Search bioRxiv⌕ Search

Biology subjects

Howden, B. P.

Publications and source records attributed to Howden, B. P..

At least 19 recordsLinked to original sources

A synthetic MALDI-TOF MS atlas for microbial classification across hospitals

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (MS) is central to routine clinical microbiology for species identification. Its combination with machine learning is constrained by fragmented public datasets, species imbalance and acquisition-related variability. Synthetic datasets could provide more training data, but they must preserve the spectral information needed to make predictions on real samples. Here, we present an open, synthetic MALDI-TOF MS atlas comprising 4.3 million spectra across 43 clinically relevant bacterial species. To construct the atlas, we harmonised public repositories from three countries and trained a species- and institution-conditioned concept-bottleneck generative variational autoencoder (CBGM-VAE) on 179,086 measured spectra. We assessed the atlas as both a stand-alone training resource and a complement to real data, using bacterial species classification as a functional benchmark. Evaluation comprised 7,269 real spectra from more than 7 countries, 24 institutions and multiple acquisition platforms, covering: temporal, technical, biological and combined shifts. A neural network trained only on synthetic spectra achieved F1 scores ranging from 0.788 to 1.000 across external cohorts. When synthetic spectra were combined with real training data, classification performance improved or matched using real data only. These findings demonstrate that the atlas retains transferable information and can complement measured data. The openly released atlas provides a reusable resource for supervised learning and benchmarking. It can also be explored for pretraining representation-learning and foundation models in MALDI-TOF MS research.

microbiology↗

Loss of GdpP function in Staphylococcus aureus confers β-lactam-specific antibiotic tolerance and promotes invasive infection

The emergence of antibiotic tolerance in Staphylococcus aureus reduces antibiotic efficacy by allowing bacterial survival despite prolonged antibiotic exposure, the molecular basis of which remains poorly understood. Moreover, the phenotypic indistinguishability of tolerant isolates in antimicrobial susceptibility testing impedes effective diagnosis and therapy. Increased concentration of the second-messenger, cyclic-di-AMP (CDA), has recently been implicated in tolerance to {beta}-lactams as well as other cell-wall-reactive antibiotics. Using the ScanLag assay, Tolerance-Disk test, and traditional methodologies and employing isogenic mutagenized strains, we demonstrate that loss of GdpP function, a phosphodiesterase that hydrolyzes CDA, confers tolerance specifically to {beta}-lactam antibiotics independent of their class. The extent of {beta}-lactam tolerance correlated directly with the intracellular CDA concentration and inversely with the inhibition of bacterial cell-wall synthesis. {Delta}gdpP mutants caused higher mortality than wild-type strains in the Galleria mellonella infection model upon {beta}-lactam treatment, suggesting GdpP-mediated tolerance could lead to {beta}-lactam treatment failure. Large-scale within-host evolution analysis demonstrated that MRSA and MSSA strains isolated from patients acquire GdpP loss-of-function mutations during invasive infections but not during nasal carriage. Overall, this study highlights the clinical relevance of gdpP mutations, frequently selected in persistent S. aureus infections, as key mediators that could promote treatment failure due to {beta}-lactam tolerance.

microbiology↗

Antifungal resistance mechanisms and nosocomial transmission of Nakaseomyces glabratus: genomic investigation and observational study in Melbourne, Australia

Nakaseomyces glabratus (Candida glabrata) is a WHO high-priority fungal pathogen associated with fungal antimicrobial resistance (fAMR). Given nosocomial transmission occurs sporadically, resistant strains could be transmitted, a concern for critically ill patients. We conducted a genomic investigation and retrospective observational study of N. glabratus to identify any nosocomial transmission of fAMR and understand resistance mechanisms and clinical and demiological factors among patients at a quaternary hospital in Melbourne, Australia. We selected stored N. glabratus with and without fAMR associated with similar patient clinical characteristics and performed whole genome sequencing. Clinical and epidemiological data were extracted from medical records. Phylogenetic, mutational, copy-number variation (CNV) and mitochondrial genomic analyses were performed, with a focus on the fAMR gene PDR1. Of 54 isolates collected over seven years, 20 (37%) were fluconazole-resistant and four (7%) had elevated flucytosine minimum inhibitory concentrations (MICs) (range 2-32 g/ml). There were no significant clinical differences between patients with and without fluconazole resistance. Most (55%) fluconazole-resistant isolates carried PDR1 mutations. Resistance was distributed throughout the phylogeny suggesting predominantly independent acquisition. However, a cluster of four resistant isolates with the same PDR1 mutation suggested nosocomial transmission. One probable ERG11 gene duplication, and two petite variants with apparent mitochondrial genomic deletions, were seen in association with fluconazole resistance. In this study, we identified a small probable nosocomial fAMR transmission cluster, and novel variants in PDR1, ERG11 and FCY2 associated with fAMR phenotypes. Future study should confirm functional impacts and systematically investigate for nosocomial transmission of resistance, including colonisation states.

microbiology↗

Flex-It: A global standardised genotyping framework for Shigella flexneri

Shigella flexneri is the leading causative agent of shigellosis globally. The public health threat posed by S. flexneri is compounded by its emergence as a sexually transmissible infection, importance of international travel in driving dissemination, and the increasing prevalence of antimicrobial resistance (AMR). A rapid and robust computational method is needed to enhance genomic surveillance and systematically explore features of the population structure of this WHO priority pathogen, which is scalable and readily implementable across jurisdictions, particularly as vaccine development efforts are underway. Here, we present Flex-It, a genomic framework and genotyping scheme implemented in Mykrobe for S. flexneri serotypes 1-5, X & Y, compatible with previous approaches used to describe S. flexneris population structure. To develop Flex-It, we curated a retrospective dataset of 5,819 publicly available S. flexneri genomes. We characterised the global population structure for S. flexneri, exploring geographical and temporal traits, and showed the granular diversity of AMR and serotype profiles. We applied Flex-It to >13,000 genomes routinely generated by public health laboratories from Australia, the UK and the USA across a ten-year period. We found significant genotype diversity in all three locations, with the emergence of genotypes with converged resistance to all major drugs currently used for treatment. Flex-It provides an open-source, novel genotyping method that rapidly characterises S. flexneri and its ciprofloxacin resistance determinants in <1 minute from both short and long whole-genome sequencing reads. Flex-It provides the community with a standardised nomenclature to monitor the emergence and spread of S. flexneri lineages.

microbiology↗

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↗

Comprehensive public health surveillance identifies novel human cryptosporidiosis subtypes and genetic diversity associated with swimming pool outbreaks in Australia, Victoria (2018-2024)

Cryptosporidium and Giardia are major causes of gastrointestinal illness globally. In Australia, cryptosporidiosis is a nationally notifiable disease, yet molecular characterisation of clinical cases is rarely performed, limiting the capacity to identify outbreaks, trace sources and assess zoonotic risk. During 2024 there was a 273% cases increase in Australia, the third country with the highest increase. We present the first comprehensive molecular investigation of human Cryptosporidium and Giardia infections in the state of Victoria, Australia. We analysed faecal samples collected between 2018 and 2024. Positive samples were subtyped and parasite load was estimated. Of the 2,330 samples tested, 225 were positive for Cryptosporidium and nine for Giardia. Seven Cryptosporidium species and 24 subtypes were identified, including multiple novel or regionally unique subtypes. C. hominis was the predominant species (85%), and three subtypes associated with 11 recreational water outbreaks in 2024. Based on spatiotemporal overlap and subtypes, 52 cases were inferred to represent undetected outbreak-associated infections. Several C. parvum subtypes reflected probable zoonotic transmission, two subtypes were associated with a childcare and camp outbreak. Six C. hominis subtypes and eight subtypes overall were reported for the first time in Australia. Globally novel subtypes of C. occultus, C. fayeri and C. meleagridis in human hosts is reported. The study reveals high diversity of Cryptosporidium subtypes in human infections in Victoria. This study demonstrates the potential for molecular surveillance to inform public health interventions when integrated with epidemiological data. These findings support One Health approaches to outbreak detection and source attribution. IMPORTANCECryptosporidium is a nationally notifiable pathogen in Australia, yet routine genotyping is not performed, limiting outbreak detection and source attribution. This study represents the first large-scale molecular surveillance of human Cryptosporidium and Giardia infections in southern Australia. By integrating species- and subtype-level typing with epidemiological metadata, we demonstrate the substantial diversity of infective lineages, including several novel or zoonotic genotypes. Our findings highlight the critical role of molecular tools in tracking transmission pathways, supporting outbreak investigation and informing public health responses. These data provide a foundation for incorporating routine genotyping into national surveillance strategies for parasitic enteropathogens. This study presents the first epidemiological study of the 2024 outbreaks in Australia. The global comparison of cases during this period also highlights potential large-scale disease dynamic of public health importance.

molecular biology↗

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↗

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↗

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 [&le;]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↗

Population structure and antimicrobial resistance of Corynebacterium diphtheriae in Victoria, Australia

Corynebacterium diphtheriae, the main aetiological agent of diphtheria, is a re-emerging bacterial pathogen of public health concern, yet remains largely understudied globally. In this study, we analysed the population structure and antimicrobial resistance (AMR) of 210 C. diphtheriae isolates from Victoria, Australia including 103 historical (1950-1970) and 107 contemporary clinical isolates (2004-2023), using whole-genome sequencing and phenotypic susceptibility testing. The diphtheria toxin gene (tox) was detected in 89 isolates, the majority of which (n=83; 93.3%) were historical. Population structure comprised two primary phylogenetic lineages, Mitis and Gravis, each containing multiple sublineages. Multi-locus sequence type (MLST) analysis revealed a highly diverse population structure with multiple novel MLST profiles and alleles. When placed within a global phylogenetic framework, Australian isolates were broadly distributed, reflecting substantial genetic diversity. Phenotypic susceptibility testing against eleven antimicrobials revealed that several contemporary isolates were resistant to multiple agents, including penicillin and erythromycin, first-line treatments of Corynebacterium infections. Eight contemporary isolates were multidrug-resistant (resistant to [&ge;]3 antimicrobial classes), including five with resistance to both penicillin and erythromycin. Genomic analysis identified multiple genes and mutations conferring resistance among contemporary isolates. In contrast, no antimicrobial resistance phenotypes or genotypes were observed in historical isolates. Analysis of historical genomes provides valuable insights into a period of heightened diphtheria activity in Victoria prior to widespread immunisation. Overall, these findings establish a baseline for ongoing genomic surveillance in the face of increasing global outbreaks, support informed empiric treatment strategies, and contribute to the knowledge of global population structure of C. diphtheriae. Impact statementCorynebacterium diphtheriae is a re-emerging pathogen of public health concern with limited genomic data available to support surveillance and public health interventions. This study provides a contemporary understanding of C. diphtheriae from Australia in the era of genomic surveillance and helps us better understand baseline genomic diversity and antimicrobial resistance. Additionally, these findings enhance preparedness for potential future incursions or outbreaks, including those involving drug-resistant strains, as recently observed in parts of Africa and Europe. Data summaryGenome sequences are deposited in GenBank under BioProject PRJNA870170. Sample data and accession numbers are included in the Supplementary Table S1. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

Uncovering bacterial pseudaminylation with pan-specific antibody tools

Pseudaminic acids (Pse) are a family of carbohydrates found within bacterial lipopolysaccharides, capsular polysaccharides and glycoproteins that are critical for the virulence of human pathogens. However, a dearth of effective tools for detecting and enriching Pse has restricted study to only the most abundant Pse-containing glycoconjugates. Here, we devise a synthesis of - and {beta}-O-pseudaminylated glycopeptides to generate pan-specific monoclonal antibodies (mAbs) that recognise - and {beta}-configured Pse and its C8 epimer (8ePse) presented within glycans or directly linked to polypeptide backbones. Structural characterisation reveals the molecular basis of Pse recognition across a range of diverse chemical contexts. Using these mAbs, we establish a glycoproteomic platform to provide unprecedented depth in mapping the Pse glycome of Helicobacter pylori, Campylobacter jejuni, and Acinetobacter baumannii strains. Finally, we demonstrate that the mAbs recognise diverse capsule types in multidrug-resistant Acinetobacter baumannii and enhance phagocytosis to eliminate infections in mice.

biochemistry↗

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↗

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↗

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↗

Dynamics of antimicrobial resistance and virulence of Salmonella enterica serovar Dublin

Salmonella Dublin is a host-adapted, invasive non-typhoidal Salmonella (iNTS) serovar that causes bloodstream infections in humans and demonstrates increasing prevalence of antimicrobial resistance (AMR). Using a global dataset of 1,303 genomes, coupled with in vitro assays, we examined the evolutionary, resistance, and virulence characteristics of S. Dublin. Our analysis revealed strong geographic associations between AMR profiles and plasmid types, with highly resistant isolates confined predominantly to North America, linked to IncC plasmids co-encoding AMR and heavy metal resistance. By contrast, Australian isolates were largely antimicrobial-susceptible, reflecting differing AMR pressures. We identified two phylogenetically distinct Australian lineages, ST10 and ST74, with a small number of ST10 isolates harbouring a novel hybrid plasmid encoding both AMR and mercuric resistance. Whereas the ST10 lineage remains globally dominant, the ST74 lineage was less prevalent. ST74 exhibited unique genomic features including a larger pan genome compared to ST10 and the absence of key virulence loci including SPI-19 which encodes a type VI secretion system (T6SS). Despite these genomic differences, the ST74 lineage displayed enhanced intracellular replication in human macrophages and induced less pro-inflammatory responses compared with ST10, suggesting alternative virulence strategies that may support systemic dissemination of ST74. The Vi antigen was absent in all ST10 and ST74 genomes, highlighting challenges for serotyping and vaccine development, and has implications for current diagnostic and control strategies for S. Dublin infections. Collectively, this study represents the most comprehensive investigation of S. Dublin to date and importantly, has revealed distinct adaptations of two genotypes within the same serovar, leading to different epidemiological success. The regional emergence and evolution of distinct S. Dublin lineages highlights the need to understand the divergence of intra-serovar virulence mechanisms which may impact the development of effective control measures against this important global pathogen.

microbiology↗

Genomic epidemiology and phenotypic characterisation of Salmonella entericaserovar Panama in Victoria, Australia

Salmonella enterica serovar Panama, a causative agent of non-typhoidal salmonellosis (NTS), is one of several serovars that causes invasive NTS disease (iNTS) in humans. S. Panama is an understudied pathogen, with its pathobiology poorly understood. It is a predominant iNTS serovar in Australia, a high-income country with high rates of salmonellosis, where S. Panama has been documented to have a high odds ratio for causing iNTS. This study investigates the genomic epidemiology and antimicrobial resistance profiles of all S. Panama isolates recovered in Victoria, Australia, between 2000 and 2020. We examined the infection dynamics of S. Panama in seven isolates, representing the genetic diversity of the study population. Two sub-lineages, encompassed within a previously described Asian lineage, were identified. Multi-drug resistance (resistance to [&ge;]3 drug classes) was detected in 46 (51.7%) Australian isolates. The plasmid-mediated colistin resistance gene, mcr1.1, was detected in one Australian S. Panama isolate, carried by an IncI plasmid previously reported in Salmonella and Escherichia coli isolates collected from poultry in South-East Asia. Examination of the intracellular replication dynamics of S. Panama isolates demonstrated diverse phenotypes. In THP-1 derived macrophages, despite low host cell uptake, S. Panama showed higher replication rates over time compared to S. enterica serovar Typhimurium. However, a causative genotype could not be identified to explain this observed phenotype. This study provides insights into the S. Panama isolates imported into Australia over two-decades, showing MDR was common in this iNTS serovar, and colistin resistance reported for the first time. It provides the first data on the host-pathogen interactions of S. Panama in Australia, which will aid our collective understanding of the pathobiology of S. Panama and iNTS serovars more broadly. Author SummaryIn Australia, non-typhoidal Salmonella (NTS) cases have been on the rise since the 1970s; characterised by self-limiting enteritis, some NTS infections can result in systemic infections called invasive NTS disease. Salmonella enterica serovar Panama is a leading iNTS serovar in Australia. This study characterised the genomic epidemiology of S. Panama, identifying two lineages circulating in Australia over two decades and placing them within a global context. It also investigated the antimicrobial resistance (AMR) mechanisms of S. Panama, with multi-drug resistance commonly observed. Further, it identified the first plasmid-mediated colistin-resistant S. Panama in Australia. We additionally examined the characteristics of S. Panama-mediated host-pathogen interactions in both epithelial and macrophage cells lines, providing the first insight into the infection dynamics of this understudied pathogen. Thus, this study combines genomics and in vitro infection experiments to understand the pathogenic behaviour of the neglected iNTS S. Panama.

microbiology↗

High-throughput method rapidly characterizes hundreds of novel antibiotic resistance mutations

A fundamental obstacle to tackling the antimicrobial resistance crsisis is identifying mutations that lead to resistance in a given genomic background and environment. We present a high-throughput technique - Quantitative Mutational Scan Sequencing (QMS-Seq) - that enables quantitative comparison of which genes are under antibiotic selection and captures how genetic background influences resistance evolution. We compared four E. coli strains exposed to ciprofloxacin, cycloserine, or nitrofurantoin and identified 975 resistance mutations, many in genes and regulatory regions not previously associated with resistance. QMS-Seq revealed that multi-drug and antibiotic-specific resistance are acquired through categorically different types of mutations, and that minor genotypic differences significantly influence evolutionary routes to resistance. By quantifying mutation frequency with single base pair resolution, QMS-Seq informs about the underlying mechanisms of resistance and identifies mutational hotspots within genes. Our method provides a way to rapidly screen for resistance mutations while assessing the impact of multiple confounding factors.

microbiology↗

Bridging genomic gaps: A versatile SARS-CoV-2 benchmark dataset for adaptive laboratory workflows

Genomic sequencings adoption in public health laboratories (PHLs) for pathogen surveillance is innovative yet challenging, particularly in the realm of bioinformatics. Low- and middle-income countries (LMICs) face increased difficulties due to supply chain volatility, workforce training, and unreliable infrastructure such as electricity and internet services. These challenges also extend to high-income countries (HICs) where bioinformatics is nascent in PHLs and hampered by a lack of specialized skills and computational infrastructure. This underlines the urgency for flexible and resource-aware strategies in genomic sequencing to improve global pathogen surveillance. In response to these challenges, the present research was conducted to identify and analyse key variables influencing the quality and accuracy of amplicon sequence data. An extensive benchmark dataset was developed that encompassed a diverse collection of isolates, viral loads, primer schemes, library preparation methods, sequencing technologies, and basecalling models, totalling 750 sequences. This dataset was analysed with bioinformatic workflows selected for varying levels of technical capacity. The evaluation focused on quality metrics, consensus accuracy, and common genomic epidemiological indicators. The analysis uncovers complex interactions between multiple parameters in laboratory and bioinformatic processes. emphasising resource-constrained PHLs, practical guidelines are proposed. Insights from the benchmark dataset aim to guide the establishment of specific laboratory and bioinformatics protocols for amplicon sequencing in these settings. The findings can also be used to guide the creation of specialised training curricula, further advancing genomic equity. The benchmark dataset itself allows laboratories to customise and evaluate workflows, catering to their distinct requirements and capacities. Such a holistic approach is imperative to build the capacity to monitor pathogens worldwide. Author summaryThis study marks a step toward equity in the field of pathogen genomics, especially for resource-constrained PHLs. It develops and evaluates a comprehensive amplicon sequencing benchmark dataset, offering vital insights for PHLs engaged in genomic surveillance. In particular, the study finds that the choice of basecaller model has a minimal impact on the quality and accuracy of consensus sequences derived from ONT data, which is crucial for labs with limited computational resources. It also highlights the effectiveness of longer amplicons in ensuring consistent coverage and reducing amplicon dropouts at higher viral loads. While Illumina remains a gold standard for data quality, the combination of the Midnight primer scheme with ONTs Rapid library preparation is shown to be a viable alternative, reducing costs, procedural complexity, and hands-on time. The study synthesises these findings into practical guidelines to aid in the development of amplicon sequencing workflows for SARS-CoV-2 with implications for other pathogens.

genomics↗