PROBIOTIC EFFECTS OF A NEW BACILLUS STRAIN Iryna Sorokulova, Ludmila Globa, Oleg Pustovyy, Vitaly Vodyanoy Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849 INTRODUCTION Bacillus bacteria as probiotics attract a growing attention of researchers as more data indicate positive effects of bacilli on the host organism. These bacteria are the important component of the normal microbiota of animals and humans. The bacilli are resistant to acid and bile and keep viability in the gut. Under strict anaerobic conditions bacilli can use nitrate as an electron acceptor and grow anaerobically. Bacillus bacteria could play a significant role in the gut because of their high metabolic activity. They are known as producers of about 800 antibiotics that differ in the structure and spectrum of activity. Bacillus spp. produce various enzymes, including digestive and lytic enzymes, essential amino acids and vitamins. Efficacy of B. subtilis BSB3 in prevention of heat stress adverse effects. - Histological analysis of the gut. Exposure of rats to heat stress conditions resulted in changes of the gut morphology: significant reduction of villi height and total mucosal thickness (Fig.3). Pre-treatment of rats with B. subtilis BSB3 strain before heat stress prevented animals from these traumatic effects of heat. - Serum LPS concentration. Level of LPS significantly increased in serum of heat-stressed animals, which received PBS before stress exposure (Fig 5). Concentration of LPS in serum of rats, pre-treated with probiotic before heat stress, was not significantly changed in comparison with non-stressed animals. - Tight junction proteins expression. Pre-treatment of rats with probiotic resulted in significant increase of claudin expression in heat-stressed animals (Fig. 6). ZO-1 expression was also higher in stressed animals, pretreated with probiotic (Fig. 7). Figure 4. Prevention of bacterial translo-cation by B.subtilis BSB3 OBJECTIVE The main aim of this study was to evaluate probiotic activity of a newly isolated B. subtilis strain. - Prevention of bacterial translocation from the gut. All samples were sterile in control animals and in heat-stressed animals, pretreated with B. subtilis BSB3 strain. Bacterial culture were isolated from MLN and liver of heat-stressed animals (Fig.4). METHODS Isolated strain was identified by morphological, biochemical and 16S rDNA sequence analysis. The activity of Bacillus strain against pathogens was studied by the method of delayed antagonism in the solid nutrient medium. Production of biosurfactants was evaluated by standard methods. For an analysis of the protective effect of Bacillus strain during heat stress, male Sprague–Dawley rats weighing 250–300 g were used. Animals were treated by oral gavage with Bacillus strain or PBS twice a day for two days. Half of the rats of each group were exposed to heat stress (45o C, relative humidity 55% for 25 min) while the remaining rats were placed at room temperature. Bacterial translocation, histological changes in the intestine, serum lipopolysaccharide level (LPS), as well as tight junction proteins (TJ) expression were analyzed and compared between groups. Figure 3. Protective effect of B. subtilis BSB3 on gut epithelial cells Figure 5. Serum LPS concentration in animals of different groups RESULTS Identification of Bacillus strain. The microscopic study of bacterial culture showed this strain to be Gram-positive rod, less than 1µm in diameter, sporulated aerobically without swelling of the cell and produced catalase. These data indicated that tested strain belongs to Bacillus genus. Additional testing with API 50CHB kit resulted in identification of the culture as B. subtilis. Partial sequence of 16S rRNA gene confirmed the obtained results of biochemical identification. Antagonistic activity. B. subtilis BSB3 strains was highly effective against tested strains of Salmonella, Shigella and Staphylococcus (Fig.1). . Antibacterial activity of biosurfactant. B. subtilis BSB3 strain was found to produce biosurfactant with high activity against S. aureus strains, including methicillin-resistant cultures (Fig.2). Biosurfactants, isolated from other strains of B.subtilis, used as a control, did not affect all tested strains of S. aureus. Figure 6. Claudin expression in the intestine of rats Figure 7. ZO-1 expression in the intestine of rats Figure 2. Antibacterial activity of biosur-factants, isolated from Bacillus strains Figure1. Antagonistic activity of B. subtilis BSB3 CONCLUSION New B. subtilis BSB3 strain demonstrated in vitro high antagonistic activity against pathogens, including resistant to antibiotics. This strain protected animals against heat stress- related complications: alteration of gut morphology , translocation of gut bacteria into circulation, elevation of serum LPS , decrease of TJ proteins expression. ACKNOWLEDGEMENT: This work was supported by Auburn University Funds #10100021392942050