Physiological metabolism, genetic characteristics, and bacteriostatic activity diversity of 37 Bacillus subtilis from mariculture system

Abstract:

[Background] Diseases caused by aquatic pathogens continue to break out, and finding safe and effective alternatives to antibiotics is an urgent need. Probiotics are often influenced by interests and profit-making, whereas not enough attention has been paid to their safety evaluation. [Objective] To find green and safe probiotics with multiple bacteriostatic activities based on the phenotypic and genetic characteristics of Bacillus subtilis in China’s mariculture system. [Methods] Taking 37 B. subtilis isolated from China’s mariculture system from 2009 to 2021 as the research object, this study used K-B method to detect the resistance of B. subtilis to different antibiotics. The amylase, protease, and hemolytic capacity of B. subtilis were determined by medium plate method. Polymerase chain reaction (PCR) was used to detect the risk of carrying genes related to hemolysis in B. subtilis. The Oxford cup method was used to determine its bacteriostatic effects on six pathogens, including Vibrio parahaemolyticus, Vibrio algaelyticus, Edwardsiella tarda, Vibrio harveyi, Pseudoalteromonas sp., and Photobacterium damselae. The safety of candidate probiotic B. subtilis was evaluated. [Results] The results of drug susceptibility test showed that 37 isolates of B. subtilis showed strong resistance to trimethoprim, pipemidic acid, and streptomycin, moderate resistance to sulfadiazine, low resistance to cefotaxime, ciprofloxacin, and sulbactam, and complete sensitivity to clarithromycin, norfloxacin, florfenicol, flumequine, cotrimoxazole, and tetracycline. The test results of protease and amylase activity showed that 37 isolates of B. subtilis hydrolyzed casein and starch to varying degrees. The hemolytic test results showed that 4/37 isolates of B. subtilis had hemolytic phenomenon, while 8 hemolysis-related genes were detected in 37 isolates of B. subtilis. The analysis of the correlation between hemolytic phenotype and detection gene showed that there was no direct correlation between the strain that produced hemolysis and its hemolytic gene carrier. The analysis of bacteriostatic experiments showed that all isolates of B. subtilis had inhibitory effect on 2 or more pathogenic bacteria, and 2 isolates (strains Bs4 and Bs7) had good bacteriostatic effects on 6 pathogenic bacteria. Safety experiments on Litopenarus vanmamei showed that strain Bs4 had high safety against L. vanmamei, and the survival rate of 7 d was 100%. [Conclusion] Through the comparative analysis of the physiological metabolic phenotype, genetic characteristics, and pathogen antagonism characteristics of B. subtilis isolates, it is revealed that B. subtilis has diversified phenotypes and genetic characteristics in China’s mariculture system. An ecologically safe and probiotic B. subtilis with multiple bacteriostatic activities is screened out, which provides a theoretical basis and technical support for the prevention and control of aquaculture diseases, the development of bacteriostatic microecological preparations, and the healthy and green development of aquaculture industry.

Key words:Bacillus subtilis;antibiotic susceptibility;hemolytic;enzyme-producing capacity;bacteriostasis;ecological security