Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Describing skin health and disease in urban-living Aboriginal children: co-design, development and feasibility testing of the Koolungar Moorditj Healthy Skin pilot project.

BACKGROUND: Indigenous children in colonised nations experience high rates of health disparities linked to historical trauma resulting from displacement and dispossession, as well as ongoing systemic racism. Skin infections and their complications are one such health inequity, with the highest global burden described in remote-living Australian Aboriginal and/or Torres Strait Islander (hereafter respectfully referred to as Aboriginal) children. Yet despite increasing urbanisation, little is known about the skin infection burden for urban-living Aboriginal children. More knowledge is needed to inform service provision, treatment guidelines and community-wide healthy skin strategies. In this pilot study, we aimed to test the feasibility and design of larger multi-site observational studies, provide initial descriptions of skin disease frequency and generate preliminary hypotheses of association. METHODS: This project has been co-designed with local (Noongar) Elders to provide an Australian-first description of skin health and disease in urban-living Aboriginal children. In collaboration with an urban Aboriginal Community Controlled Health Organisation (Derbarl Yerrigan Health Service), we conducted a week-long cross-sectional observational cohort study of Aboriginal children (0-18 years) recruited from the waiting room. Participants completed a questionnaire, skin examination, clinical photos, and swabs and received appropriate treatment. We assessed the feasibility and impact of the pilot study. RESULTS: From 4 to 8 October 2021, we recruited 84 Aboriginal children of whom 80 (95%) were urban-living. With a trusted Aboriginal Health Practitioner leading recruitment, most parents (or caregivers) who were approached consented to participate. Among urban-living children, over half (45/80, 56%) of parents described a current concern with their child's skin, hair and/or nails; and one-third (26/80, 33%) reported current itchy skin. Using a research-service model, 27% (21/79) of examined urban-living participants received opportunistic same-day treatment and 18% (14/79) were referred for later review. CONCLUSIONS: This co-designed pilot study to understand skin health in urban-living Aboriginal children was feasible and acceptable, with high study participation and subsequent engagement in clinical care observed. Co-design and the strong involvement of Aboriginal people to lead and deliver the project was crucial. The successful pilot has informed larger, multi-site observational studies to more accurately answer questions of disease burden and inform the development of healthy skin messages for urban-living Aboriginal children.

Antibiotic consumption for sore throat and the potential effect of a vaccine against group A Streptococcus: a systematic review and modelling study.

BACKGROUND: Antibiotic consumption can lead to antimicrobial resistance and microbiome imbalance. We sought to estimate global antibiotic consumption for sore throat, and the potential reduction in consumption due to effective vaccination against group A Streptococcus (Strep A). METHODS: We reviewed and analysed articles published between January 2000 and February 2022, identified though Clarivate Analytics' Web of Science search platform, with reference to antibiotic prescribing or consumption, sore throat, pharyngitis, or tonsillitis. We then used those analyses, combined with assumptions for the effectiveness, duration of protection, and coverage of a vaccine, to calculate the estimated reduction in antibiotic prescribing due to the introduction of Strep A vaccines. FINDINGS: We identified 101 studies covering 38 countries. The mean prescribing rate for sore throat was approximately 5 courses per 100 population per year, accounting for approximately 5% of all antibiotic consumption. Based on 2020 population estimates for countries with empiric prescribing rates, antibiotic consumption for sore throat was estimated to exceed 37 million courses annually, of which half could be attributable to treatment for Strep A. A vaccine that reduces rates of Strep A infection by 80%, with 80% coverage and 10 year's duration of protection, could avert 2.8 million courses of antibiotics prescribed for sore throat treatment among 5-14 year-olds in countries with observed prescribing rates, increasing to an estimated 7.5 million averted if an effective vaccination program also reduced precautionary prescribing. INTERPRETATION: A vaccine that prevents Strep A throat infections in children may reduce antibiotic prescribing for sore throat by 32-87% depending on changes to prescribing and consumption behaviours. FUNDING: The Wellcome Trust, grant agreement number 215490/Z/19/Z.

Outcome and racing performance following standing fracture repair in 245 horses.

BACKGROUND: Repair of sagittal proximal phalanx (P1) and parasagittal metacarpal/metatarsal III (MC/MTIII) fractures has evolved over recent decades from a procedure carried out solely under general anaesthesia, to one commonly performed under standing sedation. To date, standing fracture repair has not been evaluated for large cohorts. OBJECTIVES: To determine short-term (survival to discharge) and long-term (return to racing) outcomes of horses undergoing standing repair of MC/MTIII and P1 fractures, and to compare pre-surgical and post-surgical racing performance. STUDY DESIGN: Single-centre retrospective cohort study. METHODS: Retrospective clinical record review of 245 cases undergoing standing repair of MC/MTIII or P1 fractures, 1 January 2007-30 June 2021. Data on signalment, fracture configuration and complications were collected and full race records were retrieved from the Racing Post Database (wwww.racingpost.com). Chi-squared and Mann-Whitney U tests were used to determine any difference in variables between horses that raced after surgery compared to those that did not. McNemar change and Wilcoxon signed-rank tests were used to compare pre- and post-surgical racing performance, p ≤ 0.05. RESULTS: Ninety-eight percent [95% confidence interval (CI): 96.2%-99.7%] of horses survived hospital discharge, and 75.1% (95% CI: 68.9%-81.4%) raced after surgery, a median of 241 days later. Horses that raced post-surgery were significantly less likely to have suffered from complications during hospitalisation than those that did not race again [17.3% (95% CI: 11%-24%) vs. 36.5% (95% CI: 23%-50%), p = 0.005]. Comparing pre- and post-operative racing performance, there was no significant difference in earnings per start [median £628.00, interquartile range (IQR) 115.90-1934.80 vs. £653.20, 51.00-1886.40, p = 0.7] or proportion of horses winning [51% (95% CI: 41%-61%) vs. 54% (95% CI: 44%-64%), p = 0.8] or being placed first-third [77% (95% CI: 68%-85%) vs. 71% (95% CI: 62%-80%, p = 0.5] in at least one race. MAIN LIMITATIONS: Retrospective nature of study with reliance on clinical records and public databases, limiting data available for analysis. CONCLUSIONS: Standing fracture repair is a viable treatment option for MC/MTIII or P1 fractures that returns horses to the racetrack within an acceptable time frame and is capable of restoring pre-surgical athletic ability.

Searching for Strep A in the clinical environment during a human challenge trial: a sub-study protocol.

Streptococcus pyogenes (also known as group A Streptococcus , Strep A) is an obligate human pathogen with significant global morbidity and mortality. Transmission is believed to occur primarily between individuals via respiratory droplets, but knowledge about other potential sources of transmission via aerosols or the environment is limited. Such knowledge is required to design optimal interventions to control transmission, particularly in endemic settings. We aim to detail an experimental methodology to assess the transmission potential of Strep A in a clinical environment. We will examine potential sources of transmission in up to 20 participants recruited to the Controlled human infection for penicillin against Streptococcus pyogenes (CHIPS) Trial. Three approaches to understanding transmission will be used: the use of selective agar settle plates to capture possible droplet or airborne spread of Strep A; measurement of the possible distance of Strep A droplet spread during conversation; and environmental swabbing of personal and common high-touch items to detect the presence of Strep A on hard and soft surfaces. All methods are designed to allow for an assessment of transmission potential by symptomatic, asymptomatic and non-cases. Ethical approval has been obtained through Bellberry Human Research Ethics Committee (approval 2021-03-295). Trial registration number: ACTRN12621000751875. Any results elicited from these experiments will be of benefit to the scientific literature in improving our knowledge of opportunities to prevent Strep A transmission as a direct component of the primordial prevention of rheumatic fever. Findings will be reported at local, national and international conferences and in peer-reviewed journals.

Analysis of first responder-involved traffic incidents by mining news reports.

Roadside service and incident response personnel face the risk of being killed or severely injured by passing vehicles when performing their duties on or along a road. This study investigated 5,113 responder-involved event news reports to understand the characteristics of first responder-involved incidents. Through text mining, this study examined and compared the characteristics of three types of responder-involved incidents: near-miss incidents, struck-by incidents, and line-of-duty-deaths (LODD). A higher proportion of struck-by and LODD incidents are associated with law enforcement agencies. In terms of the time of day, morning and night incidents are frequently reported in the news. Driving under the influence (DUI) or driving while intoxicated (DWI) is a major cause of LODD incidents. Compared to struck-by incidents, LODD incidents have a larger portion related to out-of-control vehicles. Further, this study built a logistic regression model to relate the incident characteristics to the odds of an incident being a LODD incident. The modeling result shows that tow truck drivers are associated with a greater likelihood of being involved in a news-reported LODD incident than other responders. LODD incidents are more likely to occur on early morning. Compare to entering/leaving/staying at the scene, responders are more likely to be involved in LODD event when assisting. The results offer insights into understanding the characteristics and possible reasons for first responder-involved incidents so that potential countermeasures could be developed to improve responder safety.

Are first responders prepared for electric vehicle fires? A national survey.

Transitioning to electric vehicles (EVs) will create both opportunities and challenges. Although some programs and resources related to EVs have been made available to first responders, it remains unknown whether our first responders are well prepared for traffic incidents that involve EVs and whether there are any organizational and geographic disparities in preparedness. To answer these questions, a national survey was conducted to collect feedback on responders' incident management knowledge and training experiences related to EVs, as well as their attitudes and concerns towards EVs. Over 1000 first responders across the country participated in the survey, and the survey had representation from all 50 states and Washington DC. Over 40% of first responders reported never receiving EV-related safety training. Paramedics or EMS are associated with the highest odds of not receiving EV-related training, followed by law enforcement. Geographically, FEMA Region 8 (e.g., Montana and North Dakota) is associated with the highest percentage of not receiving EV training. Regarding EV fire tactics, more than half (57%) of law enforcement officers said they do not know any; responders from towing & recovery also have little knowledge compared to firefighters. Statistical modeling was conducted to explore correlates of responders' EV safety training and knowledge of EV fire tactics. The survey also provided insights about the challenges and risks of managing EV-involved incidents. In summary, responders are greatly concerned about the risks that EVs can pose to their community, and actions must be taken now.

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter.

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.

Standardization of Epidemiological Surveillance of Group A Streptococcal Pharyngitis.

Pharyngitis, more commonly known as sore throat, is caused by viral and/or bacterial infections. Group A Streptococcus (Strep A) is the most common bacterial cause of pharyngitis. Strep A pharyngitis is an acute, self-limiting disease but if undertreated can lead to suppurative complications, nonsuppurative poststreptococcal immune-mediated diseases, and toxigenic presentations. We present a standardized surveillance protocol, including case definitions for pharyngitis and Strep A pharyngitis, as well as case classifications that can be used to differentiate between suspected, probable, and confirmed cases. We discuss the current tests used to detect Strep A among persons with pharyngitis, including throat culture and point-of-care tests. The type of surveillance methodology depends on the resources available and the objectives of surveillance. Active surveillance and laboratory confirmation is the preferred method for case detection. Participant eligibility, the surveillance population and additional considerations for surveillance of pharyngitis are addressed, including baseline sampling, community engagement, frequency of screening and season. Finally, we discuss the core elements of case report forms for pharyngitis and provide guidance for the recording of severity and pain associated with the course of an episode.

In vitro antibacterial activity of Western Australian honeys, and manuka honey, against bacteria implicated in impetigo.

Impetigo is a contagious skin disease caused by Staphylococcus aureus and Streptococcus pyogenes. Without treatment, impetigo may be recurrent, develop into severe disease, or have serious, life-threatening sequelae. Standard treatment consists of topical or systemic antibiotic therapy (depending on severity), however, due to antibiotic resistance some therapies are increasingly ineffective. In this study we evaluated the potential for honey as an alternative treatment for impetigo. A broth microdilution assay in 96-well microtitre trays was used to determine the minimum inhibitory concentrations (MICs) of six monofloral honeys (jarrah, marri, red bell, banksia, wandoo, and manuka), a multifloral honey and artificial honey against S. aureus (n = 10), S. pyogenes (n = 10), and coagulase-negative staphylococci (CoNS) (n = 10). The optical density (OD) of all microtitre tray wells was also determined before and after assay incubation to analyse whether sub-MIC growth inhibition occurred. Jarrah, marri, red bell, banksia, and manuka honeys were highly effective at inhibiting S. aureus and CoNS, with MIC50 values ranging from 4 to 8% w/v honey. S. pyogenes was also inhibited by these same honeys, albeit at higher concentrations (8-29% w/v). Wandoo and multifloral honeys had the least antibacterial activity with MICs of >30% (w/v) for all isolates. However, OD data indicated that sub-MIC concentrations of honey were still partially restricting bacterial growth. Our pre-clinical data indicate that honey may be a potential therapeutic agent for the routine treatment of mild impetigo, and we suggest that clinical trials would be appropriate to further investigate this.

Penicillin G concentrations required for prophylaxis against Group A Streptococcus infection evaluated using a hollow fibre model and mathematical modelling.

BACKGROUND: Acute rheumatic fever (ARF), an autoimmune reaction to Group A Streptococcus (Streptococcus pyogenes; Strep A) infection, can cause rheumatic heart disease (RHD). New formulations of long-acting penicillins are being developed for secondary prophylaxis of ARF and RHD. OBJECTIVES: To evaluate the penicillin G concentrations required to suppress growth of Strep A. METHODS: Broth microdilution MIC and MBC for Strep A strains M75611024, M1T15448 and M18MGAS8232 were determined. All strains were studied in a hollow fibre model (initial inoculum 4 log10 cfu/mL). Constant penicillin G concentrations of 0.008, 0.016 and 0.05 mg/L were examined against all strains, plus 0.012 mg/L against M18MGAS8232. Viable counts were determined over 144 h. Subsequently, all penicillin G-treated cartridges were emptied, reinoculated with 5 log10 cfu/mL and counts determined over a further 144 h. Mathematical modelling was performed. RESULTS: MIC and MBC were 0.008 mg/L for all strains; small subpopulations of M75611024 and M1T15448, but not M18MGAS8232, grew at 1× MIC. Following the first inoculation, 0.008 mg/L achieved limited killing and/or stasis against M75611024 and M1T15448, with subsequent growth to ∼6 log10 cfu/mL. Following both inocula, concentrations ≥0.016 mg/L suppressed M75611024 and M1T15448 to <1 log10 cfu/mL from 6 h onwards with eradication. Concentrations ≥0.008 mg/L suppressed M18MGAS8232 to <1 log10 cfu/mL from 24 h onwards with eradication after both inoculations. Mathematical modelling well described all strains using a single set of parameter estimates, except for different maximum bacterial concentrations and proportions of bacteria growing at 1× MIC. CONCLUSIONS: In the absence of validated animal and human challenge models, the study provides guidance on penicillin G target concentrations for development of new penicillin formulations.

From the past to the future: Modeling the temporal instability of safety performance functions.

In roadway safety management, safety performance functions (SPFs) are widely used by state and local agencies to predict crashes for base site conditions. SPFs are developed based on historical traffic safety data and are used to make predictions for anticipated site characteristics in the future. An underlying assumption in SPF development is that the relationships between crash frequency and site conditions are stationary from the past (when the model data were collected) to the future (for which SPFs are applied). The assumption using the past to represent the future could be fundamentally problematic. This study proposes a modeling framework that can relax this assumption. Specifically, this framework integrates temporal modeling with time-series analysis to strengthen the current SPF estimation methods. The temporal modeling approach is Temporally Weighted Negative Binomial Regression (TWNBW), and the time-series analysis is tried by employing the Seasonal Autoregressive Integrated Moving Average (SARIMA) and Artificial Neural Networks (ANN) methods. The temporal modeling is to uncover the temporal variations of SPFs and the time-series analysis explains and predicts the relationship between the SPF's temporal variation and time. The outcome of the framework is a set of Future SPFs that capture the temporal unobserved heterogeneity in safety data and describe the predicted relationships between safety performance and site characteristics in the future. A case study using six-year safety datasets from Georgia was conducted to illustrate the key components of the modeling framework. The temporal modeling results showed significant variations in SPFs across time. The parameters for traffic volume, i.e., Average Annual Daily Traffic (AADT), and segment length are associated with an increasing trend with time, and for access point density there is a descending trend. The SPF parameters are found to have a strong seasonality. Both time-series modeling methods appear to be appropriate to explain the temporal variations of SPF parameters, and the models are able to predict SPF parameters with acceptable errors smaller than 1% on average. Future SPFs can be used to support the roadway safety management that affects future traffic safety performance.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

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.

Searching for a technology-driven acute rheumatic fever test: the START study protocol.

INTRODUCTION: The absence of a diagnostic test for acute rheumatic fever (ARF) is a major impediment in managing this serious childhood condition. ARF is an autoimmune condition triggered by infection with group A Streptococcus. It is the precursor to rheumatic heart disease (RHD), a leading cause of health inequity and premature mortality for Indigenous peoples of Australia, New Zealand and internationally. METHODS AND ANALYSIS: 'Searching for a Technology-Driven Acute Rheumatic Fever Test' (START) is a biomarker discovery study that aims to detect and test a biomarker signature that distinguishes ARF cases from non-ARF, and use systems biology and serology to better understand ARF pathogenesis. Eligible participants with ARF diagnosed by an expert clinical panel according to the 2015 Revised Jones Criteria, aged 5-30 years, will be recruited from three hospitals in Australia and New Zealand. Age, sex and ethnicity-matched individuals who are healthy or have non-ARF acute diagnoses or RHD, will be recruited as controls. In the discovery cohort, blood samples collected at baseline, and during convalescence in a subset, will be interrogated by comprehensive profiling to generate possible diagnostic biomarker signatures. A biomarker validation cohort will subsequently be used to test promising combinations of biomarkers. By defining the first biomarker signatures able to discriminate between ARF and other clinical conditions, the START study has the potential to transform the approach to ARF diagnosis and RHD prevention. ETHICS AND DISSEMINATION: The study has approval from the Northern Territory Department of Health and Menzies School of Health Research ethics committee and the New Zealand Health and Disability Ethics Committee. It will be conducted according to ethical standards for research involving Indigenous Australians and New Zealand Maori and Pacific Peoples. Indigenous investigators and governance groups will provide oversight of study processes and advise on cultural matters.

Streptolysins are the primary inflammasome activators in macrophages during Streptococcus pyogenes infection.

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes an array of infectious diseases in humans. Accumulating clinical evidence suggests that proinflammatory interleukin (IL)-1ß signaling plays an important role in GAS disease progression. The host regulates the production and secretion of IL-1ß via the cytosolic inflammasome pathway. Activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome complex requires two signals: a priming signal that stimulates increased transcription of genes encoding the components of the inflammasome pathway, and an activating signal that induces assembly of the inflammasome complex. Here we show that GAS-derived lipoteichoic acid can provide a priming signal for NLRP3 inflammasome activation. As only few GAS-derived proteins have been associated with inflammasome-dependent IL-1ß signaling, we investigated novel candidates that might play a role in activating the inflammasome pathway by infecting mouse bone marrow-derived macrophages and human THP-1 macrophage-like cells with a panel of isogenic GAS mutant strains. We found that the cytolysins streptolysin O (SLO) and streptolysin S are the main drivers of IL-1ß release in proliferating logarithmic phase GAS. Using a mutant form of recombinant SLO, we confirmed that bacterial pore formation on host cell membranes is a key mechanism required for inflammasome activation. Our results suggest that streptolysins are major determinants of GAS-induced inflammation and present an attractive target for therapeutic intervention.

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins.

Genetic Manipulation of Group A Streptococcus-Gene Deletion by Allelic Replacement.

Genetic manipulation of Streptococcus pyogenes (Group A Streptococcus, GAS) has historically been a challenging process, with considerable variation in efficiency between different strains. Here, we outline an optimized, rapid method for creating markerless isogenic mutations that combines Gibson assembly cloning with a new temperature-sensitive plasmid, pLZts. This method is highly efficient and reduces the time needed to create GAS mutants to ~2-3 weeks, with the ability to prepare multiple mutants simultaneously.

SToP (See, Treat, Prevent) skin sores and scabies trial: study protocol for a cluster randomised, stepped-wedge trial for skin disease control in remote Western Australia.

INTRODUCTION: Skin is important in Australian Aboriginal culture informing kinship and identity. In many remote Aboriginal communities, scabies and impetigo are very common. Untreated skin infections are painful, itchy and frequently go untreated due to under-recognition and lack of awareness of their potential serious complications. We hypothesise that the skin infection burden in remote Aboriginal communities can be reduced by implementing streamlined training and treatment pathways integrated with environmental health and health promotion activities, tested in the See, Treat, Prevent (SToP skin sores and scabies) trial. METHODS AND ANALYSIS: SToP will evaluate a skin control programme using a stepped-wedge, cluster randomised trial design with three intervention components (the 'SToP activities'): (1) seeing skin infections (development of training resources implemented within a community dermatology model); (2) treating skin infections (employing the latest evidence for impetigo, and scabies treatment); and (3) preventing skin infections (embedded, culturally informed health promotion and environmental health activities). Four community clusters in the remote Kimberley region of Western Australia will participate. Following baseline data collection, two clusters will be randomly allocated to the SToP activities. At 12 months, the remaining two clusters will transition to the SToP activities. The primary outcome is the diagnosis of impetigo in children (5-9 years) at school-based surveillance. Secondary outcome measures include scabies diagnosis, other child health indicators, resistance to cotrimoxazole in circulating pathogenic bacteria, determining the economic burden of skin disease and evaluating the cost effectiveness of SToP activities. ETHICS AND DISSEMINATION: This study protocol was approved by the health ethics review committees at the Child and Adolescent Health Service (Approval number RGS0000000584), the Western Australian Aboriginal Health Ethics Committee (Reference number: 819) and the University of Western Australia (Reference RA/4/20/4123). Study findings will be shared with community members, academic and medical communities via publications and presentations, and in reports to funders. Authorship for all publications based on this study will be determined in line with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals published by the International Committee of Medical Journal Editors. Sharing results with organisations and communities who contributed to the study is paramount. The results of the SToP trial will be shared with participants in a suitable format, such as a single summary page provided to participants or presentations to communities, the Kimberly Aboriginal Health Planning Forum Research Subcommittee and other stakeholders as appropriate and as requested. Communication and dissemination will require ongoing consultation with Aboriginal communities to determine appropriate formats. TRIAL REGISTRATION NUMBER: ACTRN12618000520235.

Detection of Epidemic Scarlet Fever Group A Streptococcus in Australia.

Sentinel hospital surveillance was instituted in Australia to detect the presence of pandemic group A Streptococcus strains causing scarlet fever. Genomic and phylogenetic analyses indicated the presence of an Australian GAS emm12 scarlet fever isolate related to United Kingdom outbreak strains. National surveillance to monitor this pandemic is recommended.

Group A Streptococcus co-ordinates manganese import and iron efflux in response to hydrogen peroxide stress.

Bacterial pathogens encounter a variety of adverse physiological conditions during infection, including metal starvation, metal overload and oxidative stress. Here, we demonstrate that group A Streptococcus (GAS) utilises Mn(II) import via MtsABC during conditions of hydrogen peroxide stress to optimally metallate the superoxide dismutase, SodA, with Mn. MtsABC expression is controlled by the DtxR family metalloregulator MtsR, which also regulates the expression of Fe uptake systems in GAS. Our results indicate that the SodA in GAS requires Mn for full activity and has lower activity when it contains Fe. As a consequence, under conditions of hydrogen peroxide stress where Fe is elevated, we observed that the PerR-regulated Fe(II) efflux system PmtA was required to reduce intracellular Fe, thus protecting SodA from becoming mismetallated. Our findings demonstrate the co-ordinate action of MtsR-regulated Mn(II) import by MtsABC and PerR-regulated Fe(II) efflux by PmtA to ensure appropriate Mn(II) metallation of SodA for optimal superoxide dismutase function.