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Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND (Genome-derived Ribosomal Operon Database), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.

This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing 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 them suitable for broad implementation in clinical and public health laboratories. 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.

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is usually 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 read depth; (2) most of the benefit of short-read polishing occurs by 25× depth; (3) Polypolish-careful almost 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 (<5×), Polypolish-careful and Pypolca-careful when depth is low (5-25×), and Polypolish-default and Pypolca-careful when depth is sufficient (>25×).

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

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 revolutionized 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 maximizes the use of genomic variation data and is thus well-suited for supporting Legionnaires' disease outbreak investigations.IMPORTANCEIdentifying the sources of Legionnaires' disease outbreaks is crucial for effective control. Current genomic methods, while useful, often fall short due to the complex ecology and population structure of Legionella pneumophila, the causative agent. Our study introduces a high-performing machine learning approach for more accurate geographical source attribution of Legionnair

UNLABELLED: A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans. IMPORTANCE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.

Streptococcus pneumoniae is a major human pathogen with a high burden of disease. Non-invasive isolates (those found in non-sterile sites) are thought to be a key source of invasive isolates (those found in sterile sites) and a reservoir of anti-microbial resistance (AMR) determinants. Despite this, pneumococcal surveillance has almost exclusively focused on invasive isolates. We aimed to compare contemporaneous invasive and non-invasive isolate populations to understand how they interact and identify differences in AMR gene distribution. We used a combination of whole-genome sequencing and phenotypic anti-microbial susceptibility testing and a data set of invasive (n = 1,288) and non-invasive (n = 186) pneumococcal isolates, collected in Victoria, Australia, between 2018 and 2022. The non-invasive population had increased levels of antibiotic resistance to multiple classes of antibiotics including beta-lactam antibiotics penicillin and ceftriaxone. We identified genomic intersections between the invasive and non-invasive populations and no distinct phylogenetic clustering of the two populations. However, this analysis revealed sub-populations overrepresented in each population. The sub-populations that had high levels of AMR were overrepresented in the non-invasive population. We determined that WamR-Pneumo was the most accurate in silico tool for predicting resistance to the antibiotics tested. This tool was then used to assess the allelic diversity of the penicillin-binding protein genes, which acquire mutations leading to beta-lactam antibiotic resistance, and found that they were highly conserved (≥80% shared) between the two populations. These findings show the potential of non-invasive isolates to serve as reservoirs of AMR determinants.

The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.

Outcomes of severe bacterial infections are determined by the interplay between host, pathogen, and treatments. While human genomics has provided insights into host factors impacting Staphylococcus aureus infections, comparatively little is known about S. aureus genotypes and disease severity. Building on the hypothesis that bacterial pathoadaptation is a key outcome driver, we developed a genome-wide association study (GWAS) framework to identify adaptive mutations associated with treatment failure and mortality in S. aureus bacteremia (1,358 episodes). Our research highlights the potential of vancomycin-selected mutations and vancomycin minimum inhibitory concentration (MIC) as key explanatory variables to predict infection severity. The contribution of bacterial variation was much lower for clinical outcomes (heritability <5%); however, GWASs allowed us to identify additional, MIC-independent candidate pathogenesis loci. Using supervised machine learning, we were able to quantify the predictive potential of these adaptive signatures. Our statistical genomics framework provides a powerful means to capture adaptive mutations impacting severe bacterial infections.

Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR, an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI's AMRFinderPlus, as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.

Streptococcus pneumoniae is a major human pathogen and can cause a range of conditions from asymptomatic colonization to invasive pneumococcal disease (IPD). The epidemiology and distribution of IPD-causing serotypes in Australia has undergone large changes following the introduction of the 7-valent pneumococcal conjugate vaccine (PCV) in 2005 and the 13-valent PCV in 2011. In this study, to provide a contemporary understanding of the IPD causing population in Victoria, Australia, we aimed to examine the population structure and prevalence of antimicrobial resistance using whole-genome sequencing and comprehensive antimicrobial susceptibility data of 1288 isolates collected between 2018 and 2022. We observed high diversity among the isolates with 52 serotypes, 203 sequence types (STs) and 70 Global Pneumococcal Sequencing Project Clusters (GPSCs) identified. Serotypes contained in the 13v-PCV represented 35.3 % (n=405) of isolates. Antimicrobial resistance (AMR) to at least one antibiotic was identified in 23.8 % (n=358) of isolates with penicillin resistance the most prevalent (20.3 %, n=261 using meningitis breakpoints and 5.1 % n=65 using oral breakpoints). Of the AMR isolates, 28 % (n=101) were multidrug resistant (MDR) (resistant to three or more drug classes). Vaccination status of cases was determined for a subset of isolates with 34 cases classified as vaccine failure events (fully vaccinated IPD cases of vaccine serotype). However, no phylogenetic association with failure events was observed. Within the highly diverse IPD population, we identified six high-risk sub-populations of public health concern characterized by high prevalence, high rates of AMR and MDR, or serotype inclusion in vaccines. High-risk serotypes included serotypes 3, 19F, 19A, 14, 11A, 15A and serofamily 23. In addition, we present our data validating seroBA for in silico serotyping to facilitate ISO-accreditation of this test in routine use in a public health reference laboratory and ha

Invasive Staphylococcus aureus infections are common, causing high mortality, compounded by the propensity of the bacterium to develop drug resistance. S. aureus is an excellent case study of the potential for a bacterium to be commensal, colonizing, latent or disease-causing; these states defined by the interplay between S. aureus and host. This interplay is multidimensional and evolving, exemplified by the spread of S. aureus between humans and other animal reservoirs and the lack of success in vaccine development. In this Review, we examine recent advances in understanding the S. aureus-host interactions that lead to infections. We revisit the primary role of neutrophils in controlling infection, summarizing the discovery of new immune evasion molecules and the discovery of new functions ascribed to well-known virulence factors. We explore the intriguing intersection of bacterial and host metabolism, where crosstalk in both directions can influence immune responses and infection outcomes. This Review also assesses the surprising genomic plasticity of S. aureus, its dualism as a multi-mammalian species commensal and opportunistic pathogen and our developing understanding of the roles of other bacteria in shaping S. aureus colonization.

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

Staphylococcus aureus is an opportunistic bacterial pathogen that often results in difficult-to-treat infections. One mechanism used by S. aureus to enhance survival during infection is the stringent response. This is a stress survival pathway that utilizes the nucleotides (p)ppGpp to reallocate bacterial resources, shutting down growth until conditions improve. Small colony variants (SCVs) of S. aureus are frequently associated with chronic infections, and this phenotype has previously been linked to a hyperactive stringent response. Here, we examine the role of (p)ppGpp in the long-term survival of S. aureus under nutrient-restricted conditions. When starved, a (p)ppGpp-null S. aureus mutant strain ((p)ppGpp0) initially had decreased viability. However, after 3 days we observed the presence and dominance of a population of small colonies. Similar to SCVs, these small colony isolates (p0-SCIs) had reduced growth but remained hemolytic and sensitive to gentamicin, phenotypes that have been tied to SCVs previously. Genomic analysis of the p0-SCIs revealed mutations arising within gmk, encoding an enzyme in the GTP synthesis pathway. We show that a (p)ppGpp0 strain has elevated levels of GTP, and that the mutations in the p0-SCIs all lower Gmk enzyme activity and consequently cellular GTP levels. We further show that in the absence of (p)ppGpp, cell viability can be rescued using the GuaA inhibitor decoyinine, which artificially lowers the intracellular GTP concentration. Our study highlights the role of (p)ppGpp in GTP homeostasis and underscores the importance of nucleotide signaling for long-term survival of S. aureus in nutrient-limiting conditions, such as those encountered during infections. IMPORTANCE Staphylococcus aureus is a human pathogen that upon invasion of a host encounters stresses, such as nutritional restriction. The bacteria respond by switching on a signaling cascade controlled by the nucleotides (p)ppGpp. These nucleotides function to shut down ba

Nocardia are opportunistic human pathogens that can cause a range of debilitating and difficult to treat infections of the lungs, brain, skin, and soft tissues. Despite their close relationship to the well-known secondary metabolite-producing genus, Streptomyces, comparatively few natural products are known from the Nocardia, and even less is known about their involvement in the pathogenesis. Here, we combine chemistry, genomics, and molecular microbiology to reveal the production of terpenomycin, a new cytotoxic and antifungal polyene from a human pathogenic Nocardia terpenica isolate. We unveil the polyketide synthase (PKS) responsible for terpenomycin biosynthesis and show that it combines several unusual features, including "split", skipped, and iteratively used modules, and the use of the unusual extender unit methoxymalonate as a starter unit. To link genes to molecules, we constructed a transposon mutant library in N. terpenica, identifying a terpenomycin-null mutant with an inactivated terpenomycin PKS. Our findings show that the neglected actinomycetes have an unappreciated capacity for the production of bioactive molecules with unique biosynthetic pathways waiting to be uncovered and highlights these organisms as producers of diverse natural products.

BACKGROUND: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans. BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. METHODS: We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. RESULTS: Over two sampling campaigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans-specific DNA. Using the spatial scanning statistical tool SaTScan, we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year's human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area

Staphylococcus aureus strain JKD6159 represents a prominent community-acquired methicillin-resistant S. aureus (MRSA) clone in Australia. Here, we report an improved assembly of the original S. aureus JKD6159 genome sequence. By using deep sequencing with multiple technologies combined with carefully curated assembly and polishing, we believe the assembly to contain zero errors.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship.

BACKGROUND: High testing rates and rapid contact tracing have been key interventions to control COVID-19 in Victoria, Australia. A mobile laboratory (LabVan), for rapid SARS-CoV-2 diagnostics, was deployed at sites deemed critical by the Victorian State Department of Health as part of the response. We describe the process of design, implementation, and performance benchmarked against a central reference laboratory. METHODS: A BSL2 compliant laboratory, complete with a class II biological safety cabinet, was built within a Mercedes-Benz Sprinter Panel Van. Swabs were collected by on-site collection teams, registered using mobile internet-enabled tablets and tested using the Xpert® Xpress SARS-CoV-2 assay. Results were reported remotely via HL7 messaging to Public Health Units. Patients with negative results were automatically notified by mobile telephone text messaging (SMS). FINDINGS: A pilot trial of the LabVan identified a median turnaround time (TAT) from collection to reporting of 1:19 h:mm (IQR 0:18, Range 1:03-18:32) compared to 9:40 h:mm (IQR 8:46, Range 6:51-19:30) for standard processing within the central laboratory. During deployment in nine rural and urban COVID-19 outbreaks the median TAT was 2:18 h:mm (IQR 1:18, Range 0:50-16:52) compared to 19:08 h:mm (IQR 5:49, Range 1:36-58:52) for samples submitted to the central laboratory. No quality control issues were identified in the LabVan. INTERPRETATION: The LabVan is an ISO15189 compliant testing facility fully operationalized for mobile point-of-care testing that significantly reduces TAT for result reporting, facilitating rapid public health actions. FUNDING: This work was supported by the Department of Health, Victoria State Government, Australia.

BACKGROUND: Australia's response to the coronavirus disease 2019 (COVID-19) pandemic relies on widespread availability of rapid, accurate testing and reporting of results to facilitate contact tracing. The extensive geographical area of Australia presents a logistical challenge, with many of the population located distant from a laboratory capable of robust severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. A strategy to address this is the deployment of a mobile facility utilizing novel diagnostic platforms. This study aimed to evaluate the feasibility of a fully contained transportable SARS-CoV-2 testing laboratory using a range of rapid point-of-care tests. METHOD: A 20 ft (6.1 m) shipping container was refurbished (GeneWorks, Adelaide, South Australia) with climate controls, laboratory benches, hand-wash station and a class II biosafety cabinet. Portable marquees situated adjacent to the container served as stations for registration, sample acquisition and personal protective equipment for staff. Specimens were collected and tested on-site utilizing either the Abbott ID NOW or Abbott Panbio rapid tests. SARS-CoV-2 positive results from the rapid platforms or any participants reporting symptoms consistent with COVID-19 were tested on-site by GeneXpert Xpress RT-PCR. All samples were tested in parallel with a standard-of-care RT-PCR test (Panther Fusion SARS-CoV-2 assay) performed at the public health reference laboratory. In-laboratory environmental conditions and data management-related factors were also recorded. RESULTS: Over a 3 week period, 415 participants were recruited for point-of-care SARS-CoV-2 testing. From time of enrolment, the median result turnaround time was 26 min for the Abbott ID NOW, 32 min for the Abbott Panbio and 75 min for the Xpert Xpress. The environmental conditions of the refurbished shipping container were found to be suitable for all platforms tested, although humidity may have produced condensation within the c

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation.

Enterococcus faecalis and Enterococcus faecium are Gram-positive commensal gut bacteria that can also cause fatal infections. To study clinically relevant multi-drug resistant E. faecalis and E. faecium strains, methods are needed to overcome physical (thick cell wall) and enzymatic barriers that limit the transfer of foreign DNA and thus prevent facile genetic manipulation. Enzymatic barriers to DNA uptake identified in E. faecalis and E. faecium include type I, II and IV restriction modification systems and CRISPR-Cas. This review examines E. faecalis and E. faecium DNA defence systems and the methods with potential to overcome these barriers. DNA defence system bypass will allow the application of innovative genetic techniques to expedite molecular-level understanding of these important, but somewhat neglected, pathogens.

Latent HIV-1 provirus in infected individuals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3' LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo samples. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5' LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.

During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues and is exposed to new selective pressures, thus, potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation; however, a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr-mediated adaptation, we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.

Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4+ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4+ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4+ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4+ T cell responses. Interestingly, lack of CD4+ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4+ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4+ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.

In recent years reported cases of Buruli ulcer, caused by Mycobacterium ulcerans, have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how M. ulcerans circulates in the environment and transmits to humans we analyzed environmental samples collected from 115 properties of recent Buruli ulcer cases and from 115 postcode-matched control properties, for the presence of M. ulcerans. Environmental factors associated with increased odds of M. ulcerans presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums (Pseudocheirus peregrinus) and overhead powerlines. However, only overhead powerlines and the absence of the native plant Melaleuca lanceolata were associated with Buruli ulcer case properties. Samples positive for M. ulcerans were more likely to be found at case properties and were associated with detections of M. ulcerans in ringtail possum feces, supporting the hypothesis that M. ulcerans is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with M. ulcerans positive properties versus case properties indicates the involvement of human behavior or the influence of other environmental factors in disease acquisition that requires further study.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rare but chronic debilitating skin and soft tissue disease found predominantly in West Africa and Southeast Australia. While a moderate body of research has examined the distribution of M. ulcerans, the specific route(s) of transmission of this bacterium remain unknown, hindering control efforts. M. ulcerans is considered an environmental pathogen given it is associated with lentic ecosystems and human-to-human spread is negligible. However, the pathogen is also carried by various mammals and invertebrates, which may serve as key reservoirs and mechanical vectors, respectively. Here, we examine and review recent evidence from these endemic regions on potential transmission pathways, noting differences in findings between Africa and Australia, and summarising the risk and protective factors associated with Buruli ulcer transmission. We also discuss evidence suggesting that environmental disturbance and human population changes precede outbreaks. We note five key research priorities, including adoption of One Health frameworks, to resolve transmission pathways and inform control strategies to reduce the spread of Buruli ulcer.

BACKGROUND: A cornerstone of Australia's ability to control COVID-19 has been effective border control with an extensive supervised quarantine programme. However, a rapid recrudescence of COVID-19 was observed in the state of Victoria in June, 2020. We aim to describe the genomic findings that located the source of this second wave and show the role of genomic epidemiology in the successful elimination of COVID-19 for a second time in Australia. METHODS: In this observational, genomic epidemiological study, we did genomic sequencing of all laboratory-confirmed cases of COVID-19 diagnosed in Victoria, Australia between Jan 25, 2020, and Jan 31, 2021. We did phylogenetic analyses, genomic cluster discovery, and integrated results with epidemiological data (detailed information on demographics, risk factors, and exposure) collected via interview by the Victorian Government Department of Health. Genomic transmission networks were used to group multiple genomic clusters when epidemiological and genomic data suggested they arose from a single importation event and diversified within Victoria. To identify transmission of emergent lineages between Victoria and other states or territories in Australia, all publicly available SARS-CoV-2 sequences uploaded before Feb 11, 2021, were obtained from the national sequence sharing programme AusTrakka, and epidemiological data were obtained from the submitting laboratories. We did phylodynamic analyses to estimate the growth rate, doubling time, and number of days from the first local infection to the collection of the first sequenced genome for the dominant local cluster, and compared our growth estimates to previously published estimates from a similar growth phase of lineage B.1.1.7 (also known as the Alpha variant) in the UK. FINDINGS: Between Jan 25, 2020, and Jan 31, 2021, there were 20 451 laboratory-confirmed cases of COVID-19 in Victoria, Australia, of which 15 431 were submitted for sequencing, and 11 711 met all quality co

BACKGROUND: In Australia, COVID-19 diagnosis relies on RT-PCR testing which is relatively costly and time-consuming. To date, few studies have assessed the performance and implementation of rapid antigen-based SARS-CoV-2 testing in a setting with a low prevalence of COVID-19 infections, such as Australia. METHODS: This study recruited participants presenting for COVID-19 testing at three Melbourne metropolitan hospitals during a period of low COVID-19 prevalence. The Abbott PanBioTM COVID-19 Ag point-of-care test was performed alongside RT-PCR. In addition, participants with COVID-19 notified to the Victorian Government were invited to provide additional swabs to aid validation. Implementation challenges were also documented. FINDINGS: The specificity of the Abbott PanBioTM COVID-19 Ag test was 99.96% (95% CI 99.73 - 100%). Sensitivity amongst participants with RT-PCR-confirmed infection was dependent upon the duration of symptoms reported, ranging from 77.3% (duration 1 to 33 days) to 100% in those within seven days of symptom onset. A range of implementation challenges were identified which may inform future COVID-19 testing strategies in a low prevalence setting. INTERPRETATION: Given the high specificity, antigen-based tests may be most useful in rapidly triaging public health and hospital resources while expediting confirmatory RT-PCR testing. Considering the limitations in test sensitivity and the potential for rapid transmission in susceptible populations, particularly in hospital settings, careful consideration is required for implementation of antigen testing in a low prevalence setting. FUNDING: This work was funded by the Victorian Department of Health and Human Services. The funder was not involved in data analysis or manuscript preparation.

The ability to detect SARS-CoV-2 is critical to implementing evidence-based strategies to address the COVID-19 global pandemic. Expanding SARS-CoV-2 diagnostic ability beyond well-equipped laboratories widens the opportunity for surveillance and control efforts. However, such advances are predicated on the availability of rapid, scalable, accessible, yet high-performance diagnostic platforms. Methods to detect viral RNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP) show promise as rapid and field-deployable tests; however, the per-unit costs of the required diagnostic hardware can be a barrier for scaled deployment. Here, we describe a diagnostic hardware configuration for LAMP technology, named the FABL-8, that can be built for approximately US$380 per machine and provide results in under 30 min. Benchmarking showed that FABL-8 has a similar performance to a high-end commercial instrument for detecting fluorescence-based LAMP reactions. Performance testing of the instrument with RNA extracted from a SARS-CoV-2 virus dilution series revealed an analytical detection sensitivity of 50 virus copies per microliter-a detection threshold suitable to detect patient viral load in the first few days following symptom onset. In addition to the detection of SARS-CoV-2, we show that the system can be used to detect the presence of two bacterial pathogens, demonstrating the versatility of the platform for the detection of other pathogens. This cost-effective and scalable hardware alternative allows democratization of the instrumentation required for high-performance molecular diagnostics, such that it could be available to laboratories anywhere-supporting infectious diseases surveillance and research activities in resource-limited settings.

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.

Staphylococcus aureus is a major opportunistic human pathogen that is globally prevalent. Although S. aureus and humans may have co-evolved to the point of commensalism, the bacterium is equipped with virulence factors causing devastating infections. The adoption of an intracellular lifestyle by S. aureus is an important facet of its pathogenesis. Occupying a privileged intracellular compartment permits evasion from the bactericidal actions of host immunity and antibiotics. However, this localization exposes S. aureus to cell-intrinsic processes comprising autophagy, metabolic challenges and clearance mechanisms orchestrated by host programmed cell death pathways (PCDs), including apoptosis, pyroptosis and necroptosis. Mounting evidence suggests that S. aureus deploys pathoadaptive mechanisms that modulate the expression of its virulence factors to prevent elimination through PCD pathways. In this review, we critically analyse the current literature on the interplay between S. aureus virulence factors with the key, intertwined nodes of PCD. We discuss how S. aureus adaptation to the human host plays an essential role in the evasion of PCD, and we consider future directions to study S. aureus-PCD interactions.

Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella, allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R 2 = 24.64% and R 2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.

BACKGROUND: Mosquito control interventions are widely used to reduce mosquito-borne diseases. It is unclear what combination of interventions are most effective in reducing human disease. A novel intervention study for Buruli ulcer targeting mosquito vectors was proposed for a Buruli ulcer-endemic area of Victoria, Australia. The local community expressed a preference for avoiding widespread residual spraying of pyrethroids. To inform the design of a future cluster randomised control study (cRCT) for Buruli ulcer prevention in Victoria, we conducted a systematic literature review. AIMS: The aim was to describe cRCT designs which investigated interventions other than non-targeted insecticide for reducing mosquito-borne disease transmission, and comment on the strengths and weaknesses of these study designs. METHODS: Five medical research databases were searched for eligible literature from the earliest available sources up to 5 July 2019 (Medline, Embase, Web of Science, EBM Reviews, CAB Direct). Reference lists of identified studies were hand searched. Eligible studies were cRCTs using targeted chemical or biological mosquito control interventions, or mosquito breeding source reduction, with the occurrence of mosquito-borne disease as an outcome. RESULTS: Eight eligible cRCTs, conducted between 1994-2013 were identified in a variety of settings in the Americas and Asia. Interventions to reduce dengue transmission were mass adult trapping and source reduction. Interventions to reduce malaria transmission were largescale larvicide administration and (topical and spatial) repellent use. Three studies showed the intervention was associated with statistically significant reductions in the disease of interest and entomological indicators. High community engagement with the intervention were common to all three. In two studies, large buffer zones reduced contamination between study arms. Heterogeneity was reduced through increasing study cluster numbers, cluster matching a

Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii's success and its ongoing adaptation to the human host.

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in S. aureus and should dramatically improve the scalability of gene-function studies.

Healthcare associated infections caused by vancomycin-resistant Enterococcus faecium (VREfm) have a major impact on health outcomes. VREfm is difficult to treat because of intrinsic and acquired resistance to many clinically used antimicrobials, with daptomycin being one of the few last line therapeutic options for treating multidrug-resistant VREfm. The emergence of daptomycin-resistant VREfm is therefore of serious clinical concern. Despite this, the impact that daptomycin-resistant VREfm have on patient health outcomes is not clearly defined and knowledge on the mechanisms and genetic signatures linked with daptomycin resistance in VREfm remains incomplete. To address these knowledge gaps, phenotypic daptomycin susceptibility testing was undertaken on 324 E. faecium isolates from Australia and New Zealand. Approximately 15% of study isolates were phenotypically resistant to daptomycin. Whole genome sequencing revealed a strong association between vanA-VREfm and daptomycin resistance, with 95% of daptomycin-resistant study isolates harbouring vanA. Genomic analyses showed that daptomycin-resistant VREfm isolates were polyclonal and carried several previously characterised mutations in the liaR and liaS genes as well as several novel mutations within the rpoB, rpoC, and dltC genes. Overall, 70% of daptomycin-resistant study isolates were found to carry mutations within the liaR, rpoB, rpoC, or dltC genes. Finally, in a mouse model of VREfm bacteraemia, infection with the locally dominant daptomycin-resistant clone led to reduced daptomycin treatment efficacy in comparison to daptomycin-susceptible E. faecium. These findings have important implications for ongoing VREfm surveillance activities and the treatment of VREfm infections.

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.