Streptococcus dysgalactiae Infections in Fish Are Caused by Two Distinct Host-Specific Lineages

Taylor I. Heckman1, Hasan C. Tekedar2, Julio C. García3, Alejandro Perretta4, Diem Thu Nguyen1, Eileen E. Henderson5, Alexander Chow1, Adrián López Porras6, Matt J. Griffin2,  Benjamin R LaFrentz3, Craig Shoemaker3, John Hawke7, Esteban Soto1

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California – Davis, Davis, CA, USA; 2College of Veterinary Medicine, Mississippi State University, MS, USA; 3USDA-ARS, Aquatic Animal Health Research Unit, AL, USA; 4Instituto de Investigaciones Pesqueras, Universidad de la República, Uruguay; 5California Animal Health and Food Safety Lab, School of Veterinary Medicine, University of California, Davis, CA, USA; 6UiO: Biosciences, University of Oslo, Oslo, Norway; 7Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, LA, USA

ABSTRACT

Streptococcus dysgalactiae is an established pathogen of terrestrial mammals and an emerging agent of streptococcosis in fish. The species is traditionally divided into two subspecies based on host specificity and bacterial hemolysis profiles. Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is generally α-hemolytic and associated with disease in ruminants and other animals, while S. dysgalactiae subsp. equisimilis (SDSE) is β-hemolytic and associated with human disease. Previous cases in fish were originally attributed to SDSD on the basis of hemolysis or single gene sequencing, but recent genetic studies have supported a distinct fish clade more closely related to SDSE. To better understand the unique characteristics of fish-associated S. dysgalactiae, we investigated the phylogenetic, phenotypic, and pathogenic characteristics of S. dysgalactiae isolates recovered from outbreaks of streptococcosis in cultured tilapia (Oreochromis spp.) and sturgeon (Acipenser guendelstaedtii and A. baerii) in North and South America. Multilocus sequence analysis and whole genome sequencing were used to determine phylogenetic relationships between these isolates and reference strains from other fish, terrestrial animals and human clinical cases. Phenotypic and virulence characteristics were compared between fish strains by biochemical, antimicrobial, and histological assays, and by challenges in relevant host species. Both the API STREP 20 and Vitek miniaturized kits identified the isolates as SDSE. Whole genome analysis demonstrated that while all recent and historical fish isolates are more closely related to SDSE than to SDSD, they are genetically distinct from either established terrestrial subspecies. The isolates from diseased sturgeon cultured in Uruguay also formed a separate clade to the fish-associated isolates from the USA and Asia. The average nucleotide identity between the two fish clades was approximately 98%, the established cutoff for subspecies delineation, while the fish and closest non-fish SDSE isolates shared between 96-97.5%. There were also relevant differences in phenotype and virulence between isolates from the fish clades. This work supports the existence of two fish-specific lineages, representing at least one novel subspecies of S. dysgalactiae.