Identification of two novel bacterial strains as the putative cause of black mouth disease in farmed Strongylocentrotus intermedius.

Black mouth disease (BMD) serves as the principal bacterial affliction of the cultivated sea urchin Strongylocentrotus intermedius, commonly breaking out in spring and winter. In January 2024 (winter), two predominant bacterial strains (SIBMPM04 and SIBMIT01) closely associated with BMD were isolated from the peristomial membrane and intestine of diseased S. intermedius from a farming area in the Yellow Sea of China. Characterization analyses, including Gram staining, morphology determination, artificial infection tests, and metabolic characterization, were then performed on SIBMPM04 and SIBMIT01. Both SIBMPM04 and SIBMIT01 belonged to the genus Vibrio, and 16S rRNA identification and phylogenetic analysis showed that SIBMPM04 shared a 98.68 % similarity with Vibrio bathopelagicus and that SIBMIT01 shared a 98.93 % similarity with Vibrio atypicus. Antimicrobial susceptibility tests indicated that both SIBMPM04 and SIBMIT01 were sensitive to chloramphenicol, florfenicol, imipenem, kanamycin, levofloxacin and polymyxin B. SIBMPM04 was sensitive to gentamycin and amikacin, and SIBMIT01 was sensitive to norfloxacin and sulfamethoxazole. Metabolic characterization data showed that SIBMPM04 and SIBMIT01 shared similar metabolic characteristics. Laboratory-based artificial infection tests showed that sea urchins infected with either SIBMPM04 or SIBMIT01 alone exhibited typical pathological symptoms of BMD in a dosage-dependent manner, and synergistic pathogenicity was observed when both bacterial strains were present. The observations from this study not only enrich information on these pathogens in sea urchins but also support the hypothesis that BMD may be induced by the synergistic action of multiple bacterial strains in sea urchins, leading to the rapid progression of disease symptoms.