POTENCY AND CHALLENGE UTILIZATION OF Vibrio AS PROBIOTIC IN MARINE AQUACULTURE POTENSI DAN TANTANGAN PEMANFAATAN Vibrio SEBAGAI PROBIOTIK PADA BUDIDAYA PERIKANAN LAUT

  

POTENCY AND CHALLENGE UTILIZATION OF Vibrio AS PROBIOTIC

  

IN MARINE AQUACULTURE

POTENSI DAN TANTANGAN PEMANFAATAN Vibrio SEBAGAI

  

by

Faturrahman

  Departement of Biology, Mataram University Jl. Majapahit No 62, Mataram Lombok NTB 83125; Hp : 08197210290, E-mail :

  

nayla_fatur@yahoo.co.id

  Presented at :

  th th th

  

The 5 Indonesia Biotechnology Conference: An International Forum. July 4 -7 , Mataram

Lombok Island Indonesia

  

POTENCY AND CHALLENGE UTILIZATION OF Vibrio AS PROBIOTIC

  Faturrahman Departement of Biology, Mataram University, Jl. Majapahit No 62, Mataram Lombok NTB

  83125, Hp : 08197210290, E-mail : nayla_fatur@yahoo.co.id

  

Abstract

The availability of seed have a certain quality, well-being reason and increased resistence case

pathogen towards antibiotic is the root cause decreased it use antibiotic in mari-culture industry. The

using of probiotic looked at as correct strategy, besides can overcome seed mortality, can promote

animal growth, also friendly environment. Vibrio spp be one of bacteria group that applied as

probiotic aquaculture. One other thing, a V. alginolyticus strain used as a probiotic in a commercial

shrimp hatchery in Ecuador has been applied in a bath treatment to Atlantic salmon (21 g) maintained

in freshwater and led to a reduction in mortality after exposure to A. salmonicida and to a lesser extent

after exposure to V. anguillarum and V. ordalii. However, as needed carefulness in this bacteria use,

remember a large part pathogen sea organism comes from this group.

  Key word : aquaculture probiotic, Vibrio spp, antibiotic Introduction

  Industrialization marine aquaculture requires continuous availability of seeds in sufficient quantity and quality. One of the critical success factors in the provision of quality eggs and larvae are mikrobiologisnya status (Irianto, 2003). During the microbial control of eggs and larvae are still largely relies on the use of antibotik or disinfectant. This method is not always beneficial because it causes changes in the composition of microbes and microbial pathogens may be precisely that will dominate the surface of eggs and larvae (Olafsen, 1998). In addition, strengthening the case for health reasons and pathogen resistance to antibiotics is a major cause decrease in the use of antibiotics in the farming industry (Balcazar et al. 2006).

  One strategy that can be done to lower the mortality rate of larvae is to manipulate and suppress microbial composition and the presence of opportunistic bacteria or pathogens ( Skjermo et al. 1997) through the administration of probiotics (Olafsen, 1998). Probiotics for aquaculture is defined as live microbes that benefit the host by modifying the microbial community relations associated with the host or the environment, increase the use of feed or nutritional value, spurring the host response to the disease, or by improving the quality of the aquatic environment (Verschuere et al. 2000).

  Most probiotics proposed as biocontrol agents in aquaculture was originally included into the group of lactic acid bacteria, then evolved into a broader Vibrio genus, the genus Bacillus or the genus Pseudomonas (Verschuere et al. 2000), yeast and microalgae (Abidi, 2003) .

  Research on the use of a probiotic strain of Vibrio have been widely reported . The reason is due to the utilization of this bacterial group has a number of characters required by a system of probiotics in aquaculture, among others, are able to carry out a competition with other bacteria (Riquelme et al. 1997), adds nutrients to provide essential nutrients (Thompson

  

et al. 2004), can improve the digestibility by removing the essential enzymes (Tanaka et al.

  2001), has the ability to colonize the gastrointestinal tract and the body of the host (Sawabe

  

et al. 1998; Sawabe et al. 2003; Thompson et al. 2004) , and may produce substances that

inhibit the growth of opportunistic pathogenic bacteria (Verschuere et al. 2000).

  Some Vibrio species are reported as probiotics proved to be pathogenic /opportunistic against marine animals. An example is the use of a strain of V. alginolyticus in shrimp and fish farming (Verschuere et al. 2000). V. alginolyticus though widely known as a pathogen and can cause mass mortality in several species of marine animals such as shellfish. Although limited in scale significantly improve the survival rate of shrimp larvae and fish, caution is required before the strains of Vibrio applied on a broad scale. Therefore, in this paper the authors will review the potential and challenges of the use of Vibrio species as Probion on marine aquaculture.

  Vibrios and its Role in Water

  In Bergey 's Manual of Determinative Bacteriology, Vibrio (Vibrionaceae is a family of -proteobacteria) belongs to the group of negative rod-shaped motile, mesophilic kemoorganotrof , is fermenting facultative metabolism, mainly in aquatic habitats as well as in the form of associations with eukaryotes. In general, they can be grown on marine agar and thiosulfate - citrate - bilesalt - sucrose agar (TCBS) and are mostly positive oxidase (Thompson et al. 2004).

  Vibrio was found very abundant in the aquatic environment, including estuaries, sediments and seawater, as well as widespread aquaculture environment (Barbieri et al. 1999). This group of bacteria showed a high density of marine organisms such as corals, fish, mollusks, seaweed, sponge, shrimp and zooplankton (Thompson et al. 2003; Thompson et

  al. 2004).

  Phylogenetic analysis based on 16S rRNA gene sequences showed that Vibrio distributed into 4 different families, namely Salinivibrionaceae (consisting of the genus Salinivibrio), Enterovibrionaceae (consisting of the genera Enterovibrio and Grimontia), Photobacteriaceae (composed of a genus Photobacteria) and Vibrionaceae (including all Vibrio species except V. fischeri group) (Thompson et al. 2004).

  In 2004 there were 74 species of Vibrio. Some of Vibrio species are known as major pathogens for aquatic organisms such as fish, crustaceans, corals, shellfish and molluscs. Nevertheless, there are indications that the Vibrio play an important role in nutrient cycling in aquatic environments through organic matter breakdown. Vibrio provide unsaturated fatty acids (polyunsaturated fatty acids) essential for the aquatic food chain, which is where many of the aquatic organisms are not able to produce it (Nichols, 2003). Vibrio can also degrade chitin, a homopolymer of N - acetyl glucosamine , which is one of the largest pool of amino sugars in the ocean (Rieman and Azam, 2002). Among the marine bacterium Vibrio is an important producer of antibiotics. Inhibitor compounds produced by Vibrio isolates can reduce certain other members of the community such as alpha-proteobacteria and Alteromonas (Long and Azam, 2001).

  Vibrio been exploited for various purposes. This organism has been used for biomonitoring aquatic environments because it can produce a N-acyl autoinduser homoserine lactone which can control the infection and the formation of biofilms (Nivens et al. 2004). In addition, certain species of bacteria used for the production of vaccines and probiotics, and can perform bioremediation polyaromatic hydrocarbons (Thompson et al. 2004).

  Probiotics Aquaculture and Its selection

  Fuller (1986) stated that probiotics as a mixed microbial life as a feed supplement in favorable host by improving the balance of microbial populations in the gastrointestinal tract. According Verschuere et al. (2000), submitted an application definition Fuller probiotics for aquaculture however requires some consideration. In contrast to humans and terrestrial animals, the presence of microorganisms in the digestive tract of aquatic organisms are not present as a separate entity, but is constantly in touch with the microorganisms in the environment. Therefore, the presence of bacteria in the aquatic environment influence the composition of the microbiota of the gastrointestinal tract and vice versa.

  At the beginning of the terrestrial animal bacterial colonization in children comes from contact with the mother , while aquatic species the eggs are laid in the water so that the contacts. Moreover, larvae or aquatic animal newborn percernaannya system has not developed and does not yet have a good microbial communities in the gastrointestinal tract , the skin and gills. Therefore, the early stages of larval development depends aquatic microbiota derived from primary cultured aquatic environment (Verschuere et al. 2000).

  Characteristics of microbial probiotics is its ability to colonize the gastrointestinal tract of the host, but the gastrointestinal tract microbiota in aquatic animals due to the rapidly changing constantly in touch with the flow of microbes from water and food (Abidi, 2003). Based on the above statement that the interaction between the microbiota, including probiotics, and the host is not restricted to the gastrointestinal tract. Probiotic bacteria should be active on the gills or skin of its host, but also active in the marine environment.

  Thus , probiotics for aquatic environments are defined as live microbes that benefit the host by modifying the microbial community relations associated with the host or the environment, increase the use of feed or nutritional value, spurring the host response to the disease, or by improving the quality of the aquatic environment (Verschuere et al. 2000).

  Based on the above definition includes probiotics can prevent the proliferation of pathogenic microbes in the digestive cavity, the surface structure, the marine environment, ensure optimal use of feed to help the digestive system of the host, improving water quality, or stimulate the host immune system (Verschuere et al. 2000).

  According Verschuere et al. (2000) there are several possible mechanisms of action (mode of action) of an aquaculture probiotic for yielding beneficial effects for the host is as follows: 1) produce a chemical substance that is bactericidal or bacteriostatic which can suppress or inhibit the growth of pathogens; 2) competition chemical or energy sources; 3) competition at the attachment site. Mechanisms to prevent colonization of pathogens is competition for attachment sites on the surface of the abdomen or body tissues; 4) can trigger a host immune response through the production of immunostimulatory compounds; 5) improve water quality through the conversion of organic compounds to CO , or the reduction

  2 of toxic compounds such as nitrites.

  In general, the procedure of selection and development of probiotics for aquaculture consists of 6 stages (Gomez - Gil et al. 2000), namely (1) the collection of information from the literature as well as in the field, such as ponds or tanks operational information hatchery (hatchery), production management and disease control, (2) the collection or isolation of potential probiotic candidates from the pool or the best source (indigenous/putative probiotics), ie from the host, as well as from the natural food cultivation environment, applied, (3) selection and evaluation of potential d evaluation of potential probiotic candidate 's ability to probiotic candidate 's ability to compete with pathogenic strains, (4) assessment assessment of potential probiotic pathogenicity, (5) laborato (5) laboratory -scale testing includes looking at the effect the effect of probiotic candidates towards in vivo immunolog vivo immunological variables, survival rate and variability and variability of the host, and pathogen challenge test challenge test with, (6) economic analysis (cost / benefit analysis ) ( Figure 2.4 ). t / benefit analysis ) ( Figure 2.4 ).

  Here are the steps that should that should flow through to the selection of probiotics probiotics that will be developed for the commercial marine aquaculture by Balc for the commercial marine aquaculture by Balcazar et al ( 2006) : Figure 1. Diagram of selection of of probiotics as biocontrol agents in aquaculture aquaculture (Balcazar et

  al. 2006) The Potential of Vibrio as Probiotics Probiotics aquaculture

  One approach that is widely us widely used to select candidates probiotics is to perform to perform in vitro antagonism test, where the pathogen pathogen isolates presented with candidates or extracellular products in liquid or solid medium medium (Balcazar et al. 2006).

  On the basis of the ability ability of Vibrio strains can produce substances that substances that inhibit the growth of pathogenic bacteria / opportunistic opportunistic This is the amount of research directed research directed to utilize Vibrio strains as probiotics for marine marine aquaculture (Verschuere et al. 2000).

  Verschuere et al. (2000) mentions a number of Vibrio strains that have been applied as aquaculture probiotics, including a strain of V. alginolyticus isolated from the Pacific Ocean and then applied to the tank enlargement L. Vannamei shrimp larvae can improve survival and weight and can reduce postlarva V. parahaemolyticus population. Other strains used as probiotic V. alginolyticus and commercialized shrimp hatcheries in Ecuador and then applied to Atlantic salmon, after 21 days of application of this bacterium is found in the digestive tract, it also lowers mortality probiont salmon after being challenged with A.

  salmonicida.

  Here are a few application of Vibrio strains that has been used as an aquaculture probiotics, summarized from Verschuere et al. (2000) (Table 1). Table 1. application of Vibrio strains as a probiotic aquaculture

  

Putative Source of Observation Methods Mode of action

probiotics isolates V. salmonicida- Ikan Meningkatkan sintasan larva halibut Penambahan pada imunostimulasi like air kultur

  V. pelagius Copepoda- Menurun mortalitas larva turbot Penambahan pada

pakan larva setelah ditantang dengan A. Caviae air kultur ?

turbot

  Strain E (V. Larva turbot Menurun mortalitas larva turbot Pengayaan Kompetisi besi alginolyticus-like sehat setelah ditantang dengan Vibrio rotifera strain P, juga memicu laju pertumbuhan larva

  V. alginolyticus Hatchery Menurun mortalitas juvenil ikan Perendaman Antagonisme

udang salmon atlantik setelah ditantang dalam suspensi

komersil di dengan

V. salmonicida,

V. bakteri Ekuador anguillarum, V. ordalii

  V. alginolyticus Air Lautan Meningkatkan sintasan dan berat L. Penambahan pada Antagonisme Pasifik Vannamei ,mereduksi populasi V. air kultur parahaemolyticus pada udang

  Vibrio strain 11 Mikroalga Menurun mortalitas larva scallop Perendaman Antagonisme pada setelah ditantang dengan V. dalam suspensi hatcheri Anguillarum bakteri scallop

  V. alginolyticus ? Menurun mortalitas Artemia nauplii Penambahan pada C14 setelah ditantang dengan V. air kultur ? Parahaemolyticus

  Vibrio strain 11 were used to control the cultivation of pathogenic V. Angillarum on bivalves and can significantly decrease the mortality of larvae scallop shells (Riquelme et al. 1997). The use of a strain of V. alginolyticus in shrimp and fish farming significantly improve the survival rate of larvae of both commodities (Verschuere et al. 2000).

  One of the characteristics of microbial probiotics is its ability to colonize the gastrointestinal tract of the host , but the gastrointestinal tract microbiota in aquatic animals due to the rapidly changing constantly in touch with the flow of microbes from water and food (Abidi, 2003). In this case, Vibrio halioticoli a major microflora inhabiting the stomach of some shellfish species Haliotis (Sawabe et al. 1998; Tanaka et al. 2002). The presence of

  

Vibrio halioticoli ranged between 40 -64 % of the total heterotrophic bacterial communities

  3

  7

  that can be cultured (culturable) with a cell count between 10 up to 10 CFU / g stomach Haliotis shells (Sawabe et al. 2003; Thompson et al. 2004).

  Another hallmark of a probiotic is to add nutrients to provide essential nutrients and increase digestibility by removing the essential enzymes. Vibrio halioticoli can produce acetic acid and formic acid in large quantities, which is thought to be used as an energy source or precursor protein synthesis by abalone (Thompson et al. 2004) thus considered that there is a mutual relationship between Vibrio halioticoli with abalone (Hooper and Gordon, 2001). Additionally Tanaka et al. (2001) reported that V. halioticoli produce polyguluronate lyase enzyme, an enzyme that catalyzes the degradation of marine algae which is the main food Haliotis.

  Vibrio challenge as Probiotics Aquaculture

  Selection and development of probiotics that can be applied commercially in aquaculture and requires a multistep process involving multidisciplinary basic and empirical research, pilot-scale testing, pilot and field-scale application of economic valuation (Verschuere et al. 2000).

  Gomez-Gil (2000) states that the selection of probiotic bacteria is usually an empirical process based on little scientific evidence. Many failures occur because the probiotics study mistakenly select microorganisms. Selection process had been determined, but this stage still needs to be adjusted according to the host species and the environment. Common criteria for selection is primarily determined by biological safety considerations, methods of production and processing, the method of administration of probiotics, as well as the location in the body where the expected active probiotic bacteria.

  The use of probiotic selection criteria based on test activity in vitro antimicrobial compounds often can not be used to predict the likely effects in vivo (Gram et al. 1999). For example, in vitro antagonism test of P. fluorescens against A. salmonicida can not protect salmon Atlantic against furunculosis, but the probiotics effectively provide protection against vibriosis in rainbow trout (Gram et al. 2001). Therefore, important to know the origin and host isolates, security, and the ability of strains to survive transit through the gastrointestinal tract and its host.

  Bacterial isolates were shown to inhibit pathogens in vitro should be tested against the host pathogenicity and other vulnerable animals. Pathogenicity testing of candidate probiotic isolates against other animals is often not done. Though this criterion is important, especially when aimed at the cultivation of sea -based, sea-based aquaculture, given the magnitude of the contact opportunities between probiotics with other organisms in waters very large.

  The current criteria for the introduction of new probiotic strains is their susceptibility to antibiotics used by humans and livestock. Therefore, bacteria can develop resistance to antimicrobial agents, this can be a threat to human health. The emergence of drug-resistant bacteria may be one of the important clues distribute antibiotic resistance of opportunistic pathogens, where these resistant bacteria may increase the potential threat through the transfer of resistance to human pathogenic bacteria through the gastrointestinal tract or the environment. Therefore, it must be taken into account the importance of the selection of bacteria to be used as probiotics, which bacteria do not show resistance to standard antibiotics used by humans and animals (Brashear et al. 2003).

  It is important to increase the prudence before the introduction of probiotics derived from pathogenic strains (see vibrio) commercialized so as to avoid chances of the spread of antibiotic resistance and the induction of genes that are silent pathogenicity through quorum sensing coupled with the emergence of cases of foodborne disease.

  Conclusion

  Opportunities of Vibrio spp utilization as a probiotic is enormous considering the extent of the role and capabilities of the group of bacteria is high in nature. However, it requires great care and caution the use of this bacterium as a probiotic. The selection process should be done properly. Control and periodic evaluation to see the impact of the release or application held to be important in the field of probiotics.

  References

Abidi R. 2003. Use Probiotics In Larval Rearing of New Candidate Species. Aquaculture Asia. Vol.

  VIII No.2

Balcazar JL., I. De Blas, I. Ruiz-Zarzuela, D. Cunningham, D. Vendrell and JL. Muzquiz, 2006. The

role of probiotics in aquaculture. Veterinary microbiology 114: 173-186 Fuller R. 1989. A Review : probiotics in man and animals. J. Appl. Bacteriol. 66:365-378

  

Gomez-Gill B., A. Roque, and JF. Trunbull, 2000. The use and selection of probiotic bacteria for

use in the culture of larval aquatic organisms. Aquaculture 191: 259-270

Hooper LK and JI. Gordon, 2001. Commensal host-bacterial relationships in the gut. Science

292:1115-1118 Irianto 2003. Probiotik untuk Akuakultur. PT. Penebar Swadaya, Jakarta

  

Long RA., and F. Azam. 2001. Antagonistic interactions among marine pelagic bacterial. Appl.

  Environ. Microbiol. 67:4975-4983

Nichols DS. 2003. Prokariotes and the input of polysaturated fatty acids to the marine food web.

FEMS Microbiol. Lett. 219:1-7 Nivens DE., TE. McKnight, SA. Moser, SJ. Osbourn, ML. Simpson and GS. Sayler, 2004.

  

Olafsen JA. 2001. Interactions between fish larvae and bacteria in marine aquaculture. Aquaculture

200:223-247 Prescott LM, A. Klein, D. Harley, JP. 2002. Microbiology. McGraw-Hill Higher Education

Riemann L. and F. Azam, 2002. Widespread N-acetyl-D-glucosamine up-take among marine

pelagic bacteria and its ecological implictions. Appl. Environ. Microbiol. 68:5554-5562

Riquelme C, R. Araya, N. Vergara, A. Rajas, M. Guaita, and M. Candia.1997. Potensial probiotic

strain in the culture of the Chilean scallop Argepecten purpuratus (Lamarck, 1918).

  Aquaculture 154:17-26

Sawabe T, I. Sugimura, M. Ohtsuka, K. Nakano, K. Tajima, Y. Ezura, and R. Christen. 1998. Vibrio

halioticoli sp. Nov., a motile alginolytic marine bacterium isolated from the gut of abalone Haliotis discus hannai. Int J Syst Bacteriol, 48:573-580

Sawabe T, N. Setogushi, S. Inoue, R. Tanaka, M. Ootsubo, M. Yoshimizu, and Y. Ezura. 2003. Acetic

acid production of Vibrio halioticoli from alginate: a possible role for establishment of abalone-V. halioticoli association. Aquaculture 219:671–679.

  

Skjermo J., I. Salvesen, G. Oie, Y. Olsen, and O. Vadstein. 1997. Microbially matured water: a

technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae. Aquacult. Int. 5:13–28..

  

Tanaka R., T. Sawabe, K. Tajima, J. Vandenberghe, and Y. Ezure. 2001. Identification of Vibrio

halioticoli using 16S rDNA PCR/RFLP (rectriction fragment lenght polymorphism) analysis. Fisheries Science 67:185-187.

  

Tanaka R., T. Sawabe, M. Yoshimizu, and Y. Ezure. 2002. Distribution of Vibrio halioticoli around

an Abalone-farming Center in Japan. Microbes and Environments. 17(1):6-9

Thompson FL., Y. Li, K. Vandemeulebroecke, P. Sorgeloos, B. Gomez-Gil, G. S. Rupp, and J.

  Swings. 2003. Vibrio neptunius sp. nov., Vibrio brasiliensis sp. nov. and Vibrio xuii sp. nov., isolated from the marine aquaculture environment (bivalves, fish, rotifers and shrimps). Int J Syst Bacteriol, 53:245-252

Thompson FL., T. Ida, and J. Swings. 2004. Biodiversitas of Vibrios. Microbiol and Mol Biol Rev.

  68(3):403-431

Verschuere L., G. Rombout, P. Sorgeloos, and W. Verstraete. 1997. Probiotic Bacteria as biological

control agents in aquaculture. Microbiol and Mol Biol Rev. 64(4):655-671