POTENCY AND CHALLENGE UTILIZATION OF Vibrio AS PROBIOTIC

IN MARINE AQUACULTURE

POTENSI DAN TANTANGAN PEMANFAATAN Vibrio SEBAGAI

PROBIOTIK PADA BUDIDAYA PERIKANAN LAUT

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 :

The 5th Indonesia Biotechnology Conference: An International Forum. July 4th-7th, Mataram
Lombok Island Indonesia
 POTENCY AND CHALLENGE UTILIZATION OF Vibrio AS PROBIOTIC
IN MARINE AQUACULTURE

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
primary colonization on the eggs and larvae originating from environmental microbes
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 CO2, or the reduction
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,
making habitat isolates of the organism in order to avoid lag adaptation of probiont when
applied, (3) selection and
d evaluation of potential probiotic candidate 's ability to compete with
pathogenic strains,, (4) assessment of potential probiotic pathogenicity, (5) laboratory -scale
testing includes looking at the effect of probiotic candidates towards in vivo immunological
immunolog
variables,, survival rate and variability of the host, and pathogen challenge test with, (6)
economic analysis (cost / benefit analysis ) ( Figure 2.4 ).
Here are the steps that should flow through to the selection of probiotics that will be
developed for the commercial marine aquaculture by Balcazar et al ( 2006) :

Figure 1. Diagram of selection of probiotics as biocontrol agents in aquaculture (Balcazar et

al. 2006)

The Potential of Vibrio as Probiotics aquaculture

One approach that is widely used to select candidates probiotics is to perform in vitro
antagonism test, where the pathogen isolates presented with candidates or extracellular
products in liquid or solid medium (Balcazar et al. 2006).
On the basis of the ability of Vibrio strains can produce substances that inhibit the
growth of pathogenic bacteria / opportunistic This is the amount of research directed to utilize
Vibrio strains as probiotics for 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
that can be cultured (culturable) with a cell count between 103 up to 107 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.

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