Balami

Genomic Analysis Identifies a Discrete Fish-associated Lineage of Streptococcus dysgalactiae subsp. dysgalactiae

1Sujita Balami, 1Lester Khoo, 2Caitlin Older, 1Cynthia C. Ware, 2Geoffrey C. Waldbieser,

3John Hawke, 5Adrián López-Porras, 4Taylor Heckman, 4Esteban Soto, 6Craig Shoemaker,

7Al Camus and 1Matt J. Griffin

1Thad Cochran National Warmwater Aquaculture Center, Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS; 2Thad Cochran National Warmwater Aquaculture Center, Warmwater Aquaculture Research Unit, Agricultural Research Service, United States Department of Agriculture, Stoneville, MS; 3Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA; 4Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, CA; 5Department of Biosciences, University of Oslo, Oslo, Norway; 6Aquatic Animal Health Research Unit, Agricultural Research Service, United States Department of Agriculture, Auburn, AL; 7Department of Pathology, College of Veterinary Medicine, University of Georgia

ABSTRACT

Lancefield group C Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been associated with fish kills in various cultured fish species. It was first isolated from diseased farmed amberjack (Seriola dumerili) and yellowtail (Seriola quinqueradiata) in Japan. Later, it was reported from marine fish in Taiwan, Malaysia and China. In 2021, the Louisiana Aquatic Diagnostic Laboratory isolated SDSD from a fish kill event of feral silver carp in the Mississippi River and SDSD has been reported from outbreaks in other freshwater fish in the US, namely tilapia. In 2022, a 10-pound channel catfish broodfish was submitted to the Aquatic Diagnostic Laboratory of the Thad Cochran National Warmwater Aquaculture Center in Stoneville, MS. The fish had a red vent and a swollen abdomen, which was distended with straw colored ascitic fluid. Isolation cultures from brain and kidney on Mueller-Hinton blood agar produced pure cultures of small, white non-hemolytic colonies of gram-positive bacteria. The isolates were identified by 16S rRNA sequencing as S. dysgalactiae, which warranted further investigation. A recently developed multi-locus sequence analysis (MLSA) scheme confirmed the bacteria as Streptococcus dysgalactiae, grouping with the aforementioned SDSD from fish in Asia. Additional isolates were obtained from collaborators at the USDA-Aquatic Animal Health Research Unit at Auburn, Alabama and the Louisiana Aquatic Diagnostic Laboratory at Louisiana State University in Baton Rouge, Louisiana. Complete genomes were obtained using a combination of short read (Illumina) and long read (Nanopore) technology. Phylogenetic comparisons of fish isolates to other S. dysgalactiae isolates obtained from NCBI’s GenBank revealed the fish-associated isolates form a discrete phyletic group, sharing 99-100% identity across MLSA targets; and are genetically distinct from animal and human isolates. Further, comparisons of the sodA gene reveal fish isolates from the US share high homology (99.8-100% homology; 435 bp) to the SDSD isolates from Japan, Taiwan, China, Malaysia and India; while sharing < 99.5% homology to terrestrial isolates from other geographic origins. While divergent from terrestrial SDSD isolates from animals and humans, average nucleotide identity (ANI) and digital DNA-DNA Hybridization (dDDH) estimations indicate the fish isolates are largely clonal (ANI > 99%; dDDH > 95%); but do not warrant their own subspecies designation. Interestingly, initial comparative assessments indicate multiple areas of divergence between the catfish isolate and other fish isolates from the US and Asia, which is an ongoing avenue of investigation. Herein, we report the first incidence SDSD in moribund channel catfish. While SDSD was previously thought to be limited to marine fish, isolation of these bacteria from freshwater systems indicates SDSD is a potential threat to US freshwater aquaculture, including farm-raised catfish.