ISOLATION OF ACTINOMYCETES FROM THE SEDIMENTS OF PICHAVARAM MANGROVE FOREST, SOUTH INDIA AND ANALYSING THEIR ANTIBACTERIAL EFFICACY

Objective: The aim of the present investigation is to isolate actinomycetes from the sediments of Pichavaram mangrove forest, South India, and to screen for their antibacterial efficiency. Methods: Actinomycetes were isolated by culturing the samples in Starch Casein Agar medium; they were screened primarily for their antibacterial efficiency against Gram-positive and Gram-negative bacterial organisms. Solvent extraction was done with 50% (percentage) ethyl acetate, crude extracts of actinomycetes were prepared at different concentrations using dimethyl sulfoxide and treated against the bacterial organisms. Antibacterial assay was done in Mueller–Hinton agar medium. Results: Thirteen actinomycetes were isolated; among them, four actinomycete isolates (Pichavaram mangrove actinomycete 2 [PMA2], PMA6, PMA9, and PMA13) exhibited antibacterial activity. Conclusion: Isolate PMA2 exhibited very strong antibacterial activity and isolate PMA13 is weakly active against the tested bacterial organisms.


INTRODUCTION
Coastal region is an important region for human beings since the beginning of time. Coastal ecosystem supports coral reefs, seagrasses, marine biota, and the growth of mangrove forest. Mangroves are the most important ecosystems of coastal and marine region. Mangrove forests provide direct and indirect contributions to human beings and natural habitats in the ocean. Soil microbes are important sources for displaying great biological activity against several pathogens. Bioactive molecules are capable of modulating metabolic process and they exhibit beneficial effects such as antioxidant activity, inhibition or induction of enzymes, inhibition of receptor activities, and induction and inhibition of gene expression. They are also useful for the protection mechanism [1]. Actinomycetes have more value both economically and biotechnologically which can produce 80 % of total available antibiotics in the world. The most important genera for the production of antibiotics are Streptomyces and Micromonospora [2]. Actinomycetes exist in various places of environment, including soil, freshwater, and marine water environments. The biologically active terrestrial compounds are overexploited, so the search for the new compounds has been increased toward the marine ecosystem. Marine actinomycetes are different from terrestrial actinomycetes both phylogenetically and physiologically [3]. The marine sediments are capable of synthesizing bioactive secondary metabolites [4,5]. In 2014, the World Health Organization (WHO) reported that the resistance toward antimicrobials has developed much and it is the major challenge to overcome this problem. The WHO also promotes the medicinal sources from the traditional medicinal system [6]. Hence, the study was aimed to screen antibacterial efficiency by producing bioactive natural compounds from the actinomycetes isolated from the sediments of Pichavaram Mangrove Forest, South India.

Sample collection
The water and sediment samples were collected from the mangrove forest in Thandavarayan Sholagan Pet, Chidambaram Taluk, Cuddalore district, Tamil Nadu, South India. The geographical location is shown in Fig. 1.
The field is situated in 11.41° N (North) latitude and 79.79° E (East) longitude at an altitude of above +5.25 M (meter) mean sea level. The water samples were collected in polypropylene tubes and the sediment samples were collected in plastic bags at five different locations at a depth of one feet each [7,8]. The samples were collected at 7.00 a.m. (ante meridian) during winter season. During sample collection, the sample temperature and pH were tested. Then, the samples were brought to the laboratory and stored at 4°C [9].

Salinity test (total dissolved salts [TDS])
To find TDS, 100 ml of water sample was evaporated in a hot air oven. Then, the salt settled at the bottom was measured. The tests were done twice and the average value has been taken [10].

Isolation of actinomycetes
The samples were diluted serially in the water brought from the mangrove forest and 10 −4 diluted sample was plated on starch casein agar medium [10]. The media were supplemented with cycloheximide (25 µg/ml) and nalidixic acid (25 µg/ml) for the inhibition of fungi and Gram-negative bacteria, respectively. The plates were incubated for 7 days at 30°C [9]. The actinomycetes were used against Gram-positive and Gramnegative bacterial organisms to screen their antibacterial efficiency [7].

Test bacterial organisms
Antibacterial susceptibility was detected against ten bacterial strains of the Microbial Type Culture Collection and Gene Bank and listed in Table 1 [11]. Among ten bacterial organisms, each five bacterial organisms are Gram-positive and Gram-negative organisms [12][13][14].

Screening for the antibacterial efficiency
The antibacterial efficiencies of actinomycete isolates were determined against ten bacterial strains. The actinomycete sample

Aarthi et al.
was streaked linearly on the surface of nutrient agar medium exactly at the center and incubated for 7 days at 37°C. The bacterial organisms were inoculated on both sides perpendicularly to the actinomycetes at the distance of 5 mm (millimeter) from the actinomycete [15]. The Gram-negative organisms were streaked on one side and the Gram-positive organisms were on the other side and incubated for 48 h at 37°C [7,13].

Antibacterial assay
Solvent extraction was done with 50% ethyl acetate and diluted in dimethyl sulfoxide for the preparation of different concentrated crude extracts of all the four actinomycetes [12]. The antibacterial activities of the actinomycetes were assessed against ten bacterial organisms using well diffusion assay. Mueller-Hinton agar plates were prepared and swabbed with the bacterial organisms. Four wells were made in 6 mm diameter each [16]. The different concentrated crude extract was loaded in all wells in 100 µl volume. The plates were incubated at 37°C for overnight. The antibacterial activity was then recorded as growth free inhibition zones around the well [17]. The experiments were repeated up to 3 times to find the mean value.

Sample collection
The water and sediment samples were collected from the mangrove forest in Thandavarayan Sholagan Pet, South India. During sample collection, the temperature and pH were verified. The temperature was 28°C and the pH was 7.2.

Salinity test
To find TDS, 100 ml of water sample was evaporated in a hot air oven. The salt settled at the bottom was weighed. It was 2.3 g; therefore, TDS of the water was calculated as 2.3%.

Isolation of actinomycetes
A total of 13 actinomycetes were isolated based on different colony morphology from the samples collected at five different locations. The actinomycetes were named as isolate Pichavaram mangrove actinomycete 1 (PMA1) to isolate PMA13. The colony morphology of the actinomycete isolates was listed in Table 2. Isolate PMA13 was showing mild activity on S. epidermidis. The rest of the bacterial organisms were resistant toward the isolate PMA13. The antibacterial efficiency of actinomycetes is shown in Table 3.

Isolate PMA2
Isolate PMA2 has produced 17±0.62 mm diameter zone of inhibition on B. megaterium with 1.43% of crude extract and 25±0.7 mm diameter zone of inhibition with the same organism at the concentration of 2.86 %. It also produced 15±0.66 mm, 17±0.99 mm, and 19±0.7 mm diameter zone of inhibition on S. aureus at the concentrations of 1.43%, 2.145%, and 2.86%, respectively.

DISCUSSION
Actinomycetes exist in most of the places of nature and they possess the ability of synthesizing several biologically active compounds such as antibacterial, antifungal, antiviral, antiparasitic, herbicides, pesticides, antioxidant, and anti-tumor [1]. We examined the ability of actinomycetes from the sediments of Pichavaram mangrove forest to for their antibacterial efficacy.
In our study, a total of 13 actinomycetes were isolated from five different locations of Pichavaram mangrove forest and perpendicular streaking was done to find out the antibacterial efficiency against ten bacterial organisms. One hundred six actinomycetes were isolated from five different soil samples. These actinomycetes were cross streaked against microbial pathogens [1].
Plant sources contain good bioactive molecules and it acts as a source of antimicrobial and antioxidant agents [18][19][20][21]. Allium cepa possess bioactive natural products and might be used for the treatment of infectious diseases of bacteria [22]. The growth of Salmonella bacteria Thy 1 was inhibited in vivo and it has proved that manila extract has the capability for the inhibition [23]. Ethanol extract of turmeric, cinnamon, and clove was tested against E. coli and S. aureus. These two bacterial organisms are sensitive to all three plants; cinnamon is more active than the other two plants [24].
The primary screening for the antagonistic activity of actinomycetes showed antibacterial activity against both Gram-positive and Gram-negative bacterial organisms [3]. In the same way, the actinomycetes were streaked perpendicularly and they acted against Gram-positive and Gram-negative bacterial organisms. This revealed that the actinomycetes produce antibacterial compound against both Gram-positive and Gram-negative bacterial organisms.
Out of 13 actinomycetes isolates, only 4 (31%) actinomycetes were able to produce antibacterial compound. Thirty actinomycetes were examined, of this, only 53.3% showed a positive result against the test bacteria [25]. Twenty isolates (36.4%) of 55 were potent against the test bacterial organisms. Thirteen isolates showed a positive result to more than one genus, only one isolate showed positive result against all four species of Shigella, seven isolates showed antibacterial activity against three species of Shigella and not with Shigella sonnei [26]. Even, our actinomycetes could produce the antibacterials at less percentage; they work even at very low concentrations.
India is one of the richest wealth of long seacoast and a good source of marine wealth [27]. Marine sponge-associated bacterial isolates have been evidenced as the sources for the production bioactive compounds [28]. Antimicrobial agents contribute themselves significantly in 20 th century, especially therapeutics.
Ethyl acetate extract of the actinomycetes was used in this study, ethyl acetate extract of Bergenia ciliata showed the highest zone of inhibition (7.5 mm) against B. megaterium, moderate activities against Nocardia tenerifensis and B. subtilis with 6.2 mm and 5.5 mm zone of inhibitions, respectively [29]. India like developing countries identifies the new drugs from natural sources. Few antibiotics like tetracycline are extracted from soil actinomycetes [32]. Microorganisms are the good source of enzymes, antimicrobials and they are helpful for the production of various industrial products [8]. It is vital to develop alternative drugs for the treatment of infectious diseases [33].
The sediments of Pichavaram mangroves possess certain important chemical compounds and serve as nutraceuticals, pharmaceuticals, and antimicrobials. These antimicrobials have been recommended to treat various diseases. Natural antimicrobials have greater potential applications and contribute a significant impact on health-care system of human beings and to prevent various diseases [34].

CONCLUSION
The present findings of the study gives a scientific application of the mangrove sediment as antimicrobials and commonly used for various microbial based diseases. Further, we concluded that these natural antimicrobials are the alternatives for synthetic antimicrobial drugs.

ACKNOWLEDGMENTS
The authors are thankful to the Head, Department of Biotechnology, Principal, and Management of K. S. Rangasamy College of Arts and Science (Autonomous), Tiruchengode, for providing necessary laboratory facilities during the study period.