Genomic identification and characterization of co-occurring Harveyi Glade species following a vibriosis outbreak in Pacific white shrimp, Penaeus (litopenaeus) vannamei

Shrimp farming is one of the fastest-growing food production sectors but disease related mortality remains a limitation to industry expansion. Bacterial species belonging to the Harveyi Glade, such as Vibrio harveyi and Vibrio parahaemolyticus, have long been implicated as agents of disease in shrimp aquaculture but traditional methods cannot accurately differentiate these species. Following a vibriosis outbreak in Pacific white shrimp, Penaeus (litopenaeus) vannamei, we hypothesized that infection promoted the co-occurrence of multiple Harveyi Glade species. The shrimp were reared in a zero-exchange raceway using biofloc technology and presumptive Vibrio species were isolated from the hepatopancreases of moribund shrimp by culture on a Vibrio-selective agar. Isolates were identified and characterized by PCR-based genotyping and whole-genome sequencing. A phylogenetic tree based on a 43-genome pangenome analysis revealed the presence of five known shrimp pathogens: Photobacterium damselae, Vibrio alginolyticus, V. harveyi, V. parahaemolyticus, Vibrio rotiferianus, and a potentially novel Vibrio species (Vibrio sp. Hep-1b-8). Multilocus sequence typing revealed that P. damselae and V. parahaemolyticus isolates exhibited novel allelic profiles, although the V. parahaemolyticus isolates were closely related to strains isolated from Southeastern Asia. Further, a novel comparative analysis of 23 type VI secretion systems revealed three phylogenetically distinct systems, which suggests that co-occurring bacteria possess diverse mechanisms for interspecies competition. These results highlight whole-genome sequencing as an invaluable tool for identifying and characterizing co-occurring, infection-associated bacteria at the species, strain and molecular level.