Supplementary Materials to Beneficial Microbes: Safety assessment of Rhodobacter azotoformans SY5 for potential application in Chinese mitten crab Eriocheir sinensis
Rhodobacter species are promising beneficial microbes that can improve growth performance, immunity and antioxidant capability in aquatic crustaceans. Yet the safety of Rhodobacter azotoformans for potential application in Chinese mitten crab Eriocheir sinensis is still unclear. In the present study, R. azotoformans SY5, a potential probiotic additive that can significantly improve the growth performance, immunity, antioxidant capability, and disease resistance in E. sinensis, was evaluated for safety through whole genome sequencing, antibiotic resistance, toxic metabolites, virulence, and crab tolerance assays. The results indicated that R. azotoformans SY5 only harboured the acyl carrier protein-encoding gene (acpP) that was universal in probiotic bacteria with the function of bacterial fatty acid biosynthesis, exhibited high susceptibility to aminoglycosides, penicillins, polymyxins, polyphosphates, quinolones, and tetracyclines antimicrobials, and possessed inability to produce hemolysin, hydrogen sulphide, nitrite, ammonia, and phenylpyruvate. In addition, R. azotoformans SY5 showed no pathogenicity for E. sinensis with the seven-day acute intraperitoneal LD50 value of above 6.0×109 cfu/ml and 30-day chronic oral LD50 of above 6.0×109 cfu/g diet. To our knowledge, this is the first report on the safety of R. azotoformans for potential application in Chinese mitten crabs.