Biology subjects

Stinear, T. P.

Publications and source records attributed to Stinear, T. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Mutations in mfd cause Staphylococcus aureus mucoid hyper-biofilm phenotype in chronic rhinosinusitis

Chronic Rhinosinusitis (CRS) is a common chronic inflammation of the paranasal sinus mucosa. Staphylococcus aureus contributes to its severity through biofilm formation. In this study, we isolated eight sequential methicillin-resistant S. aureus (MRSA) isolates from a patient with severe CRS over a period of 672 days (T1-T8). The isolates were phenotypically and genomically characterised, and the extracellular biofilm proteome analysed. We identified an accumulation of mutations that included the acquisition of an IS21 family insertion sequence inactivating the icaR gene and nucleotide variants in various genes including the transcription repair coupling factor (mfd). The genomic changes were associated with a switch to a mucoid phenotype from T3 onwards (Day 178), with a significant increase in biofilm-forming capacity and the secretion of multiple enterotoxins. Targeted mutagenesis confirmed mfd is a regulator of strain mucoidy with enhanced biofilm and enterotoxin production. These findings support mfd as a target for novel anti-virulence therapies.

microbiology↗

bioRxiv2026

Southeastern Australian Montane Fens Harbour Distinct Microbial Communities Rich in Novel Diversity

Montane fens are rare and microbiologically poorly characterised wetland ecosystems in south-eastern Australia, and their microbial communities remain virtually unexplored. Here, we profile the microbiomes of Victorian montane fens using 16S rRNA metabarcoding of 12 soil cores collected along a 12-m transect and sampled across four depth horizons. Surface soils exhibited slightly higher alpha diversity than deeper layers, but the most pronounced differences occurred in community composition, with surface microbiomes significantly distinct from all subsurface depths. To contextualise these communities within global environmental diversity, we compared them with 9662 Earth Microbiome Project samples spanning 24 environmental materials processed using comparable methods. Montane fen microbiomes were one of the most diverse environmental materials analysed and compositionally distinct from all comparator biomes. Overrepresentation analysis identified signature microbial taxa, including archaeal lineages from the Crenarchaeota and Methanomicrobia and bacterial phyla such as Acidobacteria, highlighting taxa involved in ecological processes associated with acidic, saturated, and organic-rich soils. Notably, the majority of sOTUs detected in montane fens were unique to this environment - the highest proportion of source-specific taxa among all biomes analysed. Together, these findings demonstrate that southeastern Australian montane fens harbour a highly distinctive and largely uncharacterised microbial community, underscoring their ecological uniqueness and the importance of conserving these rare alpine wetlands. Data SummarySequencing data is available in SRA BioProject PRJNA1398590; accessions SRR36684598 through SRR36684645. Metadata and accessions for collected montane fen samples are included in Table S1 and metadata for Earth Microbiome Project samples included in this study are listed in Table S4. Impact StatementWetland ecosystems are increasingly recognised as important reservoirs of microbial diversity, yet many remain poorly characterised in global microbiome surveys. In this study, we provide the first characterisation of microbial communities inhabiting montane fens in southeastern Australia and place them in a global context using publicly available environmental microbiome data. We show that these fens harbour exceptionally diverse microbial communities that are compositionally distinct from other environmental sources processed using comparable methods, with a high proportion of taxa that are not present in any other sample in an existing reference dataset. By extending global comparisons to an under-sampled wetland type, this work adds to the growing body of evidence that significant microbial diversity remains undocumented in geographically and ecologically restricted environments. The findings are relevant to researchers working in microbial ecology, environmental genomics, and biogeography, as well as those interested in wetland function and conservation. While largely descriptive, this study represents an important step in expanding environmental genome catalogues and provides a baseline framework for future genomic, functional, and mechanistic investigations of montane wetland microbiomes.

microbiology↗

bioRxiv2026

A mobile ESX type VII secretion system enhances intracellular persistence in globally distributed Mycobacterium abscessus

Mycobacterium abscessus are non-tuberculous mycobacteria that are widespread in the environment and of increasing global clinical significance. Accumulating evidence shows that M. abscessus has emerged as an important pathogen, driven by highly drug-resistant lineages, enhanced transmissibility and the acquisition of specific virulence factors. In this study, we describe a previously uncharacterised ESX secretion system encoded on a 123-kbp plasmid identified in a clinical isolate of M. abscessus. This ESX system, termed ESX-pMA07, is distinct from ESX systems previously reported in M. abscessus in both sequence composition and locus organisation, characterised by a unique arrangement of core ESX components and low sequence identity to ESX-3, ESX-4 and plasmid-borne ESX-P systems. ESX-pMA07 was detected in geographically diverse clinical isolates but was restricted to particular genotypes within the global M. abscessus phylogeny. Transcriptional profiling revealed expression of ESX-pMA07 components in artificial cystic fibrosis media and during intracellular growth in macrophage cell lines. Using CRISPR interference, we show that inducible silencing of eccC, encoding the ATPase component of ESX-pMA07, significantly reduced intracellular survival of M. abscessus within macrophages. To our knowledge, this is the first characterisation of a functional, plasmid-borne ESX secretion system in M. abscessus, demonstrating that mobile genetic elements contribute to the pathogens intracellular persistence and may influence its evolving virulence. Author SummaryMycobacterium abscessus is a rapidly emerging, highly drug-resistant bacterium that causes chronic infections, particularly in people with underlying lung disease such as cystic fibrosis. The factors that enable certain M. abscessus strains to persist inside host cells are not fully understood. In this study, we identified a previously unrecognised type VII secretion system (ESX) encoded on a large plasmid in a clinical isolate of M. abscessus. This plasmid-borne ESX system, which we termed ESX-pMA07, is genetically distinct from the ESX systems normally found on the chromosome and was detected in geographically diverse clinical isolates, but restricted to specific lineages within the global M. abscessus population. We show that ESX-pMA07 genes are expressed under conditions relevant to lung infection and during intracellular growth in macrophages. Using inducible CRISPR interference to silence the ESX ATPase gene eccC, we demonstrate that ESX-pMA07 contributes to intracellular survival of M. abscessus in macrophages. These findings reveal that mobile genetic elements can encode functional secretion systems that enhance intracellular persistence, providing a mechanism for the emergence and spread of virulence traits in this important pathogen.

microbiology↗

bioRxiv2025

A Practical Resource for Multi-Omics Data Integration in Microbial Systems

The increasing availability of microbial multi-omics datasets has created new opportunities to explore complex biological systems. However, exploration remains limited by the lack of accessible, reproducible workflows that integrate multiple omics layers and deliver easily interpretable visualisations of functional and pathway-level insights. Here, we present an R-based workflow for integrated analysis and network-based pathway visualisation of microbial multi-omic data. The workflow enables microbiologists to analyse transcriptomic, proteomic, and metabolomic datasets either individually or in combination, apply univariate and multivariate approaches for biomarker discovery, and generate easily interpretable visualisations of functional and pathway-level signatures. Implemented as multi-step R Markdown, it leverages widely-used open-source tools, including mixOmics for biomarker identification and omics integration and clusterProfiler for pathway and functional enrichment analyses, with a new network-based integration and visualisation. Its flexible design supports a range of experimental structures and facilitates comparisons across strains, omics layers, and conditions, making it suitable for researchers with limited computational expertise. We demonstrate its utility using a publicly available Streptococcus pyogenes dataset, revealing both shared and strain-specific functional responses to human serum. This workflow provides a comprehensive and adaptable framework for systematic multi-omics analysis, improving accessibility and reproducibility and facilitating functional interpretation of microbial responses to diverse environments. Data summaryThe code for this workflow is available on GitHub (https://github.com/warasinee/Multiomics_Case_Study). Datasets from our previously published study (1) were used to showcase the functionality and practical utility of the workflow. The multi-omics Streptococcus pyogenes dataset used in this study is available in the following public repositories: Gene Expression Omnibus (GSE152821; GSE152822; GSE152823; GSE152824, GSE152826), Proteomics Identifications Database (PXD020863), and MetaobLights (MTBLS2324) (1). Impact StatementHigh-throughput omics technologies are transforming our understanding of how microbes adapt to diverse environments and cause disease. The integration of diverse omics layers at a systems level, combining transcriptomics, proteomics, and metabolomics data to identify signature molecules, pathways, and their interactions, remains challenging. Here, we present an R-based bioinformatic workflow designed for microbiology research, which connects existing tools and customised functions to streamline data integration and interpretation. The workflow links biomarkers to functional pathways, visualises results in an interactive network context, and is designed for flexibility and reproducibility. This practical resource lowers technical barriers to microbial multi-omics analysis, providing user-friendly access for dataset exploration and integration and supporting interpretation of system-level microbial adaptation in environmental, clinical, and industrial contexts.

systems biology↗

bioRxiv2025

Stability and Processing Impacts on Faecal Microbiota Transplant Products: An Integrated Metagenomic-Culturomic Analysis

BackgroundFaecal microbiota transplantation (FMT) is an increasingly used microbiome-based therapy, whose clinical success depends on the stability and viability of the donor microbiota. Few studies have systematically evaluated these parameters for FMT preparations. Here, we integrate metagenomic sequencing with culturing-based recovery of viable microbes to comprehensively characterise FMT preparations from healthy Australian donors. ResultsMetagenomic profiling of bacterial communities from repeat FMT faecal donations revealed high temporal stability within individual donors, supporting donor suitability for consistent and standardised FMT production. The community structure and diversity of final FMT products remained largely consistent with their source material, reflecting limited disruption during processing. Culturing and sweep metagenomics showed bacterial species representing 98% of faecal microbiota abundance was recoverable, underscoring the potential of FMTs to deliver a viable and functionally active bacterial community to a recipient. When FMT donor microbiomes were compared to >7,000 healthy gut microbiomes from 27 countries, donor samples were distinct but clustered most closely with other Westernised populations, while also exhibiting significantly higher diversity than most global cohorts. These results highlight the geographic specificity of gut microbiomes and the need to consider population context in donor selection. ConclusionsThis study strengthens the evidence base for FMT product standardisation and viability, with implications for FMT clinical use, donor screening protocols, and associated regulatory frameworks.

microbiology↗

bioRxiv2025

Integrated multi-omics reveals coordinated Staphylococcus aureus metabolic, iron transport and stress responses to human serum

Bloodstream infections caused by Staphylococcus aureus remain a leading cause of mortality worldwide. Our understanding of S. aureus survival and persistence in human serum, a cell-free fraction of blood hostile for bacteria, is still limited. Here, we applied multivariate data integration methods and network analysis to a multi-omic dataset generated from five clinically prevalent S. aureus genotypes exposed to human serum. We observed and then confirmed using isogenic mutants, the significant roles of gapdhB, sucA, sirA, sstD, and perR in bacterial survival in serum. These data show that metabolic versatility in carbon source usage, iron transport and resistance to oxidative stress are interlinked and central to S. aureus fitness in serum, representing potential S. aureus vulnerabilities that could be exploited therapeutically. IMPORTANCEBloodstream infections caused by Staphylococcus aureus are associated with mortality rates of up to 30%. However, the molecular mechanisms that enable this pathogen to survive in human serum, a nutrient-limited and immunologically hostile environment remain poorly understood. By integrating multi-omic data from five clinically relevant S. aureus genotypes and validating key signatures using mutants, we identified conserved genetic determinants critical for bacterial survival in serum. Our findings highlight the interconnected roles of carbohydrate metabolic flexibility, iron acquisition, and oxidative stress resistance in shaping S. aureus adaptation to serum. This work advances our understanding of microbial strategies to survive in the bloodstream and demonstrates the potential of multi-omic integration to uncover therapeutic vulnerabilities in bacterial pathogens.

microbiology↗

bioRxiv2025

Extensive endemic transmission of multidrug resistant Mycobacterium tuberculosis in Bhutan: A retrospective genomic-epidemiological study

Despite decreasing overall tuberculosis notifications, the proportion of multidrug-resistant tuberculosis (MDR-TB) cases are increasing in Bhutan. While most MDR-TB cases are diagnosed among patients in the bordering districts and the capital, current diagnostic tests are limited in their ability to differentiate between the recurrent introductions and local transmission. For the first time, we conducted a retrospective genomic-epidemiological study to provide insights into the population structure, genotypic resistance patterns, and explore recent transmission of drug-resistant TB in Bhutan. Whole genome sequencing was performed on randomly selected drug-resistant and drug-sensitive TB isolates from Bhutan, collected between 2018-2022 at Microbiological Diagnostic Unit Public Health Laboratory in Melbourne, Australia. We investigated drug resistance mutations, and genomic clustering of cases using different single nucleotide polymorphisms (SNPs) thresholds. Of the 203 sequences that passed the quality control, 126 (62.1%) were MDR-TB and 15 (7.4%) were isoniazid-resistant TB. There were four different circulating lineages, with most sequences belonging to lineage 2 (86.2%). Using a SNP-threshold of [≤]12 SNPs, 71% of sequences formed 12 genomic clusters. Surprisingly, the largest cluster included of 88% of all MDR-TB sequences and spanned the entire study period. These cases were highly clonal (mean pairwise SNP-distance of 10, range 0-25). Phylogenetic analysis with publicly available international sequence data showed that this MDR-TB cluster formed a distinct clade. The major burden of MDR-TB in Bhutan appears to be due to recent local transmission of cases resulting in a large, single endemic cluster. This information will be critical for TB control program in Bhutan to tackle the major burden of MDR-TB through enhanced contact tracing of this MDR-TB clade. Additionally, this genomic data will be valuable to regional neighbours to monitor for dissemination of the strain. This study highlights the significant value of investing in TB genomics in resource limited settings globally to gain actionable insights into transmission dynamics.

genomics↗

bioRxiv2025

Multi-omic dereplication of antibiotic production by diffusion chamber isolated bacteria from Australian soils

Soil bacteria are a major source of clinically useful antibiotics, yet the majority of soil-dwelling microorganisms remain uncultivable by standard laboratory methods. To access this untapped microbial diversity, we employed microbial diffusion chambers to isolate bacteria from ten Australian soil samples. A total of 1,218 bacterial isolates were recovered, representing a diverse collection spanning 61 genera from 32 families, covering the major known phyla of soil bacteria. Antibiotic activity screening revealed that 16% of isolates inhibited the growth of at least one of E. coli or S. aureus, with 120 isolates displaying activity against multidrug-resistant pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Mass spectrometry-based dereplication using GNPS identified known antibiotics in 33% of bioactive strains, including actinomycin D, nonactins, and valinomycin. Genomic analysis confirmed the presence of corresponding biosynthetic gene clusters (BGCs), while targeted analysis of selected strains uncovered production of additional antibiotics such as nigericin and streptothricin that were not initially detected by mass spectrometry. Our results demonstrate that diffusion chambers enhance bacterial recovery from soil and show the benefits of a combined pipeline including bioactivity screening, mass spectrometry, and genomics for effective antibiotic dereplication and discovery. Impact StatementThis study delivers a significant advance in natural product discovery by demonstrating that microbial diffusion chambers can dramatically improve the recovery of diverse soil bacteria with antibiotic-producing potential from Australian soil samples. By integrating in situ cultivation with high-throughput screening, mass spectrometry-based dereplication and genome mining, this study yielded more than 1,200 bacterial isolates--including many with activity against multidrug-resistant pathogens--and confirms the production of both known and previously undetected antibiotics. The discovery of streptothricin production via genomics, despite its absence from mass spectrometry data, underscores the power of a multi-layered dereplication strategy. This work not only validates the utility of diffusion chambers for unlocking the "rare biosphere" of uncultivable microbes but also highlights critical methodological refinements to diversify antibiotic-producing strains beyond Streptomyces. The findings will inform and accelerate future efforts to discover urgently needed antibiotics from environmental microbiomes. Data SummaryThe whole-genome sequences were deposited in the National Center for Biotechnology Information National Library of Medicine under BioProject accession number PRJNA1272227.

microbiology↗

bioRxiv2025

Defining new Buruli ulcer endemic areas in urban southeastern Australia using bacterial genomics-informed possum excreta surveys

Buruli ulcer (BU) in southeastern Australia is a zoonosis caused by infection with Mycobacterium ulcerans. Australian native possums are a major wildlife reservoir and infected possums shed M. ulcerans in their excreta, with mosquitoes being the major transmitting vector in this region. BU is geographically restricted and this feature, combined with an average 4.8-month incubation period, makes tracking M. ulcerans environmental spread and timely identification of new BU endemic areas challenging. While human movement complicates transmission tracing, we used the highly territorial behaviour of native possums and high-resolution pathogen genomics to confidently identify new BU endemic areas in Melbournes inner northwest and southern suburbs of Geelong. Using pathogen genomic phylodynamic modelling, we estimated that M. ulcerans was introduced to these areas 2-6 years before the emergence of human BU cases. This study shows how possum excreta surveys combined with pathogen genome data can pinpoint new BU endemic areas, thus providing critical local knowledge for targeted public health interventions to reduce exposure risk and ensure early diagnosis.

microbiology↗

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2025

Simple sequence repeats power Staphylococcus aureus adaptation

Understanding Staphylococcus aureus adaptation is foundational for addressing the challenges posed by this pathogen. Using evolutionary convergence analysis on 7099 S. aureus genomes we uncovered frequent, reversible loss-of-function (LoF) mutations caused by simple sequence repeats (SSRs). Functional assays showed SSRs are potent, length-dependent switches that activate and deactivate genes, a mechanism exemplified by frequent LoF mutations in mutL (DNA mismatch repair) that causes a hypermutator phenotype. Analysis of over 600 episodes of human S. aureus colonisation and infection confirmed laboratory findings and showed that mutL SSR hypermutability behaves like a phase-variable evolutionary capacitor that unlocks subsequent adaptive SSR variations. We also experimentally demonstrate that SSRs facilitate foreign DNA uptake (hsdR restriction endonuclease) and lead to gentamicin-resistant small colony variants that are known to cause persistent infections (menF menadione biosynthesis). Furthermore, the phenomenon is widespread, with 27 of 29 diverse human microbiome samples harbouring S. aureus with distinct SSR subpopulations, indicative of niche-specific adaptations. Thus, SSRs are potent reservoirs of previously unrecognized S. aureus genetic heterogeneity that confer rapid adaptive capacity, including in antigens targeted by past vaccine trials. A deeper understanding of SSR evolutionary trajectories has the potential to improve treatments and to anticipate S. aureus responses to new therapies.

microbiology↗

bioRxiv2025

Autocycler: long-read consensus assembly for bacterial genomes

MotivationLong-read sequencing enables complete bacterial genome assemblies, but individual assemblers are imperfect and often produce sequence-level and structural errors. Consensus assembly using Trycycler can improve accuracy, but its lack of automation limits scalability. There is a need for an automated method to generate high-quality consensus bacterial genome assemblies from long-read data. ResultsWe present Autocycler, a command-line tool for generating accurate bacterial genome assemblies by combining multiple alternative long-read assemblies of the same genome. Without requiring user input, Autocycler builds a compacted De Bruijn graph from the input assemblies, clusters and filters contigs, trims overlaps and resolves consensus sequences by selecting the most common variant at each locus. It also supports manual curation when desired, allowing users to refine assemblies in challenging or important cases. In our evaluation using Oxford Nanopore Technologies reads from five bacterial isolates, Autocycler outperformed individual assemblers, automated pipelines and other consensus tools, producing assemblies with lower error rates and improved structural accuracy. Availability and implementationAutocycler is implemented in Rust, open-source and freely available at github.com/rrwick/Autocycler. It runs on Linux and macOS and is extensively documented.

bioinformatics↗

bioRxiv2025

A refined low-dose murine model of Mycobacterium ulcerans infection to assess integrated immune networks in Buruli ulcer pathogenesis

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is a slow-growing zoonotic pathogen with distinctive pathogenesis linked primarily to its toxin mycolactone. Recent research has shown that the M. ulcerans infectious dose is very low (<10 colony forming units [CFU]). Buruli ulcer animal infection models traditionally use bacterial challenge doses in the range 104 - 106 CFU; a range orders of magnitude higher than natural infection. These large doses represent an unrealistic challenge for vaccine trials and studies of immunity. Here, we address this issue and describe a murine tail infection model in two genetically distinct mouse strains (BALB/c and C57BL/6) using quality-controlled, M. ulcerans challenge doses (10 - 20 CFU and 100 CFU). Over 24-weeks, we assessed host responses to infection by measuring >70 clinical, immunological and microbiological parameters. Principal findings included a 100% infection rate even at the lowest bacterial challenge, but with a dose-dependent delay in lesion onset and disease progression for both mouse strains. Bacterial growth kinetics were similar between mouse strains. There was a difference in immune profiles between mouse strains and between low (10 CFU) versus high (100 CFU) bacterial challenge doses. C57BL/6 mice exhibited more robust systemic cellular responses and more rapid lesion onset compared to BALB/c mice. There were dose-dependent cytokine and chemokine differences in C57BL/6 mice, while BALB/c mice displayed similar responses across both doses. Antibody responses were only detected late in the infection and were associated with the high-dose inoculum in both strains. Machine learning and other statistical analyses highlighted the importance of activated CD8+ T cells and dendritic cells in the immune response to low-dose infection in C57BL/6 mice. Murine low-dose M. ulcerans infection models provide confidence for future human Buruli ulcer challenge trials and will inform the development of effective vaccines and therapeutics.

microbiology↗

bioRxiv2025

Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a {Delta}fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

microbiology↗

bioRxiv2025

Transposon-directed insertion-site sequencing (TraDIS) analysis of Enterococcus faecium using nanopore sequencing and a WebAssembly analysis platform

Vancomycin-resistant Enterococcus faecium (VREfm) are healthcare-associated opportunistic pathogens of global significance. Genetic tools are needed to understand the molecular basis for VREfm clinically relevant phenotypes, such as persistence within the human gut or antimicrobial resistance. Here, we present a transposon-directed insertion-site sequencing (TraDIS) platform optimised for E. faecium. We engineered a transposon delivery plasmid, pIMTA(tetM), that can generate high density transposon mutant libraries, combined with Oxford Nanopore Technology amplicon sequencing to map the transposon insertion sites. We have also customised a bioinformatic analysis suite that includes a WebAssembly-powered visualisation tool called Diana, for TraDIS data exploration and analysis (https://diana.cpg.org.au/). To demonstrate performance of our platform, we assessed the impact of vancomycin exposure on a library of 48,458 unique transposon mutants. As expected, we could confirm the importance of the vanB operon for VREfm vancomycin resistance. Our end-to-end platform for running TraDIS experiments in VREfm will permit accessible, genome-scale, forward genetic screens to probe molecular mechanisms of persistence and pathogenesis. ImportanceThere are limited genetic tools specifically developed and optimized for function in Enterococcus faecium. Here, we addressed this gap through the development of a transposon-directed insertion-site sequencing (TraDIS) platform with a plasmid we engineered to specifically function in E. faecium. The application of nanopore sequencing, with a highly accessible sequence data processing and bioinformatic analysis pipeline streamlines and simplifies the methodology. These developments will allow the functional genomic analysis of important traits involved in the pathobiology of this understudied bacterium. The approach and tools we have described here are likely applicable to other Gram-positive bacteria.

microbiology↗

bioRxiv2025

Are reads required? High-precision variant calling from bacterial genome assemblies

1.Accurate nucleotide variant calling is essential in microbial genomics, particularly for outbreak tracking and phylogenetics. This study evaluates variant calls derived from genome assemblies compared to traditional read-based variant-calling methods, using seven closely related Staphylococcus aureus isolates sequenced on Illumina and Oxford Nanopore Technologies platforms. By benchmarking multiple assembly and variant-calling pipelines against a ground truth dataset, we found that read-based methods consistently achieved high accuracy. Assembly-based approaches performed well in some cases but were highly dependent on assembly quality, as errors in the assembly led to false-positive variant calls. These findings underscore the need for improved assembly techniques before the potential benefits of assembly-based variant calling - such as reduced computational requirements and simpler data management - can be realised. 2. Impact statementVariant calling is foundational to microbial genomics, yet traditional workflows rely heavily on sequencing reads, which for a typical bacterial genome can be hundreds of megabytes. In contrast, genome assemblies are far smaller - usually just a few megabytes - making them significantly easier to manage. If accurate variant calls could be made directly from assemblies, this would reduce computational demands and, in some cases, may even eliminate the need to retain raw sequencing reads. This study addresses the key question of whether variant calling from assemblies is accurate enough to replace read-based methods. Our findings show that while assembly-based variant calling can achieve high accuracy, this is only possible with error-free assemblies. Since most assemblies contain errors, assembly-based variant-calling approaches should currently be used with caution. Nevertheless, as sequencing and assembly technologies continue to advance, improved assembly accuracy may make assembly-based variant calling a viable alternative, reducing data complexity and storage demands while streamlining microbial genomic analyses. 3. Data summarySupplementary methods, data, figures and tables are available at github.com/rrwick/Are-reads-required which is also archived on Zenodo (10.5281/zenodo.14868870). Reads and assemblies are available via BioProject PRJNA1193226.

bioinformatics↗

bioRxiv2025

Dual-use virulence factors of the opportunistic pathogen Chromobacterium haemolyticum mediate haemolysis and colonization

Chromobacterium haemolyticum is an environmental bacterium that can cause severe and fatal opportunistic infections in humans and animals. Although C. haemolyticum is characterized by its strong {beta}-haemolytic activity, the molecular basis of this phenotype has remained elusive over the more than fifteen years since the species was first described. Here, we report the discovery of a family of cyclic lipodepsipeptides that are responsible for the potent haemolytic activity of C. haemolyticum. Comparative genomics of Chromobacterium spp. isolated from different environments revealed a completely conserved gene locus (chl) encoding a non-ribosomal peptide synthetase (NRPS). Metabolic profiling of C. haemolyticum DSM 19808 identified a suite of cyclic lipodepsipeptides as the products of the chl locus, with the three main congeners (jagaricin, chromolysin A and B) being elucidated by a combination of tandem mass spectrometry, chemical derivatization, and NMR spectroscopy. Significantly, a C. haemolyticum chl deletion mutant is devoid of haemolytic activity. Moreover, purified jagaricin, chromolysin A and B are haemolytic at low- micromolar concentrations in an erythrocyte lysis assay. Further bioassays demonstrated that the cyclic lipodepsipeptides are crucial for biofilm-forming and swarming behavior of C. haemolyticum. MALDI mass spectrometry imaging showed that primarily chromolysin A and B are involved in these processes in vitro. Our data shed light on the bioactivities of chromolysin A and B, specialized metabolites that likely contribute to both successful colonization of new niches and virulence potential of C. haemolyticum. ImportanceDespite the rising incidence of Chromobacterium haemolyticum as a serious opportunistic pathogen, there is limited information on whether the competitive traits that ensure its survival in its freshwater niche contribute to host infection. We present a case where bacterially produced specialized metabolites act as lynchpin chemical mediators that are not only responsible for the pronounced haemolytic phenotype of C. haemolyticum but are crucial for biofilm formation and swarming motility. These results exemplify a case of coincidental evolution, wherein the selective pressures encountered in a primary environmental niche drive the evolution of a trait impacting virulence. This knowledge provides a foundation for the development of antivirulence therapies against the emerging pathogen C. haemolyticum.

microbiology↗

bioRxiv2024

Characterisation of in vitro resistance selection against second-/last-line antibiotics in methicillin-resistant Staphylococcus aureus

SYNOPSISO_ST_ABSBackgroundC_ST_ABSThe increasing occurrence of MRSA clinical isolates harbouring reduced susceptibility to mainstay antibiotics has escalated the use of second and last line antibiotics. Hence, it is critical to evaluate the likelihood of MRSA developing clinical resistance to these antibiotics. ObjectivesOur study sought to identify the rate in which MRSA develop resistance to vancomycin, daptomycin and linezolid in vitro and further determine the mechanisms underpinning resistance. MethodsMRSA was exposed to increasing concentrations of vancomycin, daptomycin, and linezolid for 20 days, with eight replicates for each antibiotic conducted in parallel. The resulting day 20 (D20) isolates were subjected to antimicrobial susceptibility testing, whole genome sequencing, autolysis assays, and growth curves to determine bacterial fitness. ResultsExposure to vancomycin or linezolid for 20 days resulted in a subtle two-fold increase in the MIC, whereas daptomycin exposure yielded daptomycin-nonsusceptible isolates with up to 16-fold MIC increase. The MIC increase was accompanied by variable changes in relative fitness and reduced resistance to autolysis in some isolates. D20 isolates harboured mutations in genes commonly associated with resistance to the respective antibiotics (e.g. walK for vancomycin, mprF and rpoB for daptomycin, rplC for linezolid), along with several previously unreported variants. Introduction of key mutations to these identified genes in the parental strain via allelic exchange confirmed their role in the development of resistance. ConclusionsIn vitro selection against vancomycin, daptomycin, or linezolid resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.

microbiology↗