Investigating important but overlooked streptococcal agents of disease in production animals.

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The overarching goal of my research program is to advance knowledge to enable better prevention and control of infectious diseases. The strain diversity, the pathogenesis of infection, and the antimicrobial resistance (AMR) of the animal pathogens Streptocococcus dysgalactiae subsp. equisimilis (SDE, an agent of septicemia and arthritis in swine) and S. dysgalactiae subsp. dysgalactiae (SDD, an agent of bovine mastitis) are poorly known. Based on differences in hemolytic activity and Lancefield antigen grouping, it can be hypothesized that SDE and SDD isolates are genetically diverse, that their virulence arsenals vary in both content and regulation, and that there are subspecies differences in terms of AMR gene content. Here, I propose to: Aim 1) investigate the diversity and phylogenetic relationships of animal SDE and SDD isolates, Aim 2) characterize several key SDE and SDD virulence factors and regulators using host-relevant infection models, and Aim 3) elucidate the AMR repertoire of both subspecies. In Aim 1, genomics of population-based isolates will permit a thorough understanding of strain diversity in both subspecies. Overlying of clinical metadata to phylogenies sharing similar gene content and/or constellation of mutations will allow for improved virulence prediction. Aim 2 will study virulence factors and regulators. Virulence factors (i.e., M protein, streptokinase, the serine protease CspA, superantigens) will be mutated and characterized using in vitro infection models (i.e., interactions with epithelial cells, uptake and killing by phagocytic cells, cellular proliferation) and mouse infection models mimicking porcine sepsis and bovine mastitis. Regulators such as two component systems will be studied using the same methods in combination with transcriptomics. Natural gene variation data from Aim1 will be leveraged to select further strains to be tested, thus enabling a diversity-driven, comprehensive understanding of the pathogenesis of infection of both subspecies. In Aim 3, further mining of the genomics data supplemented by phenotypic susceptibility data will determine the SDE and SDD AMR repertoire. Insights into acquisition and mobilization of AMR genes, and the potential of SDE and SDD to act as AMR reservoirs will be gained by determination of mobile genetic elements (MGEs) carrying resistance genes, and comparisons to bacterial species sharing colonization niches with SDE and SDD, as well as by MGE excision tests and conjugation assays. This proposal will thus advance knowledge on two veterinary pathogens by 1) unveiling the SDE and SDD population structure, 2) characterizing in detail the mode of action and regulation of virulence factors, and 3) generating a comprehensive portrait of their AMR. The data will facilitate infection control efforts in animal production systems, will help reduce antimicrobial usage in veterinary settings and, ultimately, will improve animal well-being while decreasing economic losses.