Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon

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1 Letters in Applied Microbiology 23, 36, Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon B. Vaseeharan and P. Ramasamy Department of Biotechnology, Life Sciences Building, University of Madras, Guindy Campus, Chennai, India 22 17: received 6 June 22, revised 22 October 22 and accepted 1 November 22 ABSTRACT B. V A S E E H A R A N A N D P. R A M A S A M Y. 23. Aims: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic vibrios. Methods and Results: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease. The probiotic effect of Bacillus was tested by exposing shrimp to B. subtilis BT23 at a density of 1 6 )1 8 cfu ml )1 for 6 d before a challenge with V. harveyi at 1 3 )1 4 cfu ml )1 for 1 h infection. The combined results of long- and shortterm probiotic treatment of B. subtilis BT23 showed a 9% reduction in accumulated mortality. Conclusions: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo conditions. Significance and Impact of the Study: Results indicated that probiotic treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture. Keywords: Bacillus, P. monodon, probiotics, shrimp disease, Vibrio. INTRODUCTION Aquaculture is the world s fastest growing food production sector, with cultured shrimp and prawn growing at an annual rate of 16Æ8% between 1984 and 1995 (Subasinghe et al. 1998). However, disease outbreaks have caused serious economic losses in several countries. According to a recent World Bank report, global losses due to shrimp diseases are around US$ 3 billion (Lundin 1996). Vibrio species occur as the dominant flora in various developmental stages of Penaeus monodon and have been described as the causal pathogens (Lightner 1996; Sung et al. 21). Potential negative consequences of using antibiotics in aquaculture for the prophylactic treatment of diseases are the development of drugresistant bacteria and reduced efficacy of antibiotic treatment for human and animal diseases (Moriarty 1997). Increased Correspondence to: Dr B. Vaseeharan, Genetics and Biotechnology Division, Central Institute of Brackish Water Aquaculture, 75 Santhome High Road, R.A. Puram, Chennai 6 28, India ( bvaseeharan@yahoo.co.in). concern about antibiotic-resistant micro-organisms (Amabile et al. 1995) has led to suggestions of alternative disease prevention methods, including the use of non-pathogenic bacteria as probiotic biocontrol agents. (Austin et al. 1995; Moriarty 1997). Lactic acid bacteria have been tested as probiotics in warm-blooded animals and attempts have been made to use lactic acid bacteria as antagonists (probiotics) of shrimp pathogens (Gatesoupe 1999; Skjermo and Vadstein 1999). Bacillus spores have been used as biocontrol agents to reduce vibrios in shrimp culture facilities (Skjermo and Vadstein 1999; Rengipipat et al. 2). Bacillus constitutes a large part of the microflora of the gills, skin and intestinal tracts of shrimps (Sharmila et al. 1996). Bacillus spp. are often antagonistic against other micro-organisms, including fish and shellfish pathogenic bacteria (Gatesoupe 1999; Rengipipat et al. 2). The present study investigated the inhibitory activity of Bacillus subtilis BT23, isolated from shrimp culture ponds, against pathogenic Vibrio harveyi under in vitro and in vivo conditions. ª 23 The Society for Applied Microbiology

2 84 B. VASEEHARAN AND P. RAMASAMY MATERIALS AND METHODS Bacterial strains A virulent strain of V. harveyi, obtained from P. monodon with black gill disease (LD 5 value 1 5 cfu ml )1 under experimental infection of P. monodon juveniles), was used as a pathogenic strain and B. subtilis BT23, obtained from shrimp culture ponds, was used as an antagonistic strain. Bacillus BT21, Bacillus BT22 and B. subtilis BT23 were isolated from shrimp culture ponds. The bacteria used in this study were identified using standard morphological, physiological and biochemical plate and tube tests (Holt et al. 1994). All strains were taken from the stock culture collection of our laboratory and had been stored in Luria- Bertani (LB) broth cultures with sterile glycerol (15% v v). Antagonism assay The initial screening of antagonism by Bacillus BT21, Bacillus BT22 and B. subtilis BT23 was by the agar well diffusion plate assay method (Bauer et al. 1966). Vibrio harveyi, V. anguillarum, V. vulnificus and V. damsela were precultured in LB broth incubated at 28 C for 2 d and 5 ll of this culture were spread over the agar plates. Bacillus spp. culture (3 d old) was centrifuged at 96 rev min )1 for 15 min and the supernatant fluid filtered through a Æ22-lm membrane filter (Sartorius, Bedford, MA, USA) to obtain cell-free extracts (culture supernatant fluid). A volume (1 ll) of Bacillus cell-free extracts was introduced into the wells of the agar medium and incubated for a period of h at 28 C. Antibacterial activity was defined as the diameter (mm) of the clear inhibitory zone formed around the well. rev min )1 ) and samples withdrawn daily and the number of cfu determined. Sterile filtered supernatant fluid (2 ml) was tested by adding 1 ml of supernatant fluid to 1 ml of fresh LB broth in test tubes and inoculating it with 1 ll of V. harveyi in LB broth, yielding approx. 1 4 cfu ml )1. Controls were made by inoculating V. harveyi (1 ll) in 2 ml of LB broth without B. subtilis BT23 cell-free extracts. Each combination was tested in triplicate and the growth of the V. harveyi monitored by recording the optical density at 6 nm with a spectrophotometer. Experimental infection of shrimp and Bacillus subtilis BT23 treatment Bacillus subtilis BT23 was grown for 3 d at 28 C (15 rev min )1 ) in LB broth and V. harveyi was grown for 24 h in tryptone soy broth. Two hundred P. monodon, each approx. 5 6 g, were divided equally into eight groups, each housed in a 3-l tank. Four of the tanks were treated with B. subtilis BT23 for 5 d at a level of 1 6 cfu ml )1 at 28 C (long-term treatment) by adding the bacteria to the water. After 5 d, shrimp in all of the four tanks were infected with V. harveyi (1 4 )1 6 cfu ml )1 ) for 1 h and two of the tanks were again treated with B. subtilis BT23 (1 6 cfu ml )1 ) for 1 h along with V. harveyi (combined treatment).of the remaining four tanks, two were treated with B. subtilis BT23 (1 6 cfu ml )1 ) for 1 h and the animals were then transferred to a tank in which V. harveyi (1 4 )1 6 cfu ml )1 ) was maintained for 1 h (short-term treatment); the other two tanks were infected with V. harveyi (1 4 )1 6 cfu ml )1 ) alone for positive control. The cumulative mortality of the shrimp was recorded and analysed using analysis of variance. Co-culture experiments Bacillus subtilis BT23 and V. harveyi were precultured separately in LB broth at 28 C for 3 d. Vibrio harveyi was inoculated into LB broth at an initial cell density of approx. 1 3 cfu ml )1, whereas the initial levels of B. subtilis BT23 were 1 5,1 7,1 8 and 1 9 cfu ml )1. All combinations were performed in triplicate. The co-culture plates were incubated at 28 C and samples withdrawn daily for the determination of V. harveyi densities. The numbers of V. harveyi were estimated by preparing 1-fold serial dilutions and Æ1 ml from each dilution was inoculated into thiosulphate citrate bile salts sucrose agar plates. Effect of Bacillus subtilis BT23 cell-free extracts Bacillus subtilis BT23 was precultured in LB broth and then used to inoculate 5 ml of LB broth in the same four combinations at an initial cell density of 1 4 )1 8 cfu ml )1. The flasks were incubated at 28 C with agitation (2 RESULTS Antagonism assay The cell-free extract of Bacillus BT21, Bacillus BT22 and B. subtilis BT23 showed inhibitory activity against Vibrio spp. Of these, B. subtilis BT23 showed a higher inhibitory activity than the other two Bacillus spp. tested. BT23 showed inhibitory activity against 112 Vibrio spp., V. harveyi (39 isolates), V. anguillarum (24 isolates), V. vulnificus (3 isolates) and V. damsela (19 isolates) obtained from P. monodon culture hatcheries and ponds (Table 1). The diameters of the inhibitory zones around the growth of Vibrio spp. were about 3 6 mm (Fig. 1). Co-culture experiments The growth of pathogenic V. harveyi was inhibited by B. subtilis BT23 culture inoculated at an initial level of 1 5 )1 9 cfu ml )1 (Fig. 2). Lower concentrations of

3 EFFECT OF BACILLUS ON SHRIMP PATHOGENIC VIBRIOS 85 Table 1 Inhibitory activity of Bacillus subtilis BT23 against Vibrio harveyi, V. anguillarum, V. vulnificus and V. damsela obtained from Penaeus monodon culture hatcheries and ponds B. subtilis with inhibitory effects against Source of Vibrio spp. V. harveyi isolates V. anguillarum isolates V. vulnificus isolates V. damsela isolates Vibriosis-infected post-larval P. monodon MBV-infected post-larval P. monodon Black gill-diseased P. monodon juveniles WSSV-infected P. monodon gills Total MBV, Monodon baculovirus; WSSV, white spot syndrome virus Log (cfu ml 1 ) Time at 3 C (d) Fig. 1 Petri dishes containing cell-free extract of (a) Bacillus BT21, (b) Bacillus BT22 and (d) Bacillus subtilis BT23 showed inhibitory zones against the growth of (A) Vibrio harveyi, (B) V. anguillarum and (C) V. damsela. No inhibitory zone was found in the control (c). Note the Bacillus subtilis BT23 (d) showing the greatest inhibitory zones. The strain was identified as B. subtilis BT23 and used for further in vivo and in vitro studies B. subtilis BT23 (1 5 and 1 7 cfu ml )1 ) allowed initial growth of V. harveyi, but cfu densities never reached the level of the control. High concentrations (1 9 cfu ml )1 )of B. subtilis BT23 allowed an initial increase of V. harveyi followed by a decrease in the total viable counts (Fig. 2). Co-culture experiment results showed that, when the concentration of B. subtilis BT23 increased, the growth of V. harveyi was controlled under in vitro conditions. Effect of cell-free extracts of Bacillus subtilis BT23 Cell-free extracts of B. subtilis BT23 inhibited the growth of V. harveyi in liquid culture under aerobic conditions. The Fig. 2 Growth pattern of Vibrio harveyi at 28 C with and without Bacillus subtilis BT23 at different initial concentrations (colony-forming units; cfu). r, Without B. subtilis; j, B. subtilis 1 5 cfu ml )1 ; m, B. subtilis 1 7 cfu ml )1 ; s, B. subtilis 1 8 cfu ml )1 and d, B. subtilis 1 9 cfu ml )1 inhibitory efficiency was high in B. subtilis BT23 cell-free extracts of 1 8 cfu ml )1 and low in 1 4 cfu ml )1. Bacillus subtilis BT23 cell-free extracts did not restrict the growth of V. harveyi for 2 d and after that the growth was remarkably controlled (Fig. 3) when compared with the growth of V. harveyi without B. subtilis BT23. Experimental infection of shrimp and probiotic treatment The studies on the probiotic treatment and infection of shrimp revealed that the mortality of shrimp by V. harveyi infection was reduced by B. subtilis BT23 strains under in vivo conditions. The cumulative mortality of infected P. monodon not treated with B. subtilis BT23 reached 5% on the 9th day after infection with V. harveyi and 1% on the 17th day. However, in the case of probiotic treatment

4 86 B. VASEEHARAN AND P. RAMASAMY Optical density (6 nm) Time at 3 C (d) Fig. 3 Growth of Vibrio harveyi at 28 C with cell-free extracts of Bacillus subtilis BT23 extracted by different cell densities. With B. subtilis cell-free extracts of: r, 1 4 cfu ml )1 ; j, 1 5 cfu ml )1 ; m, 1 6 cfu ml )1 ; s, 1 7 cfu ml )1 and d, 1 8 cfu ml )1 groups, the mortality levelled off after 5 d when the cumulative mortality was 1% in the combined treatment (Fig. 4). Both the long- and short-term treatments with B. subtilis BT23 caused a decrease in cumulative mortality, to 32 and 6%, respectively. No mortality was found in control tanks which were not exposed to V. harveyi. The effect of probiotic treatment was most pronounced during the first day of infection. Average accumulated mortality (%) Time after infection (d) Fig. 4 Cumulative mortality of Penaeus monodon juveniles infected with Vibrio harveyi with and without probiotic treatment of Bacillus subtilis BT23. s, Control; m, long-term treatment; j, short-term treatment and r, combined treatment DISCUSSION The present study showed that the growth of pathogenic V. harveyi was controlled by non-pathogenic B. subtilis BT23 under in vivo and in vitro conditions. The control of fish and shellfish pathogenic Vibrio, particularly using non-pathogenic bacterial strains and disease prevention, has received much attention during the last decade (Sugita et al. 1998; Rengipipat et al. 2). Fuller (1989) defined a probiotic as a live microbial feed supplement which benefits the host animal by improving its intestinal microbial balance. Co-culture experiments showed that the inhibitory activity of B. subtilis BT23 increased with increasing density of the antagonist. A high concentration of B. subtilis BT23 (antagonist) was required to inhibit V. harveyi in the co-culture experiments. The present study showed that the antagonist must be present at significantly higher levels than the pathogen and the degree of inhibition increased with the level of antagonist. During the co-culture, 1 7 )1 9 cfu ml )1 were required to inhibit the growth of the pathogen V. harveyi. Therefore, a potential probiotic co-culture must either be supplied on a regular basis or be able to colonize and multiply on or in the host. The Bacillus spp. used as probiotics for terrestrial livestock are of telluric origin and are not autochthonous in the gastrointestinal tract but they may be active during intestinal transit (Gouthier et al. 1994). Kennedy et al. (1998) isolated a strain of B. subtilis from common snook (Centropomus undecimalis). The inoculation of this strain into the rearing water resulted in the apparent elimination of Vibrio spp. from the snook larvae. Smith and Davey (1993) reported that Pseudomonas fluorescens reduced diseases caused by Aeromonas solmonicida in fish. Austin et al. (1995) also observed a similar phenomenon, that V. alginolyticus, used as a probiotic strain, reduced the diseases caused by Aerom. solmonicida, V. anguillarum and V. ordalli in P. monodon. Maedo and Liao (1992) reported the use of a soil bacterial strain, PM-4, that promoted the growth of P. monodon nauplius, probably acting as a food source. This strain also showed an in vitro inhibitory effect against V. anguillarum. Rengipipat et al. (1998) reported that inoculation of Bacillus S11, a saprophytic strain, resulted in greater survival of the post-larval P. monodon that were challenged by pathogenic luminescent bacterial culture. These works strongly suggest the effective control of microflora in fish and shellfish in culture environments by antibiotic-producing bacteria. Purification and characterization of the antibacterial substance would help to understand the mechanism of antibacterial activity of Bacillus strains. Probiotic treatment offers a very promising alternative to the use of antibiotics in fish and shrimp aquaculture. Further study is needed to elucidate the exact mode of action of the observed beneficial effects and to understand the possibilities and limitations of microbial control in aquaculture.

5 EFFECT OF BACILLUS ON SHRIMP PATHOGENIC VIBRIOS 87 ACKNOWLEDGEMENTS The authors thank Dr Junda Lin (Florida Institute of Technology, Melbourne, FL, USA) for his comments on an earlier version of the manuscript. REFERENCES Amabile, C.F., Gardenas, G.M. and Ludger, M. (1995) Antibiotic resistance. American Sciences 83, Austin, B., Stuckey, L.E., Robertson, P.A.W., Effendi, I. and Griffith, D.R.W. (1995) A probiotic strain of Vibrio alginolyticus effective in reducing disease caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalli. Journal of Fish Diseases 18, Bauer, A.W., Kioby, W.M.M., Sherris, J.C. and Turck, M. (1966) Antibiotic susceptibility testing by a standard single disc method. American Journal of Clinical Pathology 45, Fuller, R. (1989) A review: probiotics in man and animals. Journal of Applied Bacteriology 66, Gatesoupe, F.J. (1999) The use of probiotics in aquaculture. Aquaculture 18, Gournier, C.N., Larpent, J.P., Castellanos, I. and Larpent, J.L. (1994) La microflore intestinale et son role. In Lesprobiotiques an Alimentation Animale et Humaine. pp Techniques at Documentation. Paris: Lavoisier. Holt, J.G., Krieg, N.R., Sneath, P.H.A. and Staley, J.T. (ed.) (1994) Facultatively anaerobic Gram negative rods. In Bergey s Manual of Determinative Bacteriology, 9th edn. pp Baltimore, MD, USA: Williams & Wilkins. Kennedy, S.B., Tucker, J.W., Neidig, C.L., Vermeer, G.K., Cooper, V.R., Jarrell, J.L. and Sennett, D.G. (1998) Bacterial management strategies for stock enhancement of warm water marine fish: a case study with common snook (Centropomus undecimalis). Bulletin of Marine Sciences 62, Lightner, D.V. (1996) A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. pp Baton Rouge, LA, USA: World Aquaculture Society. Lundin, G.G. (1996) Fish health and quarantine. In Global Attempts to Address Shrimp Disease. pp. 45. Marine Environmental Paper No. 4. Land, Water and Natural Habitats Division, Environment Department, World Bank, Rome. Maeda, M. and Liao, I.C. (1992) Effect of bacterial population on the growth of a prawn larva, Penaeus monodon. Bulletin of National Research Institute of Aquaculture 21, Moriarty, D.J.W. (1997) The role of microorganisms in aquaculture ponds. Aquaculture 151, Rengipipat, S., Phianphak, W., Piyatiratitivorakul, S. and Menasveta, P. (1998) Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth. Aquaculture 167, Rengipipat, S., Rukpratanporn, S., Piyatiratitivorakul, S. and Menasaveta, P. (2) Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). Aquaculture 191, Sharmila, R., Jawahar Abraham, T. and Sundararaj, V. (1996) Bacterial flora of semi-intensive pond reared Penaeus indicus (H.Milne Edwards) and the environment. Journal of Aquaculture in the Tropics 11, Skjermo, J. and Vadstein, O. (1999) Techniques for microbial control in the intensive rearing of marine larvae. Aquaculture 177, Smith, P. and Davey, S. (1993) Evidence for the competitive exclusion of Aeromonas salmonicida from fish with stress-inducible furunsculosis by a fluorescent Pseudomonad. Journal of Fish Diseases 16, Subasinghe, R., Bartly, D.M., Megladdery, S. and Barg, U. (1998) Sustainable shrimp culture development: biotechnological issues and challenges. In Advances in Shrimp Biotechnology ed. Flegel, T.W. pp Bangkok: National Centre for Genetic Engineering and Biotechnology. Sugita, H., Hirose, Y., Matsuo, N. and Deguchi, Y. (1998) Production of the antibacterial substance by Bacillus sp. strain NM 12, an intestinal bacterium of Japanese coastal fish. Aquaculture 165, Sung, H.H., Hsu, S.F., Chen, C.K., Ting, Y.Y. and Chao, W.L. (21) Relationship between disease outbreaks in cultured tiger shrimp (Penaeus monodon) and the composition of Vibrio communities in pond water and shrimp hepatopancreas during cultivation. Aquaculture 192,