Presented by: Elise King Research by: Mona M. Ismail and Waleed S. E. Soliman

1. Studies on Probiotic Effects of Lactic Acid Bacteria Against Vibrio vulnificus in freshwater Prawn Macrobrachium rosenbergii Presented by: Elise King Research by: Mona M. Ismail and Waleed S. E. Soliman

2. Topic • Current research: “Determination of Effective Concentration of Formic Acid on Vibrio spp. • Often affects seafood • Common part of our culture

3. Vibrio vulnificus • Gram-negative, non-enteric, comma shaped rod • Normal flora • opportunistic • Most numerous in shellfish infections • Colonization • Appendages and foregut • Midgut • Affects the hepatopancreas and causes a terminal septicemia

4. Vibrio vulnificus • Treatments • Chemotherapeutic agents • Vaccinations • Probiotics • Natural • Lactic acid bacteria

5. Lactic Acid Bacteria (LAB) • Gram-positive, nonsporulating bacteria that produce lactic acid • Common in some animal gastrointestinal tracts, dairy products, seafood, and plant surfaces • LAB used • Lactobacillus acidophilus • Streptococcus cremoris • Lactobacillus bulgaricus

6. Methods: Getting the Cultures • LAB strains: • Revived on enrichment media • Maintained as slant culture on MRSA • Sub cultured every week to make sure cultures were fresh • LAB strains (pure cultures) • Stored on MRSA at 4oC • Sub cultured every 3 weeks

7. Methods: Getting the Cultures • Vibrio vulnificus • Stored on TSA with 1.0% sodium chloride at 4oC • Sub cultured every week

8. Methods: Bacterial Biomass • Grew LAB strains separately in 10 ml of nutrient broth for 24 hrs. at 28oC • Transferred 5 ml of each strain into 250 ml of MRS broth • Placed tubes on rotary shaker at 150 rpm for 24 hrs. at 28oC

9. Methods: Bacterial Biomass • Centrifuged each strain at 10,000 rpm for 15 min. at 4oC • Used supernatants for the in vitro testing • Separately dispensed pellets in 5 ml of Phosphate Buffer Saline (PBS) • Used for feed

10. Methods: In Vitro • Put 5 ml of nutrient broth into 4 different test tubes • Add 2 ml of 1 strain to LAB supernatant into 3 of the tubes • Inoculate all 4 tubes with V. vulnificus • Incubated at 28oC for 48 hrs. • Measured using O.D. at 540 nm

11. Methods: In Vitro • Streaked all LAB strains on TSA plates with 1.0% sodium chloride for 48 hrs. at 28oC • Streaked freshly grown V. vulnificus culture perpendicular to the growth • Incubated at 28oC • Observed for antagonism

12. Methods: Feed Preparation • Prepared a fresh moist compounded basal diet weekly • control with PBS and oil only • Added oil to each LAB biomass • Added to the diet at 5 x 106 cells*g • Stored at 4oC • Checked viability every 24 hrs.

13. Methods: Feeding • Transported juveniles of Macrobrachium rosenbergii to laboratory • Avg. weight 0.985 + 0.1 g • Avg. length 7.5 + 1 cm • Acclimatized for a week at 28oC • Distributed into treatment groups • Fed 12% of body weight daily (2 doses per day) for 4 weeks

14. Methods: In Vivo • Injected 15 prawns from each of the treatments with 0.1 ml of an aliquot containing 3 x 109 cells*ml of Vibrio vulnificus • Kept them separate from the others, but under the same conditions • Fed with the feed every other day • Observed for a week • Dissected hepatopancreas and tails • Plated serial dilutions on TSA for identification

15. Results: In Vitro

16. Results: In Vivo

17. Results: In Vitro

18. References • Corbin, Angela. 2010. Class notes • Ismail, Mona M., & Soliman, Waleed S. E. (2010). Studies on probiotic effects of lactic acid bacteria against Vibrio vulnificus in freshwater prawn Macrobrachium rosenbergii. Journal of American Science, Retrieved from http://www.jofamericanscience.org/journals/am-sci/am0612/88_3703am0612_781_787.pdf