Abstract: The objective of the present study was to isolate and identify high antimicrobial activity producing actinomycetes strains from marine habitat, to study antimicrobial production to determine their for the inhibition of the growth of the common human pathogens. Marine actinomycetes strains were isolated from coastal water of Dhanushkodi, Ramanathapuram District, India. Out of 10 isolated actinomycetes species 3 were identified and selected for antimicrobial activity. Out of the 3 actinomycetes species, Streptomyces sp. showed the best level of antibacterial and antifungal effect against selected human pathogens of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, Klebsiella sp. and Aspergillus niger. So, Streptomyces of actinomycetes may offer the potential to understand and develop treatments for disease based on the normal physiological role of their secondary metabolites. Hence, it is anticipated that the isolation, characterization and the study on actinomycetes can be useful in the discovery of antibiotics and novel species of marine actinomycetes.
INTRODUCTION
Many natural products have been isolated from marine environments. However, only a small fraction of them was derived from marine microorganisms (Munro et al., 1999; Pomponi, 1999). Most secondary metabolites from marine microorganisms found so far were isolated from Streptomyces and Alteromonas sp. (Wagner et al., 2002) Marine actinomycetes are of considerable value as antibiotic producers and other therapeutically useful compounds with diverse biological activities (Bernan et al., 1997; Sharma and Pant, 2001). In the present investigation, it has been observed that compared to other actinomycetes, Streptomyces showed efficient antagonistic activity. Only very few reports are available on the occurrence and distribution of antagonistic Streptomyces in the marine environment (Weyland, 1969; Okazaki and Okami, 1972; Postmaster and Frietas, 1975; Lakshmanaperumalsamy et al., 1994) The objective of the present study was to isolate and identify high antimicrobial activity producing actinomycetes strains from marine habitat, to study antimicrobial production to determine their for the inhibition of the growth of the common human pathogens like Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, Klebsiella sp. and Aspergillus niger.
MATERIALS AND METHODS
The water samples were collected in clean, sanitized and autoclaved bottles and transported to the laboratory for further analysis. The medium used for the isolation and cultivation of actinomycetes was CSPY-ME medium (g L-1): K2 HPO4, 0.5 g; Casein, 3.0 g; Maize starch, 10.0 g; Peptone, 1.0 g; Malt extract, 10.0 g; Agar, 15.0 g and Marine water, 1.0 L) The pH of the medium was adjusted to 7.5. The petriplates were incubated at 37°C for 7 days. The isolated actinomycetes were identified based on the colony morphology and gram staining (Holt et al., 1989).
Identified actinomycetes colonies were incubated at 30± 2°C on rotary shaker at 220 rev/min for 10 days for the enrichment of secondary metabolite producers. Then the culture was collected and centrifuged at 6000 rpm for 15 min and the supernatant immediately transferred and it was filtered through Millipore filter (0.45 μm) to get cell free extract. Antimicrobial activities were assayed in duplicate, using 50 μL of marine bacterial samples in well diffusion assay (Schillinger and Lucke, 1989) and the test organisms were Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, Klebsiella sp. and Aspergillus niger.
RESULTS AND DISCUSSION
Three actinomycetes species were identified by morphological characteristics and by gram staining. Based on these characteristics actinomycetes were identified as Actinopolyspora sp. Nocardia sp. and Streptomyces sp. (Table 1).
Out of three selected and identified actinomycetes, Streptomyces sp. showed significant antimicrobial activity against human pathogens. In the present investigation, zone of inhibition of 57 mm was the highest antimicrobial activity showed by Streptomyces sp. against Pseudomonas aeruginosa. Other two actinomycetes of Actinopolyspora sp. and Nocardia sp. showed antifungal activity only (Table 2).
Based on several studies, among bacteria, the actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products; the Streptomyces are especially prolific (Waksman, 1961; Lachevalier, 1989; Locci, 1989; Saadown and Gharaibeh, 2003). According to Kokare et al. (2004) during the screening of the novel secondary metabolite, actinomycetes isolates are often encountered which show more active antimicrobial activity against gram positive bacteria than gram negative bacteria. But from the observed results, only Streptomyces sp. showed significant antibacterial activity against Staphylococcus aureus next to Pseudomonas aeruginosa.
Normally, Pseudomonas aeruginosa, Klebsiella sp. and Salmonella typhi are even capable of growth in some antibiotics and their resistance to more antibiotics has also been medical concern (Tortora et al., 2000). Marine Streptomyces sp. exhibited the highest antibacterial activity against Pseudomonas aeruginosa followed by Staphylococcus aureus, Klebsiella sp. and Salmonella typhi. It is interesting to note that this response represents an antibiotic potential competing microorganism against Pseudomonas aeruginosa, Salmonella typhi and Klebsiella sp. in the marine environment.
Eighty three percent of actinomycetes isolated from Sagamy Bay were found to be antifungal (Okami and Okazaki, 1972). Many marine microorganisms showed antifungal activity against Aspergillus niger but not against Candida albicans (Kokare et al., 2004). As Aspergillus niger is such a common contaminant, highly effective antibiotics are required. Hundred percent of the tested actinomycetes exhibited antifungal activity. Among the tested isolates Streptomyces sp. showed strong antifungal activity. Among microorganisms, actinomycetes particularly, Streptomyces will proved to be as fruitful as their counter parts isolated from terrestrial habitat (Pisano et al., 1989). However, the marine Streptomyces have not received much attention. Recent investigations indicate that the tremendous potential of marine actinomycetes, particularly Streptomyces sp. as a useful and sustainable source of new bioactive natural products. Thus, the results of the present investigation reveal that the marine actinomycetes from coastal environment are a potent source of novel antibiotics. It is anticipated that the isolation, characterization and the study of actinomycetes can be useful in the discovery of antibiotics and novel species of actinomycetes.
| Table 1: | Identification of Actinomycetes |
| (+) sign indicates Gram positive actinomycetes | |
| Table 2: | Antimicrobial activity of marine actinomycetes against human pathogens |
| (-) sign indicates no zone formation (no antimicrobial activity); where numbers indicates marked inhibition zone | |
ACKNOWLEDGMENTS
Thanks are due to the Department of Zoology and Microbiology, Thiagarajar College, Madurai-9, Tamil Nadu, India for providing all the facilities.