowledge has not been the subject of a previous global analysis. Using a dataset comprising all NCBI contigs containing blaGES-5 (n = 431), we developed a pangenome graph-based workflow to characterise and cluster the diversity of blaGES-5 -associated integrons. We demonstrate that blaGES-5-associated integrons on plasmids are different to those on chromosomes. Chromosomal integrons were almost all identified in P. aeruginosa ST235, with a consistent gene cassette content and order. We observed instances where insertion sequence IS110 disrupted attC sites, which might immobilise the gene cassettes and explain the conserved integron structure despite the presence of intI1 integrase promoters, which would typically facilitate capture or excision and rearrangement. The plasmid-associated integrons were more diverse in their gene cassette content and order, which could be an indication of greater integrase activity and shuffling of integrons on plasmids.

genomics↗

bioRxiv2022

Evaluation of metagenomic, 16S rRNA gene and ultra-plexed PCR-based sequencing approaches for profiling antimicrobial resistance gene and bacterial taxonomic composition of polymicrobial samples

BackgroundShotgun metagenomic sequencing is increasingly popular in taxonomic and resistome-profiling of polymicrobial samples due to its agnostic nature and data versatility. However, caveats include high- cost, sequencing depth/sensitivity trade-offs, and challenging bioinformatic deconvolution. Targeted PCR-based profiling optimises sensitivity and cost-effectiveness, but can only identify prespecified targets and may introduce amplification biases. Ultra-high multiplexed PCR is a potential compromise. As no comprehensive comparative evaluation exists, we evaluated performance of each method in taxonomic/resistome-profiling of a well-defined DNA mock sample and seven "real- world" wastewater samples. ResultsWe tested three sequencing approaches (short-read shotgun metagenomics, Illumina Ampliseq ultra-plexed AMR Research Panel, 16S rRNA gene sequencing) with seven bioinformatic pipelines (ResPipe, Illumina DNA Amplicon App, One Codex Metagenomic-/Targeted Loci classification and Ampliseq Report, DADA2, and an in-house pipeline for AmpliSeq data [AmpliSeek]). Metagenomics outperformed 16S rRNA gene sequencing in accurately reconstituting a mock taxonomic profile and optimising the identification of diverse wastewater taxa, while 16S rRNA gene sequencing produced more even taxonomic outputs which may be quantitatively inaccurate but enhance detection of low abundance taxa. Shotgun metagenomic and AmpliSeq sequencing performed equally well in profiling AMR genes present in a mock sample, but AmpliSeq identified more genes in more complex, "real-world" samples, likely related to sensitivity of detection at the metagenomic sequencing depth used. ConclusionsA complementary sequencing approach employing 16S rRNA gene or shallow-metagenomic sequencing for taxonomic profiling, and the AmpliSeq AMR panel for high-resolution resistome profiling represents a potential lower-cost alternative to deep shotgun sequencing and may also be more sensitive for the detection of low-prevalence AMR genes. However, our evaluation highlights that both the sequencing and bioinformatics approach used can significantly influence results; for AmpliSeq AMR gene profiling, we developed AmpliSeek which outperformed the other pipelines tested and is open source. Sequencing approach and bioinformatic pipeline should be considered in the context of study goals and sample type, with study-specific validation when feasible.

genomics↗

bioRxiv2022

Plasmid overlap and evolution between Enterobacterales isolates from bloodstream infections and non-human compartments

Plasmids enable the dissemination of antimicrobial resistance (AMR) in common Enterobacterales pathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution between Enterobacterales causing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is highly relevant to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets) Enterobacterales. Isolates were collected between 2008-2020 from sites <60km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared "backbones", with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid "backbones" were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies. FundingThis study was funded by the Antimicrobial Resistance Cross-council Initiative supported by the seven research councils and the NIHR, UK.

genomics↗

bioRxiv2021

Flanker: a tool for comparative genomics of gene flanking regions

Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial resistance genes. Here, we present Flanker, a Python package which performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as flank patterns, are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterised to finetune outputs by characterising upstream and downstream regions separately and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (blaOXA-48 and blaKPC-2/3) and show that it successfully identifies distinct clusters of flank patterns, including both known and previously uncharacterised structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterised using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking gene characterisation. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 and blaOXA-48 upregulation and demonstrate 17 flank patterns (compared to the 5 previously identified). More generally the demonstration in this study that flank patterns are associated with to geographical regions and antibiotic susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker. Data SummaryNCBI accession numbers for all sequencing data used in this study is provided in Supplementary Table 1. The analysis performed in this manuscript can be reproduced in a binder environment provided on the Flanker Github page (https://github.com/wtmatlock/flanker).

bioinformatics↗

bioRxiv2020

Genomic network analysis of an environmental and livestock IncF plasmid population

IncF plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum {beta}-lactamases, particularly in Enterobacteriaceae. Organising this plasmid diversity is challenging, and current knowledge is largely based on plasmids from clinical settings. Here, we present a network community analysis of a large survey of IncF plasmids from environmental (influent, effluent, and upstream/downstream waterways surrounding wastewater treatment works) and livestock settings. We use a tractable and scalable methodology to examine the relationship between plasmid metadata and network communities. This reveals how niche (sampling compartment and host genera) partition and shape plasmid diversity. We also perform pangenome-style analyses on network communities. We show that such communities define unique combinations of core genes, with limited overlap. Building plasmid phylogenies based on alignments of these core genes, we demonstrate that plasmid accessory function is closely linked to core gene content. Taken together, our results suggest that stable IncF plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The recent association of IncF plasmids with AMR likely reflects their suitability for rapid niche adaptation.

bioinformatics↗