INTRODUCTION:

Actinobacteria are most economically valuable prokaryotes which are well known to produce chemically diverse metabolites with wide range of biological activity^[^1]. Actinobacteria are unparalleled sources of bioactive metabolites including antibiotics, enzymes, vitamins, plant growth promoters, and other substances^[^2]. Ocean has been considered as a rich source of compounds with novel structures and biological activities. The marine environmental conditions are extremely different from terrestrial ecosystem; hence marine actinobacteria have different characteristics from those of terrestrial counterparts and therefore might produce different types of bioactive compounds^[^3]. Unlike their terrestrial relatives, marine actinobacteria have yet to be extensively investigated. Moreover, genetic adaptations to the marine environment appear to include novel biosynthetic capabilities^[^4,5] given the remarkable success in developing clinically useful drugs from terrestrial actinobacteria.

Among actinobacteria, around 7600 compounds are produced by Streptomyces species they can produce around 80% of the total antibiotics currently available in the market and other active secondary metabolites^[^6-8]. Disease control and management in aquaculture has become one of the major problems as the fish bacterial pathogens are becoming more and more resistant to the conventional therapeutic drugs used in the industry and thus the fish farmers suffer from heavy financial losses. Hence, there is an urgent need for the search of novel bioactive compounds with therapeutic potential which can be used to control the bacterial disease in an eco-friendly manner. So, the present study deals with the isolation and characterization of marine actinobacteria isolated from east coast region of Tamilnadu as well as screening of the their antibacterial activity against a fish pathogenic bacteria.

MATERIALS AND METHODS:

Collection of soil samples:

Marine sediment samples were collected from ten different locations of East Coast Regions (ECR) of Tamilnadu, South India into sterile polythene bags. Then the samples were dried/ pretreated in an oven at 40°C for seven to ten days to reduce the numbers of vegetative bacterial cells^[^9] and stored at 4°C for further use. The samples were serially diluted and spread plate technique was implemented for the isolation of actinobacteria on 50% sea water starch casein agar (SCA) and actinomycetes isolation agar media with cyclohexamide 25mg/ml and nalidixic acid 20mg/ml^[10] for the prevention of other microbial contaminations. All the plates were incubated at 28ºC for 7 to 15 days. After incubation, the actinobacterial colonies were purified and sub-cultured on SCA medium.

Isolation of bacterial pathogens:

The infected prawns were collected from local market, transferred to laboratory and stored at 4^oC. The infected region (muscles) was separated and homogenized using sterile mortar and pestle. One milliliter of the homogenized sample was inoculated by using pour plate technique on nutrient agar and Thiosulfate-citrate-bile salts-sucrose (TCBS) agar media and incubated at 37ºC for 24-48 h. After incubation, the bacterial colonies were identified by using morphological, cultural and biochemical characters. The identified bacterial cultures were maintained on nutrient agar slants at 4ºC for further use.

Screening of antibacterial activity of actinobacteria

Primary screening:

The antibacterial activity of the actinobacteria was primarily screened by cross streak plate method^[^11] against Vibrio alginolyticus, V. cholera, V. parahaemolyticus, Pseudomonas sp. and Aeromonas sp. The actinobacterial cultures were streaked at the corner of the Petri plates containing SCA and incubated at 28ºC for 5 days, and then 24 h old fish pathogen was inoculated opposite to the original streak of actinobacteria again the plates were incubated at 37ºC for 24 h. Based on the presence of inhibition zone, the isolates with antibacterial activity were selected for further study.

Secondary screening:

The isolates with antibacterial activity in primarily screening were evaluated further for their antibacterial efficacy by shake flask culture method^[^12]. The pure culture of actinobacteria were inoculated in to starch casein broth and incubated in shaking condition (120rpm) at 28ºC for 5 days. The production medium was centrifuged in cooling centrifuge (at 4ºC) at 8000 rpm for 30 min. The crude antibacterial compound was extracted from supernatant by sequential liquid-liquid extraction method using organic solvents like acetone, chloroform, ethyl acetate, petroleum ether and methanol. Then, the antibacterial efficacy of the crude compound was tested against Vibrio sp., Aeromonas sp. and Pseudomonas sp. by well diffusion method^[^13]. After incubation at 37ºC for 24h, the diameters of the inhibition zones were recorded.

Characterization of actinobacteria:

Actinobacteria with potential antibacterial activity were identified based on their morphological (aerial mass colour, reverse side pigment, melanoid pigments and spore chain morphology), physiological and biochemical characteristics by the standard methods of International Streptomyces Project (ISP)^[^14,15].

RESULTS AND DISCUSSION:

A total of 311 actinobacterial colonies were isolated from 11 different locations of east coast of India on SCA medium. Among 311 colonies, 82 were morphologically different (Table 1).

Table 1. No. of colonies of actinomycetes in different station.

S. No	Name of the sampling station	No. of colonies (CFU/g)		Station wise total colonies
S. No	Name of the sampling station	10^-3	10^-4
1	Marina Beach station	05	07	03
2	Neelankarai	10	06	05
3	Nedukuppam	06	13	04
4	Muttukadu	18	21	14
5	Mahapalipuram	16	09	06
6	Poonjeri	11	13	05
7	Kalapet	08	09	06
8	Pitchavaram	28	21	16
9	Annagkoil	20	14	10
10	Samiyar pet	23	19	06
11	Cuddalore	21	12	07
Total colonies		166	145	82
Overall total colonies		311		82

The isolates were produced small to medium sized, white, ash, green and brown coloured, circle shaped, powdery with regular margin colonies with grey, black, red and golden yellow coloured reverse side was formed (Table 2). Similarly, the previous reported that different colour and size of actinobacteria were isolated^[^16].

Table 2. Cultural characteristics of antagonistic actinobacterial isolates.

S. No	Isolate code	Aerial mass colour	Reverse side pigment	Melanoid pigment
1	ECR1	White	-	-
2	ECR2	Light ash	yellow	-
3	ECR4	Ash	Black	-
4	ECR5	Whitish ash	Yellow	-
5	ECR6	Ash	Yellow	-
6	ECR10	White	-	-
7	ECR11	White	Yellow	-
8	ECR13	Greenish ash	Brownish black	-
9	ECR16	Whitish gray	-	-
10	ECR20	White	-	-
11	ECR23	Grayish white	Red	Green
12	ECR28	Ash	Brown	Green
13	ECR31	Ash	Red	-
14	ECR34	Ash	-	-
15	ECR64	White	-	-
16	ECR67	Ash	-	-
17	ECR69	Ash	-	-
18	ECR75	Ash	Orange	-
19	ECR77	Gray	Brown	-
20	ECR78	Greenish ash	-	-
21	ECR81	Ash	Orange	-

= No pigment

Table 3. Antibacterial activity of actinobacteria against fish pathogens by cross streak method.

S.No	Name of Actinobacteria	Zone of inhibition (mm)
S.No	Name of Actinobacteria	*V. cholarae*	*V. parahaemolyticus*	*V. alginolyticus*	*Pseudomonas sp.*	*Aeromonas* *sp.*
1	ECR - 1	-	3	-	-	-
2	ECR - 2	-	3	-	6	-
3	ECR - 4	6	-	4	3	-
4	ECR - 5	-	-	5	-	-
5	ECR - 6	-	-	3	-	3
6	ECR - 10	5	-	3	3	-
7	ECR - 11	3	-	-	-	-
8	ECR – 13	5	2	-	4	-
9	ECR – 16	-	3	-	4	-
10	ECR - 20	4	6	-	5	-
11	ECR – 23	5	-	4	6	3
12	ECR – 28	4	-	-	-	-
13	ECR – 31	-	3	-	6	-
14	ECR – 34	-	3	-	-	-
15	ECR – 64	6	4	7	4	-
16	ECR – 67	-	3	3	-	-
17	ECR – 69	4	-	-	5	-
18	ECR – 75	-	4	-	7	-
19	ECR – 77	9	4	7	6	-
20	ECR – 78	4	-	3	-	-
21	ECR – 81	-	3	-	-	4

All the 82 isolates were tested for their antibacterial activity against fish pathogenic bacteria namely Vibrio alginolyticus, V. parahaemolyticus, V. cholera, Pseudomonas sp. and Aeromonas sp. by cross streak method. Out of 82 isolates, 21 (25.6%) isolates had antibacterial activity against fish pathogenic bacteria (Table 3).

Among the 21antibacterial isolates, 12 (57.14%) isolates had activity against both V. parahaemolyticus and Pseudomonas sp., 11 (52.38%) against V. cholera, 9 (42.85%) against V. alginolyticus and 3 (14.28%) against Aeromonas sp. in primary screening. Among 21 antibacterial compound producers, the isolates ECR77 and ECR64 showed good inhibitory activity against all the bacterial pathogens tested except Aeromonas sp. in secondary screening. Hence, these two isolates were selected for further studies. Correspondingly, the earlier study reported that, out of 68 actinobacteria from saltpan environment of Vedaranyam, India, 25 (36.8%) isolates produced antimicrobial activity against various pathogens tested: 22 (88%) with antibacterial activity, 16 (64%) with antifungal activity and 13 (52%) with both antibacterial and antifungal activities^[16]. Similarly, it was reported that, among the 10 isolates from marine sediment, the isolate ECR3 showed maximum activity against V. parahaemolyticus, V. harvei, V. alginolyticus, Pseudomonas sp. and Aeromonas hydrophilia^[17].Thus, the present and previous studies reported that, the antimicrobial potentialities of the marine actinobacteria have not been uniformly distributed. It has been greatly influenced by ecological, biological and genetic properties of the isolates and culture media used for the isolation and screening of antimicrobial activity. In the present study, among the various solvents used for the extraction of the antibacterial compounds, ethyl acetate extract of the isolate ECR77 and

ECR64 were expressed maximum activity in the range of 4-16mm and 4-11mm respectively than petroleum ether extract against fish pathogens, and acetone extract of the isolate ECR77 showed less activity and no activity was found in acetone extract of the isolate ECR64 (Table 4).

Similarly, the screening of the marine actinobacterial culture extract was carried out by the disc method, which resulted in more than 10 to 30mm diameter inhibition zone against Aeromonas hydrophila, V. harveyi, V. parahaemolyticus, V. alginolyticus and Serratia sp.^[^18]. Similar type of antimicrobial compound extraction studies from actinobacteria were reported by several workers^[^12,16]. Based on these studies it is clear that, solvent selection and extraction processes are of the important step in the antimicrobial compound production. Both the two potent isolates were showed excellent growth and formed abundant aerial mycelium on 50% sea water SCA medium. The isolate ECR77 was produced un-branched aerial mycelium, grey colored substrate mycelium with brown coloured reverse pigment, and the aerial and substrate mycelia were medium dependent with smooth and long chained spores, and ECR64 produced, white coloured aerial mycelium and does not produce reverse side pigment. The isolate produced smooth and long chained spores (Table 5).

Both the isolates did not produce any melanoid pigments. Based on the morphological properties (colony and microscopic morphology), the isolates ECR77 and ECR64 were tentatively identified as genus Streptomyces sp. ECR77 and Streptomyces sp. ECR64 (Table 5). The identification of the potential isolates were compared with the keys of Nonomura (1974)^[^19]and Bergey’s Manual of Determinative Bacteriology^[20].

Table 4. Antibacterial activity of actinobacteria against Fish pathogen by well diffusion method.

S. No	ECR77 + Solvent	Zone of inhibition (mm)
S. No	ECR77 + Solvent	*V. cholarae*	*V. parahaemolyticus*	*V. alginolyticus*	*Pseudomonas sp.*	*Aeromonas* *sp.*
1	Acetone	06	-	08	04	-
2	Ethyl acetate	13	04	16	10	-
3	Petroleum ether	07	-	10	08	-
	ECR64+Solvent
4	Acetone	-	-	-	-	-
5	Ethyl acetate	11mm	04mm	09mm	11mm	-
6	Petroleum ether	04mm	-	06mm	10mm	-

Table 5. Morphological characterization of potential actinobacterial isolates.

S. No	Characteristics	Results
S. No	Characteristics	ECR77	ECR64
*Morphological properties*
1.	Presence of aerial and substrate mycelia	+	+
2.	Spores in aerial mycelium	+	+
3.	Spores in substrate mycelium	-	-
4.	No. of spores on aerial mycelium	21-50	20-50
5.	Shape of spores	Globose	Globose
6.	Spore chain morphology	Rectiflexibles	Spirals
7.	Spore surface	smooth	smooth
8.	Aerial mycelium colour	Grey	white
9.	Reverse side colour	Brown	-
10.	Melanin pigment	-	-

CONCLUSION:

The speciation of the potential isolates and characterization of antibacterial compounds will be the future course work. The results of the present investigation reported that the marine actinobacteria from east coast of India could be an alternative potent source of eco-friendly novel antibiotics against fish pathogens instead of current drugs used in aquaculture.

REFERENCES:

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Received on 30.01.2013 Accepted on 25.04.2013

Asian J. Pharm. Res. 3(2): April- June 2013; Page 75-78