BACTERIOSTATIC ACTIVITY  

AGAR DIFFUSION ASSAY  

We investigated if the experimental probiotics could prevent growth of diverse strain AHPND/EMS Vibrio parahaemolyticus originated from different shrimp producing countries. We used 2 variants of agar diffusion (AD) assay, one using inoculation of agar with freshly grown probiotics suspensions (ADS) and another using the transfer of freshly grown probiotic colonies (ADC).  

Probiotics were pre-cultured (at the same concentrations) in tryptic soy broth (TSB) and tryptic soy agar (TSA) for 24h at 27°C. TW01 was pre-cultured in TSB for 18h at 27°C. Samples of TW01 culture suspensions were smeared (plated) over TSA plates. Samples of probiotic culture suspensions were diluted and 6 concentrations inoculated (spotted) on the agar plates containing smears of TW01. Based on the optical density, the following dilutions were plated: C1= pure culture (2x 109 CFU/ml), C2=1×109 CFU/ml,C3=1×108 CFU/ml,C4=1×107 CFU/ml,C5=1×106 CFU/ml,C6=1×105 CFU/ml. Single probiotic colonies were isolated from agar plates and inoculated (spotted) on the same agar plates containing spots of probiotic suspension and smears of TW01. Agar plates were incubated for 24–48 h at 27°C. Pictures of the plates were acquired and measurements taken with a computer software. The antibacterial activity level was defined as the length (mm) of the clear inhibition zone formed around the area where the probiotics were plated/spotted.  

RESULTS AND DISCUSSION  

No technical problems were encountered during the execution of the protocol. The results of the positive control were within the expected range. The standard protocol includes the inoculation of 6 concentrations of culture suspensions and one single solid colony. However, since the experimental products contained a mixture of bacteria, also resulting in different colony types (see Annex), an additional step was performed. Samples of the probiotic suspensions were plated across individual TSA plates and grown overnight. For each product separately, all different types of colonies were collected and mixed. This mixture of colonies was transferred to one spot on an agar plate containing a pathogen smear.  

The agar diffusion test showed inhibition in several combinations of experimental products and pathogens (Table 4-6, Figure 1-4). Next to a clear inhibition zone without pathogen growth, a zone of lower pathogen growth could be observed in some cases (annex 2). The diameter after 24h was measured for both the clear inhibition and lower pathogen growth and reported separately.  

Spots of probiotic suspension  

In general, no inhibition could be observed when probiotic suspensions were diluted further than 1 x 109 CFU/ml (C2) (Table 4 and 5 & Figure 1-3). In suspension, the experimental probiotic mixture TTA Pond did not result in clear inhibition against any of the tested pathogens. However, slight inhibition (reduced bacterial growth) was observed for all pathogens except Vibrio parahaemolyticus TW01. For TTA Gut, clear inhibition was observed in the highest concentration against Vibrio parahaemolyticus MO904 and slight inhibition was seen against 3 out of the 4 pathogens. Slight inhibition could also be observed against the same pathogens when the mixture was tested in the lower (second) concentration, although less pronounced. Probiotic CS606B did not result in any inhibition based on inoculation of the probiotic suspension.  

Colony transfer  

Based on the solid colony transfer (Table 6 & Figure 1 and 4), the experimental probiotic mixture TTA Pond and TTA Gut inhibited the growth of all four pathogens. Clear inhibition whether or not in combination with slight inhibition was observed for all pathogens except Vibrio parahaemolyticus TW01. Against the later, only a small diameter of slight inhibition was noticed. Probiotic CS606B showed a clear inhibition against Vibrio parahaemolyticus TW01. Slight inhibition was noted for all 3 Vibrio parahaemolyticus strains.  

The results indicate that all probiotics can potentially be used to help controlling diseases in shrimp or fish aquaculture. In a next step, testing the in vitro bacteriostatic effect of these probiotics in a disease challenge could give more insight in the possibility of the probiotics to inhibit AHPND/EMS outbreaks in the field. However, the differences in inhibition observed after inoculation of spots of probiotic suspension or colony transfer, suggest that the probiotic culture method (bacteria culture in suspension or on a solid surface) appears to have a distinctive effect on the bacteriostatic/antibacterial characteristics of the probiotic bacteria. This might be important to consider when determining the method of administration of these probiotic mixtures to the shrimp.