Antagonistic activity of terrestrial Streptomyce s sp . VITNK 9 against Gram negative bacterial pathogens affecting the fish and shellfish in aquaculture

A total of 72 morphologically different actinomycetes isolates were isolated from samples collected at different regions of Vellore, Tamil Nadu, India and screened for its antibacterial activity against fish and shellfish pathogens. All actinomycetes isolates were screened for antibacterial activity by cross streak method against the selected fish and shellfish bacterial pathogens including Aeromonas caviae, Aeromonas hydroplila, Edwardsiella tarda, Vibrio anguillarum and Vibrio harveyi. Secondary screening of antagonistic isolates by well diffusion method leads to the identification of potential isolate. Culture conditions for the potential isolate were optimized for maximal growth and yield of the ethyl acetate (EA) crude extract. The potential isolate was characterized by molecular taxonomy and phylogeny and identified as Streptomyces species and named as Streptomyces sp. VITNK9. The 16S rDNA nucleotide sequence was searched through the GenBank database and showed 83% similarity to Streptomyces vinaceusdrappus. The EA extract prepared from Streptomyces sp. VITNK9 showed moderate antagonistic activity accessed by the formation of zone of growth inhibition against, Aeromonas caviae (15.33 mm), Aeromonas hydrophila (17.66 mm), Edwardsiella tarda (18.33 mm), Vibrio anguillarum (14.33 mm) and Vibrio harveyi (14.33 mm). The MIC value of EA extract was ranged between 0.03-0.125 mg mL-1. The GC-MS spectrum of the ethyl acetate extract revealed the presence of two major compounds, pyrrolo [1,2-A] pyrazine1,4-Dione (56.67%) and Hexahydro-3-(2-Methylpropyl) (27.91%), respectively. The results of the study suggest that Streptomyces sp. VITNK9 is a potential source for antagonistic secondary metabolites against fish and shellfish bacterial


INTRODUCTION
Aquaculture appears to be one of the last frontiers by increasing its contributions to food security in the developing countries (Aly & Albutti 2014).Intensification of aquaculture has led to the conditions favouring the development of various fish diseases.In particular, fish bacterial diseases are responsible for heavy mortality in both wild and cultured fishes.This, in turn, leads to heavy financial losses to the fish farmers.Aquaculture management and disease control have become one of the major problems as the fish bacterial pathogens have become resistant to the conventional drugs being used in the aquaculture industry.Bacterial fish diseases such as hemorrhagic septicemia, edwardsiellosis, bacterial kidney disease, bacterial gill disease, pop eye, vibriosis, fin and tail rot were reported by different workers from different parts of the world (Selvakumar et al. 2010, Sihag & Sharma 2012).Vibriosis is a major disease caused by Vibrio sp.affecting all varieties of shrimps at all stages.The major species causing vibriosis in shrimp are Vibrio alginolyticus, V. harveyi, V. anguillarum and V. parahemolyticus (Goarant et al. 1999, Letchumanan et al. 2014, Wang et al. 2015, Santhakumari et al. 2016).Several strategies have been proposed in control of vibriosis.For example, vaccines have been developed, but generally, cannot be used as a universal disease control measure in aquaculture as they are too much time consuming and labour-intensive (You et al. 2005).So antibiotics and chemotherapeutics remain the method of choice for disease control in the aquaculture industry.Hence, there is an urgent need for the search of safe, effective and novel bioactive compounds from natural sources to deal with the fish bacterial pathogens and to control them in an eco-friendly manner (Thirumurugan & Vijayakumar 2013).Unexploited habitats still remain as a promising source for the discovery of novel bioactive compounds.
Microorganisms are playing a great role in the expansion of drug development.Especially, soil-dwelling microorganisms remain as an excellent resource for the isolation and identification of therapeutically important products.Among them, actinomycetes are extensively distributed in the soil providing many important secondary metabolites of high medical importance, commercial value, and diverse biological applications.Actinomycetes are aerobic, filamentous, spore-forming Gram-positive bacteria with high G+C (60-70%) content in DNA.They are the prime sources of novel bioactive compounds like antibiotics, enzymes and other bioactive compounds (Roshan et al. 2013).Extensive screening of terrestrial actinomycetes has also yielded many important drug leads.Actinomycetes produce more than half of the bioactive compounds in the antibiotic literature database (Lazzarini et al. 2000).The Streptomyces genera of the actinomycetes group are the dominant and primary antibiotic-producing organisms exploited by the pharmaceutical industry.
Streptomyces species are capable of forming heat and desiccation-resistant spores and also most of them are non-pathogenic to plants and animals.Hence, the Streptomyces species isolated from terrestrial origin has been considered as potential biocontrol agents (You et al. 2005).Approximately 7,600 bioactive compounds have been reported from Streptomyces species (Berdy 2005).Actinomycetes have also shown interesting activities in water such as degradation of starch and casein and production of antimicrobial agents.With such types of bioactivities, actinomycetes would play an important role in dealing with the fish bacterial pathogens (Zheng et al. 2000).There are studies on the bioactivity of actinomycetes, in particular, Streptomyces species against human and fish pathogens (Patil et al. 2001).Actinomycetes remain as one of the major natural source for novel and therapeutically bioactive compounds.Among them, many have been developed into drugs for the treatment of wide range of diseases of humans, plants and animals (Vaid & Sajeevan 2016).The objective of present investigation was the isolation, characterization and screening of actinomycetes isolated from the terrestrial regions of Vellore, Tamil Nadu, India, against fish and shellfish pathogens.

SOIL SAMPLING
In the present study, soil samples were collected from different locations of Vellore district (12.9165°N, 79.1325°E), Ambur (12.9165°N, 79.1325°E), Madhanur (11.2777°N, 76.9920°E), Gudiyatham (12.9447°N, 78.8709°E) and Katpadi (12.9796°N, 79.1375°E), Tamil Nadu, India.The soil samples were collected at a depth of 5-10 cm under aseptic conditions in a sterile polyethylene bags and sealed tightly to avoid external contamination and transported immediately to the laboratory.The soil samples were air-dried and kept at 70 ºC for 30 min which stimulates the growth of actinomycetes by eliminating the vegetative Gram negative bacterial cells (Gebreyohannes et al. 2013, Janaki et al. 2014).

TEST PATHOGENS
The fish bacterial pathogens used in this study were Aeromonas caviae (MTCC 7725), A. hydrophila (MTCC 1739), Edwardsiella tarda (MTCC 2400) and Vibrio harveyi (MTCC 7954) which were obtained from Microbial Type Culture Collection and gene bank (MTCC), IMTECH, Chandigarh, India and V. anguillarum being a clinical isolate.The test pathogens Aeromonas caviae, Edwardsiella tarda and A. hydrophila were subcultured on nutrient agar medium (HiMedia, Mumbai, India) 1 whereas, Vibrio harveyi and V. anguillarum were subcultured on nutrient agar supplemented with 2% NaCl and incubated at 37 ºC for 24 h.The test pathogens were stored in nutrient broth containing glycerol stock at -20 ºC until further use.

SCREENING OF ANTIBACTERIAL ACTIVITY OF ACTINOMYCETES AGAINST TEST PATHOGENS PRIMARY SCREENING
The antibacterial activities of the isolated actinomycetes were examined by cross streak method (Ganesan et al. 2016).The Muller Hinton Agar (MHA) plates were prepared.For determining the activity against V. harveyi and V. anguillarum the MHA plates were supplemented with 2% of NaCl.The actinomycetes were inoculated by a single streak in the center of the MHA plates and incubated at 28 ºC for 7 days.The test organisms were streaked right angles to the original actinomycetes isolate and the plates were incubated at 37 ºC for 24 h.Based on the zone of inhibition against the test pathogens, the potential actinomycetes were selected for secondary screening.

SECONDARY SCREENING
The isolates which showed promising activity in the primary screening were inoculated into ISP 1 broth (Hi Media, India) and incubated in a rotary shaker (120 rpm) at 28 ºC for 7 days.The turbidity of the test bacterial suspension was adjusted to 0.5 McFarland standards in 0.85% saline.Lawn culture was spread using sterile swabs.Wells were bored using the wellbore on the MHA plates.The cell-free culture supernatant (CFS) (100 µL) obtained from the actinomycetes isolates was used to screen antagonistic activity against test pathogens by the well diffusion method.The culture plates were incubated at 37 ºC for 24 h and the zone of inhibition formed was measured in millimeters.All the experiments were repeated 3 times and the mean of the zone of inhibition was recorded.The actinomycetes isolate with potential antibacterial activity was selected for characterization and extraction of secondary metabolites.

MORPHOLOGICAL CHARACTERIZATION
Actinomycetes with potential antibacterial activity against the selected fish and shellfish pathogens were identified up to the genus level on the basis of their aerial mass color, reverse side pigments, melanoid pigments and spore chain morphology based upon Bergey's Manual of Determinative Bacteriology (Williams et al. 1989, Sharma 2014).The arrangement of spores in the mycelium was observed under 1000x magnification by cover slip method and the slides were observed under the light microscope (Thenmozhi & Kannabiran 2011).Gram's staining was also performed.The spore surface morphology was observed under scanning electron microscope.

PHYSIOLOGICAL CHARACTERIZATION
The optimum pH, temperature and sodium chloride tolerance of the potential isolate were studied.The pH of the ISP 1 broth was adjusted to 3, 5, 7, 9 and 11.The flasks were inoculated with potential actinomycetes and incubated on a rotary shaker (120 rpm) for 7 days at 28 ºC.Four flasks (250 mL) containing ISP 1 broth were prepared and inoculated with actinomycetes and kept in the incubated rotary shaker (120 rpm) at different temperatures ranging between 25-60 ºC for a period of 7 days.The effect of sodium chloride on the growth of the isolate at various concentrations ranging between 0.5-13% was also analyzed.

UTILIZATION OF CARBON SOURCES
The carbon utilization ability of the potential isolate was studied using carbon utilization agar (ISP 9) supplemented with 1% of various carbon sources like arabinose, cellobiose, glucose, inositol, raffinose and salicin (Nonomura 1974).Carbon utilization agar is used for the characterization of Streptomyces sp. on the basis of carbon source utilized by the Streptomyces isolate.

MOLECULAR TAXONOMIC CHARACTERIZATION
The species level identification of the potential isolate was carried out by16S rRNA partial gene sequencing and construction of the phylogenetic tree.The genomic DNA of the isolate was extracted and 16S rRNA gene was amplified using a forward primer (27F-5'AGAGTTTGATCMTGGCTCAG3') and a reverse primer (1492R-3'TACGGYTACCTTGTTACGACTT5').Sequencing reactions were performed using ABI PRISM® BigDyeTM terminator cycle sequencing kits.The obtained 16S rDNA nucleotide sequence of isolate VITNK9 was searched through the NCBI GenBank data base for sequence similarity using the blast search tool.The program MUSCLE 3.7 was used for multiple sequence alignment (Edgar 2004).The program Gblocks 0.91b was used to cure the resulting aligned sequences (Talavera & Castresana 2007).The phylogeny analysis was performed using the program Mega-6 software and the phylogenetic tree was constructed using the neighbor-joining method.

SECONDARY STRUCTURE PREDICTION AND RESTRICTION SITE ANALYSIS OF 16S RRNA GENE
The secondary structure of the isolate VITNK9 was predicted using the Genebee online software and restriction sites in the 16S rRNA of the potential isolate were analyzed by NEB cutter Version 2.0 available online.

LARGE SCALE FERMENTATION
The seed culture of the isolates which exhibited maximum activity in secondary screening was prepared by inoculating it into the ISP 1 broth, as the potential isolate grew excellently in ISP 1 medium (HiMedia, India) 1 and then incubating it under shaking condition at 28 ºC for 3 days (Abirami et al. 2015).After incubation, about 10 mL of the cultures were transferred into 1L of ISP 1 broth and incubated in an orbital shaker at 28 °C for 7 days.

PREPARATION OF SOLVENTS EXTRACTS
The fermented media was filtered using Whatman no.1 filter paper and the cell-free culture supernatant (CFS) was collected in a sterile conical flask.Liquid -liquid extraction technique was used to obtain the crude extract from the cell-free culture supernatant.Various solvents, ethyl acetate, chloroform and petroleum ether was used for the preparation of extracts and each extract was concentrated using a rotary evaporator.

ANTIBACTERIAL ACTIVITY
The solvent extracts of the potential isolate VITNK9 were used to determine the antibacterial activity against the selected fish bacterial pathogens by the well diffusion method.The bacterial suspension of the test bacteria was adjusted to 10 6 CFU mL -1 and a lawn culture was made on Muller-Hinton agar plates.The crude extract of the isolate was diluted to a concentration of 100µg/100µL in dimethyl sulfoxide.Wells were bored using sterile wellbore on the agar plates and 100 µL of the solvent extracts were added to each well.Ciprofloxacin was used as positive control and respective solvents were also used as negative control.The plates were incubated at 37 ºC for 18-24 h.The zone of inhibition was measured in diameters to assess the antibacterial activity.All the experiments were performed in triplicates.

DETERMINATION OF MINIMAL INHIBITORY CONCENTRATION (MIC)
The MIC of the ethyl acetate extract of the active isolate was determined by serial dilution method in a sterilized 96 well plate.The sterile Muller-Hinton Broth (100 µL) and 100 µL of the overnight culture of the fish bacterial pathogens were added to each well.The ethyl acetate extract was serially diluted from 1 mg mL -1 to 0.01 mg mL -1 for determining the MIC of the active isolate.The MIC was considered as the lowest concentration of which showed no increase in the optimal density (OD 570) read at the microplate reader (Bio-Rad model 680 Microplate reader) 2 .

GAS CHROMATOGRAPHY AND MASS SPECTROMETRY OF THE ETHYL ACETATE CRUDE EXTRACT
The antibacterial bioactive compounds present in the ethyl acetate crude extract was identified by subjecting to the crude to GC-MS analysis using Perkin Elmer workstation Clarus 600GC coupled to a mass spectrometer Elite-5MS (30m x 0.25mm) width film depth of 250 µm capillary tube was used.The instrument had an oven initial temperature of 55 ºC for 3 min and a ramp program which elevates from 6 ºC/min up to 310 ºC further 3 min of the isothermal hold.Helium was used as a carrier gas with a flow rate split ratio of 10:1.About 2 µL of sample was injected and the injector temperature maintained at 250 ºC.Total run time was 32 min.The mass spectrum obtained was compared with spectra available in NIST-LIB 0.5 (National Institute of Standards and Technology) 3 library for matching using the in-built software of GC-MS system (Wiley GC-MS-2007).The concentration of individual compounds present was expressed as percentages through peak area presentation.

ISOLATION AND SCREENING OF ACTINOMYCETES
Based on the colony morphology, a total of 72 actinomycetes were isolated from different terrestrial samples.Distinct actinomycetes colonies were observed in culture plates at 10 -3 and 10 -4 dilutions.Actinomycetes isolate exhibited different colony morphology and characteristics (size, shape, and colour) and the colonies were small to medium sized.The colour of the mature sporulating aerial mycelium ranged from white, gray, blue, light orange to violet.Brown, black and yellow coloured substrate mycelium was also recorded (Table 1).The isolate NN25 produced melanoid pigments.AIA or SCA agar media was used for isolation of all the 72 actinomycetes isolates.All the isolates were screened for antibacterial activity against the selected fish bacterial pathogens.About 18% of the isolates showed antibacterial activity in the primary cross streak method.Among them, only 7% of the isolates had abroad range of antibacterial activity against all the selected fish bacterial pathogens.The isolate VITNK9 exhibited abroad spectrum of activity against all the tested fish bacterial pathogens (Fig. 1).Those isolates with a broad range of antibacterial activity in primary screening were subjected to secondary screening by well diffusion method.Among all the isolates, the isolate VITNK9 showed a broad spectrum of activity against all the tested bacterial 3 <https://www.nist.gov/>Table 1.Cultural characteristics of potential actinomycetes isolates / Características de posibles aislamientos de actinomicetos en cultivo pathogens.The cell-free supernatant (CFS) of the isolate VITNK9 exhibited significant activity against Aeromonas caviae (16 mm), Aeromonas hydrophila (16.5 mm), Edwardsiella tarda (17 mm), Vibrio anguillarum (15 mm) and Vibrio harveyi (14 mm).The fish bacterial pathogen E. tarda was found to be more susceptible and V. harveyi was least susceptible for CFS treatment.The potential isolate was selected for further characterization.

MORPHOLOGICAL CHARACTERIZATION
The colonies of the isolate VITNK9 were medium to large sized, powdery with irregular margin on AIA culture medium (Fig. 2a).The colour of the aerial mycelium was white to gray and substrate mycelium was pale yellow.The complete growth of the isolate was observed on the 14 th day.The isolate was Gram-positive and long chains of spores (oblong in shape) were observed under the light microscopic examination (1000X magnification).Smooth spore surface and hyphae with spiral spore chain morphology were observed under scanning electron microscope (Fig. 2b).The isolate exhibited the typical structural features and morphology of Streptomyces sp.The morphological and biochemical characteristics of the isolate are given in Table 2.

BIOCHEMICAL CHARACTERIZATION
The growth of the isolate VITNK9 on various media is given in Table 3.The isolate was characterized by the methods recommended by International Streptomyces Project.The isolate grew in abundance on ISP 3 and ISP 4 agar media.The isolate also grew excellent in AIA, ISP 1,  glucose soya meal broth, Kuster's agar and Nutrient agar.Good to moderate growth of the isolate was observed in SCA, ISP 5, ISP 6 and ISP 7. Poor growth was observed in ISP 2 and SDA media.
Different biochemical tests were carried out to identify the isolate.The isolate was found to be positive for methyl red test and citrate utilization test and negative for indole, Voges Proskauer's test and triple sugar iron test.It produced amylase, catalase, oxidase and urease, and was negative for nitrate reduction and gelatinase and lipase production.It utilized arabinose, glucose, inositol, mannitol, and salicin as carbon sources for maximal growth and cellubiose and raffinose were not used by the isolate.The culturing conditions were optimized with respect to culture media, pH, temperature and NaCl concentration.The morphological and cultural characterization of the potential isolate revealed the nature and genera of the isolate.

MOLECULAR TAXONOMIC CHARACTERIZATION
The blast search of the 16S rDNA nucleotide sequence (876 bp) of the isolate with the NCBI, GenBank database revealed 83% similarity with Streptomyces vinaceusdrappus (KF554235).The 16S rDNA nucleotide sequence of the isolate was deposited in GenBank (NCBI) with an accession number (KX894540).Based on the molecular characterization and phylogeny of the isolate, it was confirmed to belong to the genus Streptomyces and designated as Streptomyces sp.VITNK9.The phylogenetic tree of the Streptomyces sp.VITNK9 is shown in the Figure 3.

SECONDARY STRUCTURE PREDICTION AND RESTRICTION SITE ANALYSIS OF 16S RRNA GENE
The secondary structure for the isolate Streptomyces sp.VITNK9 was predicted by the greedy method.It was observed that the free energy of the thermodynamic structure was -194.8 kcal mol -1 , energy threshold was -4.0 with cluster factor 2, conserved factor 2, compensated factor 4, and conservativity 0.8.The isolate had 59 restriction enzyme sites.

ANTIBACTERIAL ACTIVITY OF THE SOLVENT EXTRACTS
The isolate VITNK9 was extracted with different solvents including ethyl acetate, petroleum ether, and chloroform.The EA extract showed maximum antibacterial activity   4. Antibacterial activity of the actinomycetes isolate VITNK9 against fish pathogens by well diffusion method / Actividad antibacteriana del aislamiento de actinomicetos VITNK9 frente a patógenos de peces por método de difusión de pozos when compared to other solvent extracts.The EA extract showed antibacterial activity against Aeromonas caviae (15.33 mm), Aeromonas hydrophila (17.66 mm), Edwardsiella tarda (18.33 mm), Vibrio anguillarum (14.33 mm) and Vibrio harveyi (14.33 mm).The antibacterial activity observed with EA extract was comparatively higher than the activity observed with CFS (Table 4).

DETERMINATION OF MINIMAL INHIBITORY CONCENTRATION (MIC)
The MIC value of the ethyl acetate extract prepared from the active isolate was ranged between 0.03 -0.125 mg mL -1 .The MIC value of A. hydrophila and E. tarda was found to be 0.03 mg mL -1 , for V. anguillarum and V. harveyi was 0.06 mg mL -1 and for A. caviae was 0.125 mg mL -1 .

GAS CHROMATOGRAPHY AND MASS SPECTROMETRY OF THE ETHYL ACETATE CRUDE EXTRACT
The partial characterization of the EA crude extract of VITNK9 was carried out by GC-MS and the chromatogram obtained is shown in the Figure 4.The GC-MS spectrum of the EA crude extract revealed the presence of two major compounds (Table 5)

DISCUSSION
Actinomycetes are one of the most indispensable and vibrant bioactive compounds producing microorganisms found in both terrestrial and marine environments.These actinomycetes are fertile producers of antibiotics and new chemical entities as intracellular/ extra cellular secondary metabolites (Hassan et al. 2017).Soil samples have been screened for decades and only very few taxa of actinomycetes have been isolated and studied (Abd-Elnaby et al. 2016).The emerging bacterial diseases have hampered the growth of aquaculture tremendously.Hence, there is a need for developing antibacterial agents from natural sources to control and management of fish and shellfish bacterial diseases.
In the present study, 72 morphologically distinct actinomycetes were isolated from the terrestrial soil samples collected from Vellore, India.Among them, the potential isolate VITNK9 exhibited a significant antibacterial activity against fish and shellfish bacterial pathogens.Isolation of actinomycetes from the terrestrial environment has been inevitably reported earlier (Vaijayanthi et al. 2012, Cholarajan & Vijayakumar 2013, Vijayakumar & Malathi 2014).The isolate VITNK9 produced whitish gray aerial mycelium and pale yellow substrate mycelium.It also had an earthy odour which is the known characteristics of the Streptomyces sp.The colonies of actinomycetes with white and greyish aerial mycelium and yellow substrate mycelium have been reported earlier (Gebreyohannes et al. 2013).The isolate also grew excellently well on ISP3, ISP4, nutrient agar, Kuster's agar and AIA.The potential actinomycetes isolate VITNK9 was characterized by morphological, biochemical, physiological and molecular characterization.The isolate was identified based on the comparison with the keys for classification and identification by Nonomura and Bergey's manual of Determinative Bacteriology (Nonomura 1974, Williams et al. 1989).The 16S rRNA gene sequencing remains as a significant and reliable tool for the identification and confirmation of the actinomycetes up to the genus and species levels.The isolate was identified as Streptomyces sp. and designated as Streptomyces sp.VITNK9 on the basis of the molecular taxonomic characterization (Thirumurugan & Vijayakumar 2015, Augustine et al. 2016, Jenifer et al. 2018).
In the present study, 18% of the isolates showed antibacterial activity against the fish and shellfish bacterial pathogens.There are many reports which illustrate the antimicrobial activity of the actinomycetes isolated from different habitats (Cho et al. 2012, Chaudhary et al. 2013, Gebreyohannes et al. 2013, Ganesan et al. 2017).Antibacterial activity of actinomycetes against human as well as fish bacterial pathogens has been reported (Babuselvam et al. 2016, Jenifer et al. 2018).Mohan et al.(2016) have reported on the antimicrobial activity of actinomycetes extracts against the fish bacterial pathogens.Antibacterial activity of actinobacterial isolates against A. hydrophila, A. sorbia and E. tarda has already been reported (Patil et al. 2001).
In our study, among the various solvents used the ethyl acetate crude extract of the potential Streptomyces sp.VITNK9 had a broad spectrum antibacterial activity against the tested fish and shell fish bacterial pathogens.In a study, the protein extracted from Streptomyces spp.KV3 showed an inhibitory activity against Vibrio harveyi (17 mm) Aeromonas hydrophila (10 mm) and Vibrio alginaticus (9 mm) (Rani & Doss 2016).Similar findings were reported where the ethyl acetate extract of actinomycetes showed antibacterial activity against fish bacterial pathogens (Patil et al. 2016a,b).The ethyl acetate crude extract of the potential isolate VITNK9 showed a broad range of antibacterial activity with the MIC value ranging between 0.03-0.125mg mL -1 against the fish and shellfish pathogens A. caviae, A. hydrophila, E. tarda, V. harveyi and V. anguillarum.The effectiveness of the antibacterial compounds does vary from one species to other, depending upon the factors like sampling site, media composition to the genetic potential of an organism.Tuan et al. (2017) have also reported the antibacterial activity shown by the endophytic actinomycetes (MTR 711,MTR 622 and MTL 121) with the MIC of 93.30 to 300 mg mL -1 against A. hydrophila, A. caviae and S. agalactiae.It was also reported that the partially purified bioactive compound (protein) showed moderate antibacterial activities against the fish bacterial pathogens A. hydrophila, V. harveyi and V. alginolyticus (Pushpa-Rani & Doss 2016).
Streptomyces sp.VITNK9 isolated from the terrestrial soil sample found to be a rich source of antibacterial compounds against the fish and shellfish pathogens.The secondary metabolites (Pyrrolo [1,2-A] pyrazine-1,4-dione and hexahydro-3-(2-Methylpropyl) present in the ethyl acetate crude extract needs to be explored further to develop as an antibacterial agent against fish and shellfish pathogens.

Figure 1 .Figure 2 .
Figure 1.Primary screening of the isolate VITNK9 against the fish bacterial pathogens by cross streak method / Cribado primario del aislado VITNK9 contra los patógenos bacterianos de los peces por el método de raya cruzada

Table 3 . Characteristics of the isolate Streptomyces sp. VITNK9 on different media /
Características del aislamiento de Streptomyces sp.VITNK9 en diferentes medios de cultivo

Table 5 . GC-MS analysis of the ethyl acetate extract of VITNK9 /
Análisis GC-MS del extracto de acetato de etilo de VITNK9