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Abstract Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

ABSTRACT Bacterial species often consist of strains with variable gene content, collectively referred to as the pangenome. Variations in the genetic makeup of strains can alter bacterial physiology and fitness. To define biologically relevant genes of a genome, genome-wide transposon mutant libraries have been used to identify genes essential for survival or virulence in a given strain. Such phenotypic studies have been conducted in four different genotypes of the human pathogen Streptococcus pyogenes, yet challenges exist in comparing results across studies conducted in different genetic backgrounds and conditions. To advance genotype to phenotype inferences across different S. pyogenes strains, we built a pangenome database of 249 S. pyogenes reference genomes. We systematically re-analyzed publicly available transposon sequencing datasets from S. pyogenes using a transposon sequencing-specific analysis pipeline, Transit. Across four genetic backgrounds and nine phenotypic conditions, 355 genes were essential for survival, corresponding to ~24% of the core genome. Clusters of Orthologous Genes (COG) categories related to coenzyme and lipid transport and growth functions were overrepresented as essential. Finally, essential operons across S. pyogenes genotypes were defined, with an increased number of essential operons detected under in vivo conditions. This study provides an extendible database to which new studies can be added, and a searchable html-based resource to direct future investigations into S. pyogenes biology.IMPORTANCEStreptococcus pyogenes is a human-adapted pathogen occupying restricted ecological niches. Understanding the essentiality of genes across different strains and experimental conditions is important to direct research questions and efforts to prevent the large burden of disease caused by S. pyogenes. To this end we systematically reanalyzed transposon sequencing studies in S. pyogenes using transposon sequencing-specific methods, integrating them into an extendible meta-analysis framework. This provides a repository of gene essentiality in S. pyogenes which was used to highlight specific genes of interest and for the community to guide future phenotypic studies.

Abstract 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.

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen that causes a wide range of diseases from pharyngitis and scarlet fever to more severe, invasive infections such as necrotising fasciitis and streptococcal toxic shock syndrome. There has been a global increase in both scarlet fever and invasive infections during the COVID-19 post-pandemic period. The aim of this study was the molecular characterisation of 17 invasive and non-invasive clinical non-emm1 GAS isolates from an Australian tertiary hospital collected between 2021 and 2022. Whole genome sequencing revealed a total of nine different GAS emm types with the most prevalent being emm22, emm12 and emm3 (each 3/17, 18%). Most isolates (14/17, 82%) carried at least one superantigen gene associated with contemporary scarlet fever outbreaks, and the carriage of these toxin genes was non-emm type specific. Several mutations within key regulatory genes were identified across the different GAS isolates, which may be linked to an increased expression of several virulence factors. This study from a single Australian centre provides a snapshot of non-emm1 GAS clinical isolates that are multiclonal and linked with distinct epidemiological markers commonly observed in high-income settings. These findings highlight the need for continual surveillance to monitor genetic markers that may drive future outbreaks.

Streptococcus pyogenes, or Group A Streptococcus (GAS), is responsible for over 500,000 deaths per year. Approximately 15% of these deaths are caused by necrotizing soft-tissue infections. In 2008, we isolated an M5 GAS, named the LO1 strain, responsible for the nosocomial transmission of necrotizing fasciitis between a baby and a nurse in Belgium. To understand this unusual transmission route, the LO1 strain was sequenced. A comparison of the LO1 genome and transcriptome with the reference M5 Manfredo strain was conducted. We found that the major differences were the presence of an additional DNase and a Tn916-like transposon in the LO1 and other invasive M5 genomes. RNA-seq analysis showed that genes present on the transposon were barely expressed. In contrast, the DNases presented different expression profiles depending on the tested conditions. We generated knock-out mutants in the LO1 background and characterized their virulence phenotype. We also determined their nuclease activity on different substrates. We found that DNases are dispensable for biofilm formation and adhesion to both keratinocytes and pharyngeal cells. Three of these were found to be essential for blood survival; Spd4 and Sdn are implicated in phagocytosis resistance, and Spd1 is responsible for neutrophil extracellular trap (NET) degradation.

ABSTRACTAmong 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.