EFFICACY OF PROBIOTIC Bacillus amyloliquefaciens CECT 5940 AGAINST EMS/AHPND Vibrio parahaemolyticus  

Alvaro Ortiz *, Pilar Honrubia, João Dantas Lima, Juan J. Mallo
NOREL S.A.
Madrid, Spain
aortiz@norel.net

A strain of Vibrio parahaemolyticus (TW01) isolated from shrimp infected with early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) capable of reproduce the disease was used to determine the efficacy of four experimental products against this pathogen. The products consisting in Bacillus amyloliquefaciens CECT 5940 (ECOBIOL®) culture supernatants were supplied by Norel S.A. with the following codification: AMY/9-13 F2 α, AMY/9-13 F2 β, AMY/1-15 F2 γ and AMY/1-15 F2 δ.

The bacteriostatic activity of the experimental products towards the experimental bacteria was determined using a minimum inhibitor concentration (MIC) protocol established at IMAQUA. Each experimental product was serially diluted in 2-fold steps in 96-well plates containing nutrient broth. Necessary controls were enclosed. For controlling the quality of the test, a serial dilution containing a standard antimicrobial (gentamycin) was inoculated with a susceptible bacterium (V. campbellii LMG21363). Except for the control wells containing pure medium (contamination control), all wells were inoculated with bacteria at a certain concentration. After, the plates were incubated at 27°C on a shaker at 90 rpm. After 12 hours, the optical density (bacterial growth) was measured using a microplate reader. The values of absorbance were used to determine the MIC for each experimental product, which is a direct measure for the level of antibacterial activity. The turbidity in the wells (bacterial growth) was also scored visually.

Summary of the MIC values obtained for each experimental product against each of the test pathogens is shown in the table below.

All the products were effective against the pathogen. Among them, AMY/1-15 F2 δ induced the highest antibacterial activity towards both tested pathogens. This supernatant could inhibit the growth of V. campbellii and V. parahaemolyticus (TW01) at a dilution of 1:16 and 1:32, respectively. This level of antibacterial activity is considered to be moderately high.

It may be concluded that the probiotic Bacillus amyloliquefaciens CECT 5940 presents a good potential for controlling infections of V. parahaemolyticus TW01 and, consequently, the mortality of shrimp due to AHPND.