Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Genomic epidemiology of Salmonella Typhi in Central Division, Fiji, 2012 to 2016.

Background: Typhoid fever is endemic in some Pacific Island Countries including Fiji and Samoa yet genomic surveillance is not routine in such settings. Previous studies suggested imports of the global H58 clade of Salmonella enterica var Typhi (Salmonella Typhi) contribute to disease in these countries which, given the MDR potential of H58, does not auger well for treatment. The objective of the study was to define the genomic epidemiology of Salmonella Typhi in Fiji. Methods: Genomic sequencing approaches were implemented to study the distribution of 255 Salmonella Typhi isolates from the Central Division of Fiji. We augmented epidemiological surveillance and Bayesian phylogenomic approaches with a multi-year typhoid case-control study to define geospatial patterns among typhoid cases. Findings: Genomic analyses showed Salmonella Typhi from Fiji resolved into 2 non-H58 genotypes with isolates from the two dominant ethnic groups, the Indigenous (iTaukei) and non-iTaukei genetically indistinguishable. Low rates of international importation of clones was observed and overall, there were very low levels an antibiotic resistance within the endemic Fijian typhoid genotypes. Genomic epidemiological investigations were able to identify previously unlinked case clusters. Bayesian phylodynamic analyses suggested that genomic variation within the larger endemic Salmonella Typhi genotype expanded at discreet times, then contracted. Interpretation: Cyclones and flooding drove 'waves' of typhoid outbreaks in Fiji which, through population aggregation, poor sanitation and water safety, and then mobility of the population, spread clones more widely. Minimal international importations of new typhoid clones suggest that targeted local intervention strategies may be useful in controlling endemic typhoid infection. These findings add to our understanding of typhoid transmission networks in an endemic island country with broad implications, particularly across Pacific Island Countries. Funding: This work was supported by the Coalition Against Typhoid through the Bill and Melinda Gates Foundation [grant number OPP1017518], the Victorian Government, the National Health and Medical Research Council Australia, the Australian Research Council, and the Fiji Ministry of Health and Medical Services.

Role of Glutathione in Buffering Excess Intracellular Copper in Streptococcus pyogenes.

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion.IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

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.

Biphasic Outbreak of Invasive Group A Streptococcus Disease in Eldercare Facility, New Zealand.

A 3-month outbreak of invasive group A Streptococcus disease at an eldercare facility, in which 5 persons died, was biphasic. Although targeted chemoprophylaxis contained the initial outbreak, a second phase of the outbreak occurred after infection control processes ended. To retrospectively investigate the genomic epidemiology of the biphasic outbreak, we used whole-genome sequencing and multiple bioinformatics approaches. Analysis of isolates from the outbreak and isolates prospectively collected during the outbreak response indicated a single S. pyogenes emm81 clone among residents and staff members. Outbreak isolates differed from nonoutbreak emm81 isolates by harboring an integrative conjugative genomic element that contained the macrolide resistance determinant erm(TR). This study shows how retrospective high-resolution genomic investigations identified rapid spread of a closed-facilty clonal outbreak that was controlled, but not readily cleared, by infection control management procedures.

Author Correction: Atlas of group A streptococcal vaccine candidates compiled using large-scale comparative genomics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Atlas of group A streptococcal vaccine candidates compiled using large-scale comparative genomics.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies.

Systematic Review of Group A Streptococcal emm Types Associated with Acute Post-Streptococcal Glomerulonephritis.

Acute post-streptococcal glomerulonephritis (APSGN) is a postinfectious immune-mediated kidney disease associated with group A Streptococcus (GAS). The prevalence of APSGN varies within and between countries and is influenced by socioeconomic, host, and bacterial factors. The disease is more prevalent in developing countries and resource-poor settings of developed countries, such as the Indigenous populations residing in tropical Australia. The M-protein is a universally present GAS surface antigen that is the focus of molecular typing and vaccine research. Early reports suggested that some M-proteins (emm types) are more likely to cause APSGN than others. Here, we present the first systematic review of the global distribution of APSGN-associated GAS emm types. There were 46 emm types among the 676 cases described in 15 reviewed articles. Only 43% APSGN cases would have had theoretical coverage from the experimental M protein-based GAS vaccine. Vaccine coverage was higher in regions such as North America (97%) and the United Kingdom (98%) than Africa (67%) and Australia (38%). Variable vaccine coverage against APSGN- associated emm types highlights the need for further research into this disease, particularly in settings of poverty, where APSGN prevalence is higher. Three GAS emm types (emm49, emm60, and emm55) consistently occur in APSGN cases around the world. Future studies would therefore benefit from examining the genomic epidemiology of these emm types to unravel potential markers of APSGN.

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.

A Controlled Human Infection Model of Group A Streptococcus Pharyngitis: Which Strain and Why?

Group A Streptococcus (GAS) is a major cause of global infection-related morbidity and mortality. A modern controlled human infection model (CHIM) of GAS pharyngitis can accelerate vaccine development and pathogenesis research. A robust rationale for strain selection is central to meeting ethical, scientific, and regulatory requirements. Multifaceted characterization studies were done to compare a preferred candidate emm75 (M75) GAS strain to three other strains: an alternative candidate emm12 (M12) strain, an M1 strain used in 1970s pharyngitis CHIM studies (SS-496), and a representative (5448) of the globally disseminated M1T1 clone. A range of approaches were used to explore strain growth, adherence, invasion, delivery characteristics, short- and long-term viability, phylogeny, virulence factors, vaccine antigens, resistance to killing by human neutrophils, and lethality in a murine invasive model. The strains grew reliably in a medium without animal-derived components, were consistently transferred using a swab method simulating the CHIM protocol, remained viable at -80°C, and carried genes for most candidate vaccine antigens. Considering GAS molecular epidemiology, virulence factors, in vitro assays, and results from the murine model, the contemporary strains show a spectrum of virulence, with M75 appearing the least virulent and 5448 the most. The virulence profile of SS-496, used safely in 1970s CHIM studies, was similar to that of 5448 in the animal model and virulence gene carriage. The results of this multifaceted characterization confirm the M75 strain as an appropriate choice for initial deployment in the CHIM, with the aim of safely and successfully causing pharyngitis in healthy adult volunteers.IMPORTANCE GAS (Streptococcus pyogenes) is a leading global cause of infection-related morbidity and mortality. A modern CHIM of GAS pharyngitis could help to accelerate vaccine development and drive pathogenesis research. Challenge strain selection is critical to the safety and success of any CHIM and especially so for an organism such as GAS, with its wide strain diversity and potential to cause severe life-threatening acute infections (e.g., toxic shock syndrome and necrotizing fasciitis) and postinfectious complications (e.g., acute rheumatic fever, rheumatic heart disease, and acute poststreptococcal glomerulonephritis). In this paper, we outline the rationale for selecting an emm75 strain for initial use in a GAS pharyngitis CHIM in healthy adult volunteers, drawing on the findings of a broad characterization effort spanning molecular epidemiology, in vitro assays, whole-genome sequencing, and animal model studies.

Characterisation of Staphylococcus felis isolated from cats using whole genome sequencing.

This study used phenotypic tests and whole genome sequencing to characterise a collection of 37 clinical Staphylococcus felis isolates from cats. Samples were isolated from a range of diseases including feline lower urinary tract disease (n = 15), otitis externa (n = 13), and ocular disease (n = 2). Isolates were identified using MALDI-TOF MS and by BLASTn analysis of S. felis-specific 16S rRNA, rpoB and nuc genes in whole genome sequence-based contigs. Phenotypic antimicrobial resistance was determined using disk diffusion and broth microdilution. Coagulase activity was assessed using feline and rabbit plasma. Genomes were screened for putative virulence and antimicrobial resistance genes using the sequences of known genes from other staphylococci as homologous references. Phylogenetic relationships were inferred using single nucleotide polymorphisms. One isolate was coagulase-positive when tested with feline plasma but all isolates were rabbit plasma coagulase-negative. No genetic determinant of coagulase activity was identified in this isolate. A range of putative virulence genes were found amongst isolates including genes associated with adhesion, toxin production and immune evasion. Ninety two percent of isolates were fully susceptible to all antimicrobials tested, which was reflected by a general absence of resistance genes. Clustering within the phylogenetic tree suggested a multiclonal population structure; this clustering did not correlate with disease syndrome or geographic origin of the isolate. Future studies of veterinary staphylococci will benefit from the publicly available S. felis draft genomes that were generated in this study.

Scarlet Fever Epidemic in China Caused by Streptococcus pyogenes Serotype M12: Epidemiologic and Molecular Analysis.

From 2011, Hong Kong and mainland China have witnessed a sharp increase in reported cases, with subsequent reports of epidemic scarlet fever in North Asia and the United Kingdom. Here we examine epidemiological data and investigate the genomic context of the predominantly serotype M12 Streptococcus pyogenes scarlet fever isolates from mainland China. Incident case data was obtained from the Chinese Nationwide Notifiable Infectious Diseases Reporting Information System. The relative risk of scarlet fever in recent outbreak years 2011-2016 was calculated using the median age-standardised incidence rate, compared to years 2003-2010 prior this outbreak. Whole genome sequencing was performed on 32 emm12 scarlet fever isolates and 13 emm12 non-scarlet fever isolates collected from different geographic regions of China, and compared with 203 published emm12 S. pyogenes genomes predominantly from scarlet fever outbreaks in Hong Kong (n=134) and the United Kingdom (n=63). We found during the outbreak period (2011-2016), the median age-standardised incidence in China was 4.14/100,000 (95% confidence interval (CI) 4.11-4.18), 2.62-fold higher (95% CI 2.57-2.66) than that of 1.58/100,000 (95% CI 1.56-1.61) during the baseline period prior to the outbreak (2003-2010). Highest incidence was reported for children 5years of age (80.5/100,000). Streptococcal toxin encoding prophage φHKU.vir and φHKU.ssa in addition to the macrolide and tetracycline resistant ICE-emm12 and ICE-HKU397 elements were found amongst mainland China multi-clonal emm12 isolates suggesting a role in selection and expansion of scarlet fever lineages in China. Global dissemination of toxin encoded prophage has played a role in the expansion of scarlet fever emm12 clones. These findings emphasize the role of comprehensive surveillance approaches for monitoring of epidemic human disease.

Case report of whole genome sequencing in the XY female: identification of a novel SRY mutation and revision of a misdiagnosis of androgen insensitivity syndrome.

BACKGROUND: The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways in utero. The cause is usually genetic and various genes are implicated. CASE PRESENTATION: Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant (p.Arg130Pro, c.389G>C) in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient's disorder of sexual development. CONCLUSION: This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development.

Presentation of m.3243A>G (MT-TL1; tRNALeu) variant with focal neurology in infancy.

The Mitochondrial tRNALeu (MT-TL1) mutation, m.3243A>G constitutes the commonest identified mitochondrial genome mutation. Characteristically, giving rise to MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), a phenotypic spectrum associated with this genetic variant is now apparent. We report on the first patient with infantile hemiparesis, without comorbid encephalopathy, attributed to this variant. This further expands the recognized disease spectrum and highlights the need to consider mitochondrial genomic mutations in cases of cryptogenic focal neurological deficit in infancy. The potential for genetic disease modifiers is additionally discussed.

Vapour-liquid-solid growth of ternary Bi2Se2Te nanowires.

: High-density growth of single-crystalline Bi2Se2Te nanowires was achieved via the vapour-liquid-solid process. The stoichiometry of samples grown at various substrate temperatures is precisely determined based on energy-dispersive X-ray spectroscopy, X-ray diffraction, and Raman spectroscopy on individual nanowires. We discuss the growth mechanism and present insights into the catalyst-precursor interaction.