Isolation of Bacteriocin Producing Lactic Acid Bacteria from Fish Gut and Probiotic Activity Against Common Fresh Water Fish Pathogen Aeromonas hydrophila
Lactic acid bacteria (LAB) produce many kinds of metabolites, which might affect the other microbes in the fish mid gut. Lactic acid produced by both homolactic acid and heterolactic strains, which will reduce pH in the luminal contents in the stomach of neonatal piglets of fish. Isolated LAB was used as a probiotic in fresh water fish tilapia (Oreochromis mossambicus) against the most common fish pathogen Aeromonas hydrophila. Higher antagonistic activity recorded from extra cellular protein (ECP) or bacteriocin compared to the intra cellular protein (ICP) against A. hydrophila. After feeding with the potential probiotics for 25 days, challenge by immersion indicated effectiveness at reducing disease caused by A. hydrophila in fishes. Tilapia exhibited no significant difference in growth, survival nor external appearance between the probiotic fed combine probiotic treatments, but significant differences (p< 0.05) occurred between probiotic and control groups. The use of LAB also enhances the production rate of rotifers, which act as biocarriers of probiotics and when fed to fish, they showed increased growth rate and weight of the animal. LAB is highly appreciated as a biological enhancers-probiotics, because of beneficial effect of live microorganisms.
Lactic acid bacteria are gram-positive, non-sporulating and catalase
negative rods or cocci that ferment various carbohydrates mainly to lactate
and acetate. Various amino acids, vitamins and minerals are essential
for their growth (Kandler and Wise, 1986). Various authors have shown
that lactic acid bacteria are also part of the normal intestinal flora
of fish (Ringo and Gatesoupe, 1998). Most of the evidence comes
from salmonid species like Arctic charr (Salvelinus alpinus) and
Atlantic salmon (Salmo salar) (Ringo et al., 1995; Gonzalez
et al., 2000). Few studies have described lactic acid
bacteria in other freshwater fish (Kvasnikov et al., 1977; Cai
et al., 1999). Kvasnikov et al. (1977) described the presence
of lactic acid bacteria, including Lactobacillus in the intestines
of various fish species at larval, fry and fingerling stages inhabiting
ponds. However, it was discussed that some human activities like artificial
feeding in ponds would have had an effect on the bacterial composition
and load in some fish, like carp (Cyprinus carpio) which showed
the highest content of lactic acid bacteria in the intestines.
Probiotics can be defined as a food (feed) or drug containing live microbes
that, when ingested, is expected to give beneficial physiologic effects
to the host animal through microbial actions (Ishibashi and Yamazaki,
2001). Most of the probiotics are marketed as foodstuffs or drug. Consideration
of the safety of probiotics is therefore of extreme importance. The safety
of the microbes that have been used traditionally in probiotics can be
conformed through a long period of experience (Mayra-Makien and Bigert,
1993). Yasuds and Taga (1980) suggested that probiotic bacteria would
be found to be useful not only as food but also as biological controllers
of fish disease and activators of nutrient regeneration. In the biological
control in aquaculture emerge and since then the research effort has continually
increased. Bacillus sp. is often antagonistic against other fresh
water fish pathogenic bacteria (Gatesoupe, 1999; Rengipipat et al.,
2000). Generally bacteria play two major roles as beneficial bacteria
and pathogenic forms, beneficial bacteria are helpful in nutrient recycling
and organic matter degradation and thus clear the environment (Moriarty,
1997). Pathogenic bacteria are the causative agents of bad water quality,
stress and diseases as they act as primary and secondary pathogens (Karunasagar
et al., 1996). Among fish pathogenic Aeromonas hydrophila
and direct cause the syptecimea diseases and indirectly affect the fish
health through developing poor water quality, high cyclone and stress
(Lightner et al., 1992).
In this present study investigated the inhibitory activity of Bacillus
sp. isolated from fish digestive tract, identify and evaluate bacteria
which could be used in disease prevention in Oreochromis mossambicus.
To test bacterial isolates for antagonistic activity against putative
fresh water pathogenic bacteria Aeromonas hydrophila.
MATERIALS AND METHODS
Lactic acid producing bacteria isolated from fish digestive tract:The
tilapia Oreochromis mossambicus fish was collected from the Cauvery,
fresh water river, Erode district, Tamilnadu, India. The fish were washed
with sterile distilled to remove the unwanted particles. Then the animals
were dissected to remove the digestive tracts by the sterilization condition.
The digestive tracts were homogenized in the same sterile distilled water
for centrifugation. After centrifugation the supernatant was taken and
serially diluted in sterile distilled water in the test tubes to 10-2,
10-3, 10-4, 10-5, 10-6, 10-7
and 10-8 dilution and were pour plated on nutrient agar
plate and incubated for 24 h at room temperature. Individual colonies
were taken and inoculated in Elliker`s broth. Then the broths were incubated
for 18-24 h at 37°C. One hundred microliter of culture was inoculated
in Bacillus selective medium Lactobacillus bulgaricus agar
plate and different colonies were selected and subjected to confirmatory
tests for (LAB).
Conformation of Bacillus sp.
Grams staining: Markedly available Gram`s staining kit purchased
from Himedia, heat-fixed smears should be stained for 1 min with crystal
violet, washed in tap water, covered with Grams iodine for 1 min re-washed,
decolorized by a few seconds in acetone-alcohol and counterstained for
30 sec in safranin. The smears are washed thoroughly and gently air-dried
and observed under the oil immersion objective.
Methyl Red test (MR): Inoculate the isolated Bacillus with
buffered glucose broth and incubate at 37°C for 48 h. After incubation
add a few drops of methyl red solution to the culture, read immediately.
Catalase test: A loopful of the culture was placed on a slide
and few drops of 10% hydrogen peroxide were added. The slides were observed
Detection of antagonistic activity: Isolated probiotic Bacillus
sp. was assayed by the agar well diffusion method according to Benkerroum
et al. (1993). The plates were examined for lysis around the wells
at different time intervals for a total of 24 h. The agar well disc diffusion
assay was used by two different isolated bacteriocin such as extra cellular
protein (ECP) and intra cellular protein (ICP), the zone of inhibition
was observed against common fish pathogen A. hydrophila.
Probiotic study-Maintenance of fish: The fish employed in this
study were common fish Oreochromis mossambicus with weight range
between 20±2 g fish was collected from the Cauvery fresh water
river, Erode district, Tamilnadu, India. The major physical and chemical
factors affect the growth of fish in a closed system. The fish were feed
with commercially available artificial diet twice a day. The parameters
were maintained, temperature (26-30°C), salinity (1-2 ppm), dissolved
oxygen (6-7 mg L-1), pH (6.5-8) and light.
Immersion study of Bacillus sp. against Aeromonas hydrophila:
Tilapias (Oreochromis mossambicus) had not been exposed to fish
diseases and were deemed specific pathogen free. The tilapias were acclimated
for one week in four tanks to laboratory conditions before the start of
the trial. After the acclimation period the average weight of the fish
was 22±2g and the fish were randomly divided into twenty 500 L
capacity polypropylene tanks with three treatments and three replicates
per treatment, each containing 50 tilapia fish. The water temperature
was held at 26-30°C during the whole trial. One treatment served as
the control and was fed with regular diet (without the probiotics) during
the entire trial period.
In order to obtain the bacterial suspensions, the probiotic strains were
grown in MRS broth in a shaking incubated at 30°C overnight. After
incubation, the cells were harvested by centrifugation (2000 rpm), washed
twice with PBS buffer and re-suspended in the same buffer. The absorbance
at 600nm was adjusted to standardize the number of bacteria (105-106CFU
mL-1). Commercial artificial feed was used as the basal diet
for the supplementation of probiotic strains. Probiotic diets were prepared
with cells resuspended in 5mL of PBS to 106 CFU mL-1
and mixed. Tilapia was fed two times daily at 5% body weight per day with
a 50% water change every day, during 25 day feeding trials and survival
was estimated visually each morning. A. hydrophila was used as
an infectious agent in the experiment. The strain was grown for 12 h at
30°C in nutrient broth. Directly immersion of the tank 3 and 4, treatments
and control were exposed to pathogenic A. hydrophila at a level
of 105-106 CFU mL-1 at 28°C by adding
the bacteria to the water. Dead fish were collected and recorded daily.
Statistical analysis: The results were analyzed using one-way
analysis of variance (ANOVA). Fisher`s range test was used to determine
differences (p<0.05) between tested groups. All statistics were performed
using SPSS 10 for Windows.
||Antagonistic activity against Aeromonas hydrophila, A: Antibiotic,
B: Bacteriocin (Extra cellular protein), I: Intra cellular protein,
||Accumulated mortality of Bacillus sp. infected with 105-106
CFU mL-1 of pathogenic A. hydrophila for 25 days
with probiotic treatment
The Bacillus sp. colonies were obtained from
the Oreochromis mossambicus fish gut by the 10-6 and
10-7 dilutions; these were conformed as the initial screening
for grams staining. Lactic acid bacteria are cocci or bacilli forms, non-motile,
gram positive, Methyl red test positive and Catalase negative. Inhibition
zone obtained the fish pathogen by antagonistic activity assay.
The inhibition zone 8 mm was observed on the plates inoculated with Aeromonas
hydrophila by Bacillus sp. indicates the production of secondary
metabolite of antimicrobial substances of bacteriocin against A. hydrophila
(Fig. 1) by agar well diffusion method. Immersion study was observed in
the four different groups of tanks contain O. mossambicus Values are
given as mean±SD for three tanks in each group, Significant at
(p<0.05), NS: Non significant, 1Tank 1. Control, 2Tank
2. Bacillus sp. (105-106CFU mL-1),
3Tank 3. Aeromonas hydrophila (105-106CFU
mL-1), 4Tank 4. Aeromonas hydrophila (105-106
CFU mL-1) + Bacillus sp. (105-106CFU
mL-1) with 105-106 CFU mL-1
cultures of Bacillus sp. and A. hydrophila and mortality
was observed (Table 1).
||Immersion study of A. hydrophila by Bacillus sp.
Isolated bacillus strains were inoculated second tank 87.5% of mortality
rate was observed by the 25 days experiment. At the same mass culture
of common fish pathogen A. hydrophila (105-106CFU
mL-1) were inoculated third tank was observed zero % of mortality
rate. Both two stains were immersed fourth tanks survival rates were increased
compare to the third tank. 65% of mortality rates were observed by probiotic
stains using fourth tank (Fig. 2).
The concept of biological control for health maintenance has received
widespread attention during the last few years, driven in large part by
consumers and the lay press. The first report of the existence in freshwater
of bacteria with an inhibitory effect against a Aeromonas sp. has
been attributed to Ochoa and Olmos (2006) Subsequently, Rosenfeld and
ZoBell (1947) described a study of antibiotic-producing marine microorganisms
and since then research has begun to develop biological control strategies
based on the application of these bacteria.
The isolated LAB species conformed by the methyl red and catalyzed negative
test by Jack et al. (1995). Gatesoupe et al. (1999) observed
that L. lactis showed an inhibitory activity against fish pathogen.
This observation was confirmed by an inhibition zone of 8 mm as observed
in this study. The culture media (supernatant) ECP were used, which showed
highest inhibition against A. hydrophila. The inhibitory effects
of Bacillus sp. may be due to production of antibiotics, bacteriocins,
lysozymes, proteases and/or hydrogen peroxide and the alteration of pH
values by the production of organic acids (Verschuere et al., 2000).
All the LAB species produced by secondary metabolite of bacteriocin compounds
against A. hydrophila (Santos et al., 1996). Adolfo (2004)
was reported to the isolated Lactobacillus sp., both homofermenters
and heterofermenters, were able to inhibit the human and fish pathogens
by acid production when using a high glucose concentration. A few strains
also inhibited both gram-positive and gram-negative fish and human pathogens
with low (0.2%) concentration of glucose in the medium. Inhibitory activities
of these strains have been usually detected against related species such
as Staphylococcus aureus, Clostridium and other fish pathogenic
bacteria (Schillinger and Lucke, 1989). But, only few Bacillus growing
at low glucose concentration have been reported to inhibit a broader range
of microorganisms, including gram negative foodborne and human pathogens.
Some of these are currently used as probiotics (Jacobsen et al.,
Bacteriocins are bactericidal or bacteriostatic peptides that are mostly
active against bacteria closely related to the producer (Klaenhammer,
1988). Immersion application against pathogenic bacteria, O. mossambicus
fish mortality rate was increased by the above experiments. A. hydrophila
produces extracelluar hemolysis compounds after 24 h 105-106
CFU mL-1 culture inoculations. In vivo condition A.
hydrophila hemolytic toxins were produced at the same time as Bacillus
sp. had produced by the secondary metabolite of extracelluar bacteriocin
compounds. The isolated Bacillus sp. produces hydrogen peroxidase,
other organic acids and bacteriocin (Klaenhammer, 1988; Daeschel, 1989).
A growing concern about the high consumption of antibiotics in aquaculture
has initiated a search for alternative methods of disease control (Gildberg
et al., 1997) and growth promotion (Byun et al., 1997).
The isolated Bacillus sp. was checked the disc diffusion assay
and to improved resistance against infectious diseases can be achieved
by the use of probiotics (Gildberg et al., 1997). Probiotic are
living preparations of microbial cells that, when ingested in high enough
concentration, beneficially affect the host`s health and growth by improving
the intestinal microbial balance (Fuller, 1989; Havenaar et al.,
1992). Selection of probiotic strains is achieved by screening procedures
for several characteristics in vitro, such as inhibitory activities
against several fish pathogens and gastric and intestinal secretions (Byun
et al., 1997; Joborn et al., 1997). Cai et al. (1998)
was reported to the similar experiments have shown that the inoculation
of some probiotic strains, mainly lactic acid bacteria, increase fish
survival after being challenged with fish pathogens while lactic acid
bacteria populations in the gut increase and in one study lactic acid
bacteria inoculation was related with an increase of fish growth rate.
The present study suggests that it is possible to maintain artificially
the gut isolated Bacillus sp. treatment of fish by the immersion
techniques proved successful as against fish pathogen of A. hydrophila.
It is apparent that lactic acid producing bacteria may be used to
control the A. hydrophila infection in fresh water fishes. Statistical
analysis displayed significant differences (p<0.05) in the mortality
of the tilapia between the treatment and control groups. Mortality in
probiotic treatments and control were observed starting on the third day
of contact with the pathogen, but the probiotic treatments were significantly
different from the control when the mortality stabilized ending on the
25th day (Fig. 2). The high dose of the Bacillus sp. did not observe
any negative side effects and this dose was associated with some improvements
in the specific growth rate. However, further studies in which dosages
of Bacillus sp. are repetitively fed to fish and to evaluate the
biological effects of such treatments.
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