Inhibitory Activity against Shrimp Pathogens of Bacterial Isolates from Shrimp Food, Shrimp Gut, Seawater and Seaweed  

Gurusamy Chelladurai1 , Mani Karthigarani2 , Iruthaiyam Vijayakumar3
1. Department of Zoology, Kamaraj College, Tuticorin, Tamilnadu, India
2. Department of Zoology, Yadava College, Madurai, Tamilnadu, India
3. Department of Zoology, SN College, Madurai, Tamilnadu, India
Author    Correspondence author
International Journal of Marine Science, 2015, Vol. 5, No. 37   doi: 10.5376/ijms.2015.05.0037
Received: 05 Apr., 2015    Accepted: 16 May, 2015    Published: 10 Jun., 2015
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Chelladurai, Karthigarani amd Vijayakumar, 2015, Inhibitory activity against shrimp pathogens of bacterial Isolates from shrimp food, shrimp gut, seawater and seaweed, International Journal of Marine Science, Vol.5, No.37 1-6 (doi: 10.5376/ijms.2015.05.0037)


The present study was developed in-vitro, to assess the inhibitory activityagainst shrimp pathogens of bacterial Isolates from shrimp food, shrimp gut, seawater and seaweed. Two types of strain of commercial probiotics were isolated from aquaculture feeds named as A and B. The identified strains were Bacillus subtilis, Lactobacillus plantarum, Pseudomonas fluorescens and Staphylococcus lactis. Both commercial and isolated probiotic organisms were identified and screened against shrimp pathogens like Vibrio parahaemolyticus, Aeromonas hydrophila, Vibrio cholerae and Vibrio harveyi by agar well diffusion method. Among eight organisms in the commercial probiotics vial A and B, only four organisms showed highest zone of inhibition against shrimp pathogens. The four isolated probiotic organisms showed highest zone of inhibition against shrimp pathogens. The measurement of zone of inhibition of isolated probiotics was greater than the inhibition zone of commercial probiotics. The isolated probiotic organisms were analyzed in survival of high pH. The results obtained were shown that all the isolated organisms were survived up to pH 14. The formulated probiotics is better for shrimp aquaculture practices than the commercial probiotics

Antibacterial activity; Biochemical test; Probiotics; pH; Shrimp

Aquaculture is one of the fastest growing food producing sectors of the world. The probiotics are harmless bacteria that promote the well beings of a host animal and contribute to the direct or indirect protection of the animals against harmful bacteria (Decamp et al., 2008). The positive results of applying certain beneficial bacteria in aquacultures are well documented and treatments with probiotics have also rapidly increased (Farzamfar, 2006). Probiotics, which act as biofriendly agents, such as lactic acid bacteria of Bacillus sp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms (Alifarzanfar, 2006). In aquaculture, antibiotics are discharged into the environment causing the occurrence of resistant bacteria on fish farms (Aoki, 1992). The control of fish and shellfish pathogenic Vibrio sp. particularly by using non-pathogenic bacterial strains and disease prevention has received much attention during the last decade (Sugita, 1998; Rengpipat et al., 1998). The bacteria isolated from the adult common clownfish have the potential of colonizing intestinal mucus and consequently may serve as prophylactic and therapeutic agents. These findings will be used to further refine a protocol for the selection of probiotics in marine fish larviculture (Vine et al., 2004). They investigated the effects of ozone with and without feeds supplemented with the probiotic Bacillus sp. on bacterial (V. harveyi) growth and shrimp (P.monodon) survival (Vaseeharan and Ramasamy, 2004)..The microbial strains such as Lactobacillus sp, V. alginolyticus, P. fluorescens and B. Subtilis are used as biological control agents in aquaculture. They are non pathogenic, non toxic and can survive in the gut and remain stable and viable for long periods under storage and field conditions. In shrimp culture, application of probiotics improve the intestinal microbial balance, leading to better growth by improving food absorption and digestive enzyme activities (Surajit et al., 2006). The present study aims specifically on the investigation of the comparison of inhibitory activity between commercial and isolated probiotics against shrimp pathogens.

1 Materials and Methods
1.1 Collection of Samples
The samples were obtained from seaweed, seawater and gut region of shrimp. Seaweed was collected in a sterile polythene bag and seawater in a sterile flask from seashore of Tuticorin coastal water. The gut region of fresh shrimp was aseptically transferred into sterile screw cap bottle containing 1% peptone broth. The commercial feeds enriched with probiotics A and B were purchased from Shrimp farm industry of Tuticorin Tamilnadu, India.
1.2 Isolation of Probiotic Bacteria from Collected Samples
The Marine Zoobell Agar (MZA) medium was employed for the enumeration of total bacterial population in aseptically collected samples. 1ml of aliquots of 10-3 to 10-7 dilution was serially diluted using sterile distilled water. Approximately 20ml of sterilized MZA medium was poured onto sterile petri plates and was allowed to solidify. After solidification of agar 0.1ml of samples was spread on the petri plates using L-rod. The plates were incubated at 370C for 24-48hours. After 48 hours, the petri plates with 30-300 bacterial population were observed and the number of colonies were counted and tabulated. Representative colony of each isolate was purified, identified and used for further analysis.
1.3Isolation of Probiotic Bacteria from Commercial Shrimp Feed
The two commercial feeds (10g) were taken and homogenized with 90 ml of distilled water; the serial decimal dilution was carried out. The diluted suspensions were plated simultaneously in to Nutrient agar (NA) and MRS lactobacillus agar using plate technique, to enumerate the total bacteria and total lactic bacteria, respectively (APHA, 1976). The nutrient agar plates were incubated at 370C for 48hours and the MRS agar plates at 30+ 20C for 48 hours. The number of colonies developed on the plates were counted and expressed as CFU/g. Representative colonies of bacteria from each feed (A-4, B-4) were picked up from NA as well as MRS agar plates and again purified on plates. The purified colonies were identified by standard biochemical tests described in Bergey’s Manual of Bacteriology (Gibson et al., 1998).
1.4Morphological Identification of Probiotics
The isolated and commercial probiotics were streaked on MZA and nutrient agar plates to purify and incubated at 370C for 24 hours. Based on the morphological appearance of isolates on slant they were confirmed and the results were noted and tabulated.
1.5 Biochemical Identification of Probiotics
The commercial and isolated bacterial colonies were identified on the basis of their morphological, physiological and biochemical character (Berg, 1996). The bacterial isolates on agar slants were sub cultured in the nutrient broth. These cultures were subjected to various biochemical tests such as gram staining, spore staining, motility, indole, methyl red, voges proskauer, citrate utilization, catalase, oxidase, casein hydrolysis and starch hydrolysis tests.
1.6 Antagonistic activity of Formulated and Commercial Probiotics
The initial screening of antagonism activity by both commercial probiotics and isolated probiotics were done by the agar well diffusion plate assay method (Rajaganapathi, 1996). The V. parahaemolyticus, V. cholerae, A. hydrophila were pre cultured in nutrient agar brothincubated at 280C for 2 days and 50μl of this culture were spread over the agar plates. The probiotics culture (3days old) was centrifuged at 9600 rpm min-1 for 15 minutes and the supernatant fluid was filtered through a membrane filter to obtain cell-free extracts (Culture supernatant fluid). A small volume (100μL) of cell-free extracts was filled in 7mm diameter wells cut in the agar medium. The positive control well containing 50 µL of Tetracycline (1mg/mL) were used. The diameters of the zone of inhibition were measured after 24 to 48 hrs at 280C of incubation.
1.7 Survival of Formulated Probiotics at Different pH level
The isolated probiotics from different sources were also tested for their survival in different alkaline pH systems by inoculating them individually on to the Nutrient broth with pH ranging from 7.0 to 14.0. The tubes were incubated at 370C for 48hours and checked for the turbidity, to record positive growth
1.8Statistical analysis
Data on the inhibitory effects of commercial and isolated probiotics was analyzed by One-way analysis of variance (ANOVA) using SPSS-16 version software followed by Duncun’s multiple range test and standard deviation. P<0.05 were considered for describing the significant levels.
2 Results
2.1 Antibacterial activity of Commercial Probiotics
The present study was done by in vitro comparison of inhibitory activity against shrimp pathogens between commercial and isolated probiotics. The vial A and B contains organisms like L .plantarum , P. fluorescens, B. subtillus and S. lactis (Table 1, 2, 3).The probiotics present in the vial A and B were screened against the shrimp pathogens like V. Parahaemolyticus, V. cholerae, A. hydrophila and V. harveyi. Among the eight organism L. plantarum and B. subtillus from vial A showed significantly highest inhibition activity (p<0.05) against V. parahaemolyticus and V. cholera and S. lactis and B. subtilis from vial Bshowed highest inhibitory activity against A. hydrophila and V. cholerae. (Table 4).

Table 1 Identification of isolated probioctic

Table 2 Identification of commercial probiotics

Table 3 Antagonistic activity of commercial probiotics

Table 4 Antagonistic activity of Isolated Probiotics

2.2 Antibacterial activity of Isolated Probiotics
The isolated probiotic organisms of B. subtillus, P. fluorescens, L. Plantarum and S. lactis were screened against shrimp pathogens. The maximum inhibition activity was recorded (25mm) against V. parahaemolyticus and minimum was recorded (4mm) against V. cholera (Table 5).

Table 5 Survival of isolated probiotics in different pH Level

2.3 Comparative study between Commercial Probiotics and Formulated Probiotics
Among the commercial probiotics of A and B, the type B probiotic showed maximum zone of inhibition (15 ±1.13mm to 2±1.18 mm). The isolated probiotics of L. plantarum showed maximum zone of inhibition against V. parahaemolyticus. The inhibitory compounds produced by B. subtillus, P. fluorescens, L. plantarum and S. lactis was significantly more effective (P<0.05) than the organisms present in commercial probiotics. The formulated probiotics were also tested for their survival in different pH (7-14). The results obtained were shown that all the isolated organisms were survived up to pH 14. Hence the formulated probiotics were more effective than commercial probiotics.
3 Discussion
The present study showed that the growth of pathogenic V. parahaemolytics, A. hydrophila, V. cholerae and V. harveyi were controlled by non pathogenic B. Subtilis, L. Plantarum, S. lactis and P. Fluorescens under in-vitro conditions. An “in- vitro” assay is not a sufficient support to establish if an isolated strain is a “probiotic” species. Nevertheless, you can speculate that it could be a “potential” probiotic strain or species). The microbiological studies have demonstrated the beneficial effect of Bacillus sp, which have the potential application for controlling Vibrio sp. in shrimp aquaculture (Balcazar and Luna rojas, 2007). The Previous microbiological studies 172. have demonstrated the beneficial effect of Bacillus sp, which have the potential application for controlling Vibrio sp. in shrimp aquaculture (Balcazar and Luna rojas, 2007). In the previous studies inoculation of Bacillus sp inhibitory effect (In-vitro) against V. parahaemolyticus and V. harveyi resulted in greater survival of P. monodon challenged with Aquatic pathogens in-vitro study cannot support in-vivo results). The control of shrimp pathogens predominantly by using non-pathogenic bacterial strains and disease prevention has received more attention during the last decade (Rangpipat et al., 1988). The probiotic as a live microbial feed supplement which benefits the host animal by improving its intestinal microbial balance (Fuller, 1989). Similarly the protective effect of probiotic diet on heamotobiochemical and histopathological changes of cat fish was observed against A. hydrophila (Gurusamy Chelladurai et al., 2013). The present study showed that the non pathogenic antagonists showed effects significantly higher (P<0.05) than the pathogen and the degree of inhibition increased with the level of antagonists. The V. alginolyticus, used as a probiotic strain which reduced the diseases caused by A.solmonicida, V.anguillarum and V.ordalli in P. monodon (Austin et al., 2005). The use of a soil bacterial strain, PM-4, that promoted the growth of P.monodon most likely acting as a food source (Maeda and Liao, 1995). The inoculation of Bacillus sp strain, resulted in greater survival of the post-larval P.monodon that were challenged by pathogenic luminescent bacterial culture (Rangpipat et al., 1998).The antagonistic effect of B. subtilis BT23 against the pathogenic Vibrios sp in P.monodon results in 90% reductionin accumulated mortality. The present study recommends the effective control of pathogenic microflora in shrimp culture environments by probiotic bacteria (Vaseeharan and Ramasamy, 2004). Purification and characterization of the antibacterial substances would help to understand the mechanism of antibacterial activity of strains like B. subtilis, L. plantarum, S. lactis and P. fluroscens. V. alginolyticus act as a probiotic strain to reduced the diseases caused by A. solmonicida, V. anguillarum and V. ordalliin in P. Monodon (Austin et al., 1995). The isolated probiotic bacteria OY15 from oysters and scallops which inhibited the growth of known scallop- pathogen bacterial strains B183 and B122 and found that it can also be incorporated into functional foods for use in shellfish hatcheries which will significantly improve larval survival (Lim et al. 2001). L. acidophilus are generally could not survive in low pH environment, as these cells were proven to be vulnerable at pH 2.0 and below. Low pH environments are thought to inhibit the metabolic activity and growth of L. acidophilus, thus reducing the probiotics’ viability (Sultana et al., 2000). The result of the present study of formulated probiotics was survival in pH 7 to14. (Mandal et al., 2006) also confirmed that the viability count of the bacteria declined tremendously when exposed to simulated gastric juice of pH 1.5 after an incubation period of 3 hours. The compositions of formulated probiotics aresomewhat different from that of commercial probiotics. The organisms involved in formulated probiotics showed more inhibitory activity than commercial probiotics and the measurement of zone of inhibition is also greater. Hence we conclude that the formulated probiotics is better for aquaculture practise than the commercial probiotics. In future study the isolated and commercial probiotics were mixed up with shrimp diet separately and study the effect of growth and immuno biochemical changes.
The authors thankful to Director and Principal of Kamaraj College, Tuticorin, Tamilnadu for Supportive and encouraged during the study period.
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