It was observed that the antibiotic production varied significantly
depending on the media constituents. Medium B supported marked
antibiotic production and hence chosen as the basal medium for
optimization. Among the selected actinobacterial strains, S26 showed
pronounced antibacterial activity to all the tested pathogens and
maximum inhibition was against V. harveyi.3.3. Molecular identification and phylogenetic analysis of the
antagonistic actinomycete S26BLASTn analysis of the 16S rRNA nucleotide sequence of S26 at NCBI,
showed 99.77 % similarity to Streptomyces variabilis.
Phylogenetic tree was constructed to study the taxonomic position of S26
with that of 16S rDNA sequences of various Streptomyces spp.
(Fig.3).3.4. Optimization of the mediumEstimation of antibacterial activity with respect to OVAT method
revealed the range of the media components that controlled the
antibiotic production significantly (Fig.4). It was observed that the
bioactivity increased with the increase in concentration of starch up to
1.5 g, beyond which the activity was found to decrease. Similar results
were obtained with the ingredient soy powder, where maximum activity was
obtained at a concentration of 1 g. With increase in concentration of
ammonium sulphate, bactericidal activity increased up to a level of 0.15
g and above that no considerable increase was observed. Absence of
bioactivity was noticed in the range 0-15 ppt salinity and steadily
increased from 15 to 30 ‰ salinity. pH 7 was found to be optimum. The
concentration for the four media components viz. starch, soy
powder, ammonium sulphate and salinity were selected based on the
results of optimization of one variable at a time approach (Fig.4).
Using CCD method, a total of 30 experiments with appropriate combination
of starch, soy powder, ammonium sulphate and salinity were conducted
(Table 3). The matrix was analyzed by
standard analysis of variance
(ANOVA) as approximate to the experimental design used. The ANOVA of the
quadratic regression model demonstrated that the model was highly
significant (Table 4) for the response concerned i.e.,
antimicrobial compound production as evident from the Fisher’s F-
test with a very low probability value [(Pmodel>F) =0.0001]. In this case, linear
coefficient B and C along with quadratic coefficients such as
B2, C2 and D2 were
significant model terms, where ‘B’ is soy powder, ‘C’ is ammonium
sulphate and ‘D’ is salinity (Fig.5). The OVAT analysis of the media
components showed the range of each media component and condition. It
gave an optimum activity when starch was at a concentration of 1
g/100ml; soy powder at 0.5 g/100ml; ammonium sulphate at 0.15 g/100ml;
salinity at 30 ppt and pH at 7.3.5. Streptomyces variabilis S26 as a biocontrol
agentGenerally, the Vibrio count showed an increasing trend over the
culture period in both the treated group and control group. But the
actinomycete (S26) treatment could significantly suppress the
proliferation of these Vibrios (Fig.6). The student t test for
the observed data showed a P value of 0.007. The percentage survival was
significantly higher in the actinomycete treated tanks compared to the
control (Fig.7).4. DiscussionScreening
for novel antimicrobial agents is a continuous process to meet the
increasing demand for therapeutants and overcome the phenomenon of drug
resistance. Actinomycetes being one of the most potential candidates for
the same have been intensively screened from various ecological niches.
Only very few reports are available on the antagonisticStreptomyces from the marine environment and their use as
biocontrol agents in aquaculture systems [18]. Antibiotics belonging
to the classes’ viz., aminoglycosides, ansamycins,
anthracyclines, glycopeptides, macrolides, peptides, tetracyclines, etc.
had been developed from Streptomyces spp. [19, 20].
The discovery of new bioactive compounds hinges upon selective and
sensitive screening methods. In the present study, of the 98 isolates
screened, 49 % showed marked activity against the pathogens. Similar
observations were made by other workers also [21, 22]. Significant
antibacterial activity by actinomycetes against the pathogensviz., S. aureus, P. aeruginosa and B. subtilis andVibrio species could be observed [23, 24, 25]. The
performance of the actinomycete isolates was found to be different in
different media in terms of its inhibitory action against the pathogens.
The composition of the media influenced the production of the active
principle. The importance of the media composition for the production of
antibiotic by marine microorganisms was demonstrated by Okazaki and
Okami [26]. Previous reports by Sujatha et al. [27] states that
the optimization of fermentation conditions can increase the production
of secondary metabolites. The production of secondary metabolites in
actinobacteria is greatly influenced by various fermentation parameters
such as available nutrients [28], pH and temperature [27]
partial pressure of oxygen (pO2) [29], agitation
[30], mineral salts [31], metal ions [32], precursors and
inducers [10], and inhibitors [33]. Actinobacteria that produce
secondary metabolites often have the potential to produce various
compounds from a single strain [34]. In the present study, we could
clearly observe significant increase in activity, with medium B
supporting the best production. The induction might be due to the
complementary interaction of various media ingredients. The strains
seemed to produce antibiotic substances after 7 days in significant
amounts. This might be due to the enhanced secondary metabolite
production during the sporulating stage or due to the rapid utilization
of the initial nutrients to increase the cell number to the threshold
level that is necessary for the production of the antibiotics. The
release of antibiotics by Streptomyces is in a defensive mode to
compete microorganisms in the environment. These are gyrase interfering
small molecules which hinders the growth and replication in competing
microbes. Streptomyces protect themselves with the help of efflux
pumps, ribosomal protection proteins and modifying enzymes.
The pH of the medium at a range of 6-8 was observed to be critical for
the growth of the organisms and the higher and lower level drastically
reduced the antibiotic production which is in agreement with the work of
Sujatha et al. [27]. In the present study, it was observed that
higher concentrations of starch and soy powder inhibited the antibiotic
production by the strain S26, though the growth was enhanced. The
presence of ready to use carbon sources and nutrients usually promote
the growth of actinomycetes but not the antibiotic production. The
effect of ammonium sulphate is due to the presence of
NH4+ that favors the formation of
glutamate in the carbohydrate metabolism, whose one or more
hydroxylation led to antibacterial activity. The absence of growth at
lower salinity was due to the test organism being marine in origin. The
growth pattern in varying pH reconfirmed the optimum pH level as neutral
for the growth of actinomycetes.
ANOVA showed that soy powder, ammonium sulphate and salinity were
controlling antibiotic production significantly. Though the interactive
models seemed to be insignificant, it cannot be avoided as it is a
hierarchical model. The response graphs showed that there is no increase
in response for starch and soy powder above 1 % in the media. It was
also evident that the suggested combinations remained within the
observed range from the 10 solutions generated by the software. As per
the experimental results, the optimum value of the medium components
that gave maximum production of antimicrobial compounds was starch, 1.38
%; soy powder, 0.88 %; ammonium sulphate, 0.16 % and salinity, 27.76
‰. Therefore, by the RSM, the concentration of the medium components
could be reduced considerably yielding better production at a lower
price.
Vibrios are opportunistic pathogens found in the marine environment and
hence under stressful conditions, the chances of infection in shrimps
are more. Since the indiscriminate use of antibiotics has led to the
development of resistance, the use of antibiotics in culture systems is
restricted, and therefore alternate methods for exclusion of diseases
especially Vibrios are essential. Application of indigenous microflora
with potential antimicrobial activity in aquaculture systems would be a
promising alternative. Defoirdt et al. [35] recommended isolating
candidate probiotics from the culture system(s), which will facilitate
their growth and establishment in the host. In the present study,Streptomyces spp. of marine origin was used. Exclusion of
luminescent Vibrios using probiotic Bacillus andStreptomyces has been already reported [36, 37, 38, 39, 40].
Kumar et al. [41] observed the antiviral effect of actinomycetes
while applied in feeds of P. monodon. Aguilera-Rivera et al.
[42] had clearly stated the application of formulated probiotic feed
that effectively reduces the load of Vibrios found in hepatopancreas and
intestine of white shrimp. The exclusion of V. parahaemolyticusby probiotic feed by white shrimp maintained in a biofloc technology was
reported by other studies also [43].
Another study by Sridevi and Dhevendaran [44] has also brought
forward 6 actinomycete candidates isolated from seaweeds that has got
probiotic potential, hence ascertaining the potential of marine
ecosystem for novel probiotics. Studies proven that the probiotic
consortium can act as an effective way for the reduction of
pathogenic Vibrio species and prevention of mortality
during Vibrio challenges [45, 46]. In the present work,
stressful immune-compromising condition was created without water
exchange and removal of excess feed or fecal matter, deteriorating the
water quality. There was significantly high survival rate in penaeid
post larvae compared to the control. The actinomycetes inhibited the
growth of Vibrios by about 63 % in comparison to that of the control.
Hence, S. variabilis S26 can be used as an anti-vibrio agent in
penaeid larval rearing systems. In the present scenario, use of
antibiotics in aquaculture practices is highly limited, necessitating
the application of antagonistic microbes in aquaculture system.
Extraction, purification and characterization of the bioactive principle
might lead to the identification of novel antimicrobial compounds which
can find potential application in medicine.5. AcknowledgementThe authors are thankful to the Department of Biotechnology (DBT), Govt.
of India for the research grant (BT/PR 13761/AAQ/03/514/2010) with which
the work was carried out. We are also grateful to Department of Marine
Biology, Microbiology and Biochemistry and National Centre for Aquatic
Animal Health (NCAAH), Cochin University of Science and Technology for
providing necessary facilities to carry out the work.6. Authors’ contributionSS carried out the experiment of the present work with support from DTB
and DA. The work was carried out under the supervision of RP. SS wrote
the manuscript. BK, MS, DTB and DK reviewed and edited the manuscript.
All authors have read and approved the manuscript.7. Compliance with ethical standards This article does not contain any studies with human participants or
animals performed by any of the authors.