Biology subjects

Stinear, T. P.

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

At least 37 records · Page 2Linked to original sources

bioRxiv2024

The complete genome sequence of the crayfish pathogen Candidatus Paracoxiella cheracis n.g. n.sp. provides insight into pathogenesis and the phylogeny of the Coxiellaceae family

The Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever, Coxiella burnetii, represents the best characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the previously referred to as Candidatus Coxiella cheraxi represents a novel genus within this family, herein denoted Candidatus Paracoxiella cheracis. Interestingly, we demonstrate that Candidatus P. cheracis encodes for a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologues of several important C. burnetii effectors including a homologue of CpeB that was demonstrated to be a Dot/Icm substrate in C. burnetii. IMPORTANCEUsing long-read sequencing technology we have uncovered the full genome sequence of Candidatus Paracoxiella cheracis, a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insight into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. Candidatus P. cheracis also represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. Candidatus P. cheracis possesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology manipulating effector proteins.

microbiology↗

bioRxiv2024

Quorum sensing and DNA methylation play active roles in clinical Burkholderia phase variation

Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions and is exemplified by variable colony morphotypes. Discrete genomic changes and modulation in gene expression occur in Burkholderia pseudomallei undergoing adaptation. Alternatively, adapted colony morphotype variants of species belonging to the Burkholderia cepacia complex (Bcc) lose a complete replicon (the pC3 virulence megaplasmid), which affects their production of virulence factors. We report that variants arising in Burkholderia ambifaria clinical isolates - with affected phenotypes - have retained their pC3, suggesting that another phase variation mechanism can take place in this Bcc species. Proteomic and phenotypic characterisation showed that morphotype variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) share similarities in phenotypes controlled by the Cep quorum sensing system. Thus, we determined the role of quorum sensing in B. ambifaria HSJ1 phase variation and confirmed that the main quorum sensing system Cep is important for the emergence of variants. As DNA methylation is one of the main epigenetic factors involved in bacterial phase variation that regulates some virulence factors of the Bcc species Burkholderia cenocepacia, we hypothesized that B. ambifaria HSJ1 phase variation could also be regulated by adenosine DNA methylation. By deleting the three putative adenosine DNA methyltransferases, we found that an orphan type II DNA methyltransferase prevents the emergence of phase variants. This is the first study to report quorum sensing and adenosine DNA methylation as two antagonistic systems independently controlling phase variation. ImportanceSome Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, such as those with cystic fibrosis, infection with Burkholderia cepacia complex (Bcc) bacteria can lead to "cepacia syndrome." In the Australian Aboriginal population, melioidosis caused by B. pseudomallei is prevalent, particularly among those with diabetes or alcoholism. Burkholderias phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group. We found that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying CMV mechanisms could lead to the development of targeted therapies against these highly antibiotic-resistant bacteria.

microbiology↗

bioRxiv2024

Exploring SNP Filtering Strategies: The Influence of Strict vs Soft Core

Phylogenetic analyses are crucial for understanding microbial evolution and infectious disease transmission. Bacterial phylogenies are often inferred from single nucleotide polymorphism (SNP) alignments, with SNPs as the fundamental signal within these data. SNP alignments can be reduced to a strict core by removing those sites which do not have data present in every sample. However, as sample size and genome diversity increase, a strict core can shrink markedly, discarding potentially informative data. Here, we propose and provide evidence to support the use of a soft core that tolerates some missing data, preserving more information for phylogenetic analysis. Using large datasets of Neisseria gonorrhoeae and Salmonella enterica serovar Typhi, we assess different core thresholds. Our results show that strict cores can drastically reduce informative sites compared to soft cores. In a 10,000-genome alignment of Salmonella enterica serovar Typhi, a 95% soft core yielded 10 times more informative sites than a 100% strict core. Similar patterns were observed in Neisseria gonorrhoeae. We further evaluated the accuracy of phylogenies built from strict and soft-core alignments using datasets with strong temporal signals. Soft-core alignments generally outperformed strict cores in producing trees displaying clock-like behaviour; for instance, the Neisseria gonorrhoeae 95% soft core phylogeny had a root-to-tip regression R2 of 0.50 compared to 0.21 for the strict-core phylogeny. This study suggests that soft-core strategies are preferable for large, diverse microbial datasets. To facilitate this, we developed Core-SNP-filter (github.com/rrwick/Core-SNP-filter), an open-source software tool for generating soft-core alignments from whole-genome alignments based on user-defined thresholds. IMPACT STATEMENTThis study addresses a major limitation in modern bacterial genomics - the significant data loss observed in large datasets for phylogenetic analyses, often due to strict-core SNP alignment approaches. As bacterial genome sequence datasets grow and diversity increases, a strict-core approach can greatly reduce the number of informative sites, compromising phylogenetic resolution. Our research highlights the advantages of soft-core alignment methods which tolerate some missing data and retain more genetic information. To streamline the processing of alignments, we developed Core-SNP-filter (github.com/rrwick/Core-SNP-filter), a publicly available resource-efficient tool that filters alignments to informative and core sites. DATA SUMMARYAll genomic sequence reads used in this study were already publicly available and accessions can be found in Supplementary Dataset 1. Supplementary methods and all code can be found in the accompanying GitHub repository: (github.com/mtaouk/Core-SNP-filter-methods).

genomics↗

bioRxiv2024

A low-cost and versatile paramagnetic bead DNA extraction method for Mycobacterium ulcerans environmental surveillance.

In Australia, native possums are an animal reservoir for Mycobacterium ulcerans, the causative agent of Buruli ulcer, a neglected tropical skin disease that can progress to extensive ulceration with deformity and disability. Surveillance of possum excreta for shedding of M. ulcerans can be used to inform geospatial modelling to predict locations at which humans are at increased risk of disease acquisition. This discovery provides opportunities to disrupt transmission pathways. However, the significant expense of commercial kits used for DNA extraction from environmental samples in large scale surveillance studies can hinder implementation of public health measures. To address this barrier, we developed a low-cost method for extraction of nucleic acids from possum excreta, possum tissue swabs, and mycobacterial cultures, using a guanidinium thiocyanate lysis solution and paramagnetic beads for DNA clean-up. In 96-well plate format for high-throughput processing, the SPRI-bead DNA extraction method for possum excreta was 3-fold less sensitive but only 1/6 the cost of a widely used commercial kit. While a SPRI-bead microtube-based extraction method for tissue swabs sampled from possums was 4-fold more sensitive and 1/5 the cost of the corresponding commercial kit. Furthermore, when used for preparing DNA from pure mycobacterial cultures, the SPRI-bead method produced genomic DNA with quality metrics comparable to more laborious techniques. The methods described here provide an economical means to continue large-scale M. ulcerans environmental surveillance that should facilitate efforts to halt the spread of Buruli ulcer in Victoria, Australia, with potential for applicability in other endemic countries. IMPORTANCEBuruli ulcer is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In this region of the world Buruli ulcer is a zoonosis, Australian native possums are a major wildlife reservoir of the causative agent, Mycobacterium ulcerans, and mosquitoes the vector to humans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.

microbiology↗

bioRxiv2024

Buruli ulcer surveillance in south-eastern Australian possums: infection status, lesion mapping and internal distribution of Mycobacterium ulcerans

Buruli ulcer (BU) is a neglected tropical disease of skin and subcutaneous tissues caused by Mycobacterium ulcerans. BU-endemic areas are highly focal, and M. ulcerans transmission dynamics vary in different settings. In the south-eastern Australian state of Victoria, BU is an endemic vector-borne zoonosis, with mosquitoes and native possums implicated in transmission, and humans as incidental hosts. Despite the importance of possums as wildlife reservoirs of M. ulcerans, knowledge of BU in these animals is limited. Opportunistic necropsy-based and active trap-and-release surveillance studies were conducted in and around Melbourne and Geelong, Victoria, to investigate BU in possums. Demographic data and biological samples were collected, and when present, cutaneous lesions suggestive of BU were mapped. Samples were tested for the presence of M. ulcerans DNA by IS2404 qPCR. The final dataset included 26 possums: 20 necropsied; 6 trapped and released. Most possums (77%) were common ringtails from inner Melbourne. Nine possums (eight ringtails, one brushtail) had skin lesions, ranging from single and mild, to multiple and severe, exposing bones and tendons in three cases. M. ulcerans was confirmed in 78% (7/9) of clinically affected possums and 65% of possums without lesions (11/17). Possums with moderate and severe disease had widespread systemic internal bacterial dissemination and were shedding M. ulcerans in their faeces. The anatomical distribution of cutaneous lesions and PCR positivity of biological samples suggests possums may be contracting BU from bites of M. ulcerans-harbouring mosquitoes, traumatic skin wounds, ingestion of an unknown environmental source, and/or during early development in the pouch. Ringtail possums appear highly susceptible to infection with M. ulcerans and are important reservoirs of the bacteria in Victoria. A One Health approach is needed to design and implement integrated interventions that reduce M. ulcerans transmission in Victoria, thereby protecting wildlife and humans from this emerging zoonotic disease. Author summaryBuruli ulcer (BU), a neglected tropical skin disease, is emerging as a public health concern in the temperate Australian state of Victoria. Here, BU is spread by mosquitoes, and native possums are wildlife reservoirs of the causative bacterium, Mycobacterium ulcerans. Possums can be infected by BU, but knowledge of infection and disease in these animals is limited. We conducted surveillance studies in the two largest cities of Victoria, examining live and deceased possums. We found skin lesions in a third of examined possums and confirmed presence of M. ulcerans in almost three-quarters of the animals. Mouth swabs were positive for the bacteria in nearly two thirds of possums, as were pouch swabs of almost half the females. We also conducted mapping of the bodily distribution of skin lesions and found that paws and the undersides of abdomens and tails were the most affected areas. Our findings add support to the concept that possums, particularly ringtails, are M. ulcerans reservoirs in Victoria, and suggest several possible routes of infection for free-living possums that warrant further research. Improved understanding of BU in possums may allow development of targeted interventions that reduce disease transmission and protect both animal and human health.

microbiology↗

bioRxiv2024

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↗

bioRxiv2024

Benchmarking reveals superiority of deep learning variant callers on bacterial nanopore sequence data

Variant calling is fundamental in bacterial genomics, underpinning the identification of disease transmission clusters, the construction of phylogenetic trees, and antimicrobial resistance prediction. This study presents a comprehensive benchmarking of SNP and indel variant calling accuracy across 14 diverse bacterial species using Oxford Nanopore Technologies (ONT) and Illumina sequencing. We generate gold standard reference genomes and project variations from closely-related strains onto them, creating biologically realistic distributions of SNPs and indels. Our results demonstrate that ONT variant calls from deep learning-based tools delivered higher SNP and indel accuracy than traditional methods and Illumina, with Clair3 providing the most accurate results overall. We investigate the causes of missed and false calls, highlighting the limitations inherent in short reads and discover that ONTs traditional limitations with homopolymer-induced indel errors are absent with high-accuracy basecalling models and deep learning-based variant calls. Furthermore, our findings on the impact of read depth on variant calling offer valuable insights for sequencing projects with limited resources, showing that 10x depth is sufficient to achieve variant calls that match or exceed Illumina. In conclusion, our research highlights the superior accuracy of deep learning tools in SNP and indel detection with ONT sequencing, challenging the primacy of short-read sequencing. The reduction of systematic errors and the ability to attain high accuracy at lower read depths enhance the viability of ONT for widespread use in clinical and public health bacterial genomics.

bioinformatics↗

bioRxiv2024

How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is still required for perfection. However, the effect of short-read depth on polishing performance is not well understood. Here, we introduce Pypolca (with default and careful parameters) and Polypolish v0.6.0 (with a new careful parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default and Polypolish-careful commonly introduce false-positive errors at low depth; (2) most of the benefit of short-read polishing occurs by 25x depth; (3) Polypolish-careful never introduces false-positive errors at any depth; and (4) Pypolca-careful is the single most effective polisher. Overall, we recommend the following polishing strategies: Polypolish-careful alone when depth is very low (<5x), Polypolish-careful and Pypolca-careful when depth is low (5-25x), and Polypolish-default and Pypolca-careful when depth is sufficient (>25x). Data SummaryPypolca is open-source and freely available on Bioconda, PyPI, and GitHub (github.com/gbouras13/pypolca). Polypolish is open-source and freely available on Bioconda and GitHub (github.com/rrwick/Polypolish). All code and data required to reproduce analyses and figures are available at github.com/gbouras13/depth_vs_polishing_analysis. All FASTQ sequencing reads are available at BioProject PRJNA1042815. A detailed list of accessions can be found in Table S1.

bioinformatics↗

bioRxiv2024

Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

microbiology↗

bioRxiv2024

A marine sponge-associated mycobacterium closely related to Mycobacterium tuberculosis

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

microbiology↗

bioRxiv2023

Season of transmission of Ross River/Barmah Forest Virus and Mycobacterium ulcerans closely align in southeastern Australia, supporting mosquitoes as the vector of Buruli ulcer

Ross River Virus and Barmah Forest Virus infections (alphaviruses) have short incubation periods and are transmitted to humans by mosquitoes. Mycobacterium ulcerans infection (Buruli ulcer) has a much longer incubation period and its mode of transmission is contested. We studied the relationship between month of notification of alphavirus infections and Buruli ulcer in the temperate Australian state of Victoria over the six-year period, 2017-2022. Using cross-correlation, a signal processing technique, we found that a five-month temporal shift in month of Buruli ulcer notification provided optimal alignment with month of alphavirus notification. This closely matches the previously determined 5-month Buruli ulcer incubation period. Inferred transmission of both conditions showed coordinated maxima in summer and autumn and coordinated minima in winter and spring. The close alignment in season of transmission of alphavirus infection and Buruli ulcer in Victoria supports mosquitoes as the primary local vector of M. ulcerans.

microbiology↗

bioRxiv2023

High performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Hepatitis A virus (HAV) infections are an increasing public health concern in low-endemicity regions due to outbreaks from foodborne infections and sustained transmission among vulnerable groups, including persons experiencing homelessness, those who inject drugs, and men who have sex with men (MSM), which is further compounded by aging, unvaccinated populations. DNA sequence characterisation of HAV for source tracking is performed by comparing small subgenomic regions of the virus. While this approach has been successful when robust epidemiological data are available, poor genetic resolution can lead to conflation of outbreaks with sporadic cases. HAV outbreak investigations would greatly benefit from the additional phylogenetic resolution obtained by whole virus genome sequence comparisons. However, HAV genomic approaches can be difficult because of challenges in isolating the virus, low sensitivity of direct metagenomic sequencing in complex sample matrices like various foods such as fruits, vegetables and molluscs, and difficulty designing highly multiplexed PCR primers across diverse HAV genotypes. Here, we introduce a proof-of-concept pan-HAV oligonucleotide hybrid capture enrichment assay from serum and frozen berry specimens that yields complete and near-complete HAV genomes from as few as four input HAV genome copies. We used this method to recover HAV genomes from human serum specimens with high C{tau} values (34{middle dot}7--42{middle dot}7), with high assay performance for all six human HAV sub-genotypes, both contemporary and historical. Our approach provides a highly sensitive and streamlined workflow for HAV WGS from diverse sample types, that can be the basis for harmonised and high-resolution molecular epidemiology during HAV outbreak surveillance. ImportanceThis proof-of-concept study introduces a hybrid capture oligo panel for whole genome sequencing (WGS) of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making it suitable for broad implementation in clinical and public health laboratories. The ability to capture small amounts of virus from complex samples is promising for passive surveillance application to environmental substrates, such as wastewater. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations, as well as our understanding of the global diversity and transmission dynamics of HAV.

genomics↗

bioRxiv2023

Tolerance to Haemophilus influenzae infection in human epithelial cells: insights from a primary cell-based model

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, unproductive inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extra- and intracellular burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intracellular bacterial populations, and appears to involve interruption of NFkB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been u. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection resulting in release of pro-inflammatory cytokines (IL8, TNF). Our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

microbiology↗

bioRxiv2023

The 3'UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions

Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3 UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3 UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3 UTR was found to repress dapE, a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3 UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3 UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3 UTR. Our findings reveal an extended regulatory network for vigR, uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate.

microbiology↗

bioRxiv2023

A transmission chain linking Mycobacterium ulcerans with Aedes notoscriptus mosquitoes, possums and human Buruli ulcer cases in southeastern Australia

In temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused by Mycobacterium ulcerans. Native possums are the major local environmental reservoir of M. ulcerans as they not only develop Buruli lesions but they also shed M. ulcerans in their excreta. However the way humans acquire M. ulcerans from possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes in M. ulcerans transmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and four ad hoc field surveys across an area of 350km2 on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns of M. ulcerans-positive mosquito occurrence with M. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstruct M. ulcerans genomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period. Culex molestus and Aedes notoscriptus were the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association between M. ulcerans and Ae. notoscriptus (p<0.0001) with a maximum likelihood estimate (MLE) of 5.88 M. ulcerans positive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fed Ae. notoscriptus showed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed shared M. ulcerans genome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate that Ae. notoscriptus likely transmit M. ulcerans in southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

microbiology↗

bioRxiv2023

High performance Legionella pneumophila source attribution using genomics-based machine learning classification

Fundamental to effective Legionnaires disease outbreak control is the ability to rapidly identify the environmental source(s) of the causative agent, Legionella pneumophila. Genomics has revolutionised pathogen surveillance but L. pneumophila has a complex ecology and population structure that can limit source inference based on standard core genome phylogenetics. Here we present a powerful machine learning approach that assigns the geographical source of Legionnaires disease outbreaks more accurately than current core genome comparisons. Models were developed upon 534 L. pneumophila genome sequences, including 149 genomes linked to 20 previously reported Legionnaires disease outbreaks through detailed case investigations. Our classification models were developed in a cross-validation framework using only environmental L. pneumophila genomes. Assignments of clinical isolate geographic origins demonstrated high predictive sensitivity and specificity of the models, with no false positives or false negatives for 13 out of 20 outbreak groups, despite the presence of within-outbreak polyclonal population structure. Analysis of the same 534-genome panel with a conventional phylogenomic tree and a core genome multi-locus sequence type allelic distance-based classification approach revealed that our machine learning method had the highest overall classification performance - agreement with epidemiological information. Our multivariate statistical learning approach maximises use of genomic variation data and is thus well-suited for supporting Legionnaires disease outbreak investigations.

bioinformatics↗

bioRxiv2023

Novel determinant of antibiotic resistance: a clinically selected Staphylococcus aureus clpP mutant survives daptomycin treatment by reducing binding of the antibiotic and adapting a rod-shaped morphology

Daptomycin is a last-resort antibiotic used for treatment of infections caused by Gram-positive antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations, however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and {beta}-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that the improved survival of the clpP mutant strain during daptomycin treatment was associated with reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10 x MIC daptomycin resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression. IMPORTANCEThe bacterium Staphylococcus aureus is a leading cause of life-threatening infections and treatment is challenged by the worldwide dissemination of methicillin-resistant Staphylococcus aureus (MRSA) that are multi-drug resistant. Daptomycin, a cell membrane-targeting cationic lipopeptide, is one of the few antibiotics with activity against MRSA, however, the killing mechanism of daptomycin and the mechanisms leading to resistance are not fully understood. Here we show than an MRSA strain, isolated from the blood of a patient treated with daptomycin, has acquired a mutation that inactivates the ClpXP protease resulting in increased resistance to several antibiotics and diminished expression of virulence genes. Super resolution microscopy showed that the mutant avoids daptomycin-elicited killing by preventing the binding of the antibiotic to the septal site and by growing into a rod-shaped morphology. In summary, this study discloses new perspectives on the mechanism of killing and the mechanism of resistance to an antibiotic of last resort.

microbiology↗

bioRxiv2023

Uncovering strain- and age- dependent differences in innate immune response to SARS-CoV-2 infection in nasal epithelia using combined short and long-read scRNA-seq

Assessing the impact of SARS-CoV-2 variants on the host is crucial with continuous emergence of new variants. We employed single-cell sequencing to investigate host transcriptomic response to ancestral and Alpha-strain SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from adults and adolescents. Strong innate immune responses were observed across lowly-infected and bystander cell-types, and heightened in Alpha-infection. Contrastingly, the innate immune response of highly-infected cells was like mock-control cells. Alpha highly-infected cells showed increased expression of protein refolding genes compared with ancestral-strain-infected adolescent cells. Oxidative phosphorylation- and translation-related genes were down-regulated in bystander cells versus infected and mock-control cells, suggesting that the down-regulation is protective and up-regulation supports viral activity. Infected adult cells revealed up-regulation of these pathways compared with infected adolescents, implying enhanced pro-viral states in infected adults. Overall, this highlights the complexity of cell-type-, age- and viral-strain-dependent host epithelial responses to SARS-CoV-2 and the value of air-liquid-interface cultures.

immunology↗