Int J Pharm Pharm Sci, Vol 6, Issue 7, 144-147Original Article
STUDY ON ANTIMYCOBACTERIAL ACTIVITY OF MARINE ACTINOMYCETES FROM COROMONDAL COASTAL REGION OF SOUTHERN INDIA
P.GAJALAKSHMI1, A.RAJA2, R. ARTHUR JAMES3
1Department of Microbiology, Dhanalakshmi Srinivasan College of Arts and Science for Women, Perambalur 621212, Tamil Nadu, India, 2Department of Microbiology, Jamal Mohamed college, Tiruchirapalli, Tamilnadu,India, 3Department of Marine Science, Bharathidasan University, Tiruchirapalli 620024, India.
Email: gaja_black@yahoo.co.in, rajajmcmicro@yahoo.co.in
Received: 19 Jun 2014 Revised and Accepted: 21 Jul 2014
ABSTRACT
Objective: The objective of the present study is to screen the novel actinomycetes from marine sediment active against mycobacterium sp.
Methods: actinomycetes were isolated by crowded plate technique and its antimycobacterial activity was determined by stroke method followed by optimized fermentation. The active fraction was extracted and bio assayed by TLC.
Results: Totally 25 actinomycetes were isolated from 9 Marine sediment samples and frequency of isolates were, 40% of Streptomyces sp (ECRST) 32% of Micromonospora(ECRMM), 16% of Micropolyspora (ECRMP) and 12% were Streptoverticillium (ECRSV). Among the 25 isolates Microployspora a rare actinobacteria designated as ECRMP 4 showed potent antimycobacterial activity against M.tuberculosis. The zone of inhibition was 20 ± 0.1mm and the percentage of relative inhibitory zone was 62 against M.tuberculosis. The bioassay of crude compound prevails that the Rf value of compound is 0.16.
Conclusion: The present study concludes that the isolated marine Micropolyspora capable to produce noval antimycobacterial compound effective against M.tuberculosis.
Keywords: Micropolyspora, M.smegmatis,M.tuberculosis, Relative zone of inhibition.
INTRODUCTION
Multi drug resistant tuberculosis strains are generally considered to be those resistant to at least two drugs, such as INH and Rifampicin most frequently in the countries of the former Soviet Union and in Asia. The current prophylaxis for TB is very long and results in significant toxicity, development of resistant strains and is unable to eliminate the latent bacilli [1]. On this view point, attempts have been made to develop new drug against MDR – TB. Marine environment covers almost 70% of the earth surface [2].
Isolation of rare actinomycetes form marine sediment targeted in drug discovery due to its diverse nature of biomolecules. The ocean remains as an unexploited source for many drugs and pharmacologically active substances [3].The role of marine actinomycetes in drug discovery dramatically increased from the past few years due to substantial improvement in screening methods. Actinobacteria are gram positive, filamentous bacteria which are supreme secondary metabolite producers [4] which shows a range of biological activities including antibacterial, antifungal, anticancer, antitumor, cytotoxic, cytostatic, anti-inflammatory, anti-parasitic, anti-malaria, antiviral, antioxidant, anti-angiogenesis, etc. Of them, many have been obtained from Streptomyces [5] and these natural products have been an extraordinary source for lead structures in the development of new drugs [6]. It is apparent that the marine environment is a potent source for finding new actinobacteria and biologically active substances [7].
The present work was undertaken to isolate potent actinobacteria from marine sample and to assess their antimycobacterial activity.
MATERIALS AND METHODS
Sample collection
Marine sediment samples were collected from east coast region starting from Pondicherry to Mahabalipuram at 10intches depth by hand core sampler in a sterilized container and transferred to the laboratory for further processing.
Isolation & identification of actinobacteria [8]
Isolation and enumeration of actinobacteria was performed on selective media, Actinomycetes Isolation Agar (AIA). The samples were pretreated with 0.1% phenol and after serial dilution one milliliter of 107 dilution samples were poured and overlayerd with sterile AIA supplemented with Amphotericin B (10 µg/ml). Plates were incubated at room temperature for 7days. The isolated actinobacteria are characterized by spore and biochemical method and the results were compared with Bergey’s manual of Systemic Bacteriology.
Test organism
The test organisms Mycobacterium smegmatis MTCC NO-6 and Mycobacterium tuberculosis were collected from Bioline laboratory and maintained in Dubos oleic agar at -20c.
Fermentation process [9]
All the actinobacterial isolates were inoculated into one litre production broth (ISP4 Media) in a bio reactor and fermentation was carried out at 200rpm at 280c for 7 days. After fermentation, the medium was harvested and centrifuged to remove cell debris. Filtrates were collected and lyophilized at 40C for further use.
Primary antimicrobial screening [10-11]
Mycobacterium smegmatis was inoculated on dubos plates. In each of these plates, wells were cut out using a sterilized gel borer. 100 µl of lyophilized filtrates were loaded into each well. Plates were incubated at 35+/- 20c for 3 days. After incubation, all the plates were examined for the presence of zone of inhibition around the Wells.
Extraction of bioactive metabolites [12 - 13]
The bioactive metabolites were collected from the harvested medium by solvent extraction method. The filtrate was mixed with ethyl acetate and shaken vigorously for 1 hour in a solvent extraction funnel. Solvent and filtrate mixture were stabilized for 24-48 hrs. After 48 hrs the solvent phase was separated from aqueous phase. The solvent extracts were concentrated and used for antibacterial activity.
Secondary screening (Disc diffusion method)
Secondary antimicrobial screening of actinobacteria was detected by disc diffusion method on dubos medium. The crude extract was re dissolved in distilled water at 1mg/ml concentration and about 25µg, 50µg and100µg of samples were loaded on the sterile disc and its activity was checked against Mycobacterium tuberculosis by stroke method. The percentage of relative zone of inhibition was calculated as follows
Thin layer chromatography [14]
10 µl of the ethyl acetate fractions were applied on Silica gel plates and the chromatogram was developed using chloroform: methanol (4:1) as solvent. The spots in the chromatogram were visualized in UV chamber and it was bio assayed. All the tests were conducted in triplicate.
RESULTS AND DISCUSSION
A total 25 actinobacteria colonies (Table-1) was isolated from nine Marine sediment samples and identified based on spore morphology. Of these nine samples, maximum number of isolates was obtained at Auroville site. Among the 25 isolates, Strptomyces was the most frequently isolated species.
Based on colony morphology and microscopic appearance the isolated strains were designated as ECRST (Streptoyces), ECRMM (Micromonospora), ECRMP (Micropolyspora), ECRSV (Strepto verticillium). The frequency of isolates were Streptomyces (40%), Micromonospora (32%), Micropolyspora(16%) and Strptoverticilium (12%). marine sediments were good sources for isolation of actinobacteria and M2 media is the most suitable agent for the purpose [15].
Colonies were 3-4 mm in diameter, initially had a smooth appearance but later developed a weft of aerial mycelium which is floccose, granular, and powdery in nature. The aerial and substrate mycelium are non fragmented (except ECRMP isolate) and showed the production of conidiospores. Monospores and arthrospore on the mycelium.
Table 1: Number of isolated actinomycetes and its sampling site
| Sample site | Offshore distance in km | Lattitide | Longitude | Depth (intches) | Number of isolates |
| Puducherry beach | 10 | 11.9300° N | 79.8300° E | 10 | 4 |
| auroville | 10 | 12.0069° N | 79.8106° E | 10 | 5 |
| Anichakuppam | 10 | 11.890° N | 79.8147° E | 10 | 2 |
| pudukuppam | 10 | 12.053276° N | 79.873057 | 10 | 2 |
| marakkanam | 10 | 12.187655° N | 79.937655 ° E | 10 | 3 |
| vennangupattu | 10 | 12.237715° N | 79.96482 ° E | 10 | 3 |
| Ammanur | 10 | 12.3648° N, | 80.0122 | 10 | 2 |
| Kalpakkam | 10 | 12.5576° N, | 80.1754° E | 10 | 2 |
| Mahabalipuram | 10 | 12.357655° N | 80.127655 ° E | 10 | 2 |
| Total | 25 |
Table 2: Colony and Spore Morphology of isolated actinomycetes
| Site | CFU | Strain Code | Colony Colour | Spore |
| Puducherry Beach | 14x107 | ECRST1 | Grey, powdery | rectiflexibiles |
| ECRMM1 | White, granular | Monospore | ||
| ECRMM2 | Ash, granular | Monospore | ||
| ECRST2 | Dark Grey, powdery | retinaculiaperti | ||
| Anichakuppam | 8 x107 | ECRST3 | Greyish white powdery | rectiflexibiles |
| ECRST4 | white,powdery | rectiflexibiles | ||
| Auroville | 16 x107 | ECRST5 | Chalky White, powdery | rectiflexibiles |
| ECRST6 | Light Ash, fine powdery | retinaculiaperti | ||
| ECRMP1 | Dark grey, leathery | Spiral | ||
| ECRSV1 | Ash, powdery | Ellipsoidal | ||
| ECRMM3 | Chalky white, powdery | Monospore | ||
| Vennangupattu | 6 x107 | ECRST7 | Ash, powdery | retinaculiaperti |
| ECRST8 | White, powdery | Spiral | ||
| ECRMM4 | Ash granular | Monospore | ||
| Pudukuppam | 6 x107 | ECRST9 | Sandal white,powdery | Spiral |
| ECRMM5 | Grey, granular | Monospore | ||
| Ammanur | 5 x107 | ECRMM6 | Ash, powdery | Monospore |
| ECRMP2 | Dark grey, granular | Spiral | ||
| Marakkanam | 7 x107 | ECRMP3 | Dark grey, granular | Spiral |
| ECRMP4 | Dark grey,granular | Spiral | ||
| ECRSV2 | Grey,leathery | Ellipsoidal | ||
| Kalpakkam | 8 x107 | ECRST10 | Dull white powdery | Spiral |
| ECRMM7 | Dull white | Monospore | ||
| Mahabalipuram | 6 x107 | ECRSV3 | Greeish Grey,leathery | Ellipsoidal |
| ECRMM8 | Ash,granular | Monospore |
Among the Strptomyces isolate 70% of them produced spiral chains of spore and less often spirally coiled, retinaculiaperti and refractile features (table 2) on substrate mycelium. Strains of Micromonospora sp were gram positive, did not make aerial mycelium, did not produce sporangia, and produced nonmotile mono spores that were borne singly off branched hyphae. Streptomyces was described as Streptomycetaceae that formed spores in chains on aerial hyphae [16]. Micropolyspora produced chain of spore are differentiated from Streptomyces by the fragmenting nature of the aerial mycelium [17]. Carbon and nitrogen utilization tests were performed according to standard methods described for actinomycetes [18]. Strains belongs to Strptomyces utilized L asparagines as nitrogen sources. Among the ten Strptomyces (ECRST1 to ECRST10) only two isolates designated as ECRST8 and ECRST9 failed to utilize carbon and nitrogen source. Similarly, isolates belongs to Streptoverticiliun (ECRSV) also failed to utilize L asparagine (table 3). In this screening, ISP4 medium was found to be the most significant medium for the growth and antibiotic production. The optimized growth condition was found to be 200 rpm at 28 ° C for 7 days on ISP4 broth (Fig 1).
Table 3: Utilization of carbon and nitrogen source by actinomycetes isolates
| Strain Code | Nitrogen | Carbon | ||||
| D-glucose | L rhamnose | D-Xylose | D-Mannitol | L-arabinose | ||
| ECRST1 | + | + | + | + | + | + |
| ECRST2 | + | + | + | + | + | + |
| ECRST3 | + | + | + | + | + | + |
| ECRST4 | + | + | + | + | + | + |
| ECRST5 | + | + | + | + | + | + |
| ECRST6 | + | + | + | + | + | + |
| ECRST7 | + | + | + | + | + | + |
| ECRST8 | - | + | + | - | - | - |
| ECRST9 | - | + | + | - | - | - |
| ECRST10 | + | + | + | + | + | + |
| ECRMM1 | + | + | + | + | + | + |
| ECRMM2 | + | + | + | + | + | + |
| ECRMM3 | + | + | + | + | + | + |
| ECRMM4 | + | + | + | + | + | + |
| ECRMM5 | + | + | + | + | + | + |
| ECRMM6 | + | + | + | + | + | - |
| ECRMM7 | + | + | + | + | + | - |
| ECRMM8 | + | + | + | + | + | - |
| ECRMP1 | + | + | + | + | + | - |
| ECRMP2 | + | + | + | + | + | + |
| ECRMP3 | + | + | + | + | + | + |
| ECRMP4 | + | + | + | + | + | + |
| ECRSV1 | - | + | + | + | + | + |
| ECRSV2 | - | + | + | + | + | + |
| ECRSV3 | - | + | + | + | + | + |
Table 4: Antimycobacterial Study of Isolated Actinomycetes against Mycobacterium sp
| Strain Code | M.smegmatis | Mycobacterium tuberculosis (disc diffusion) | |||
| Sample | PC | NC | RIZD | ||
| ECRST1 | - | - | - | - | - |
| ECRST2 | - | - | - | - | - |
| ECRST3 | - | - | - | - | - |
| ECRST4 | 8 mm | - | - | - | - |
| ECRST5 | - | - | - | - | |
| ECRST6 | - | - | - | - | - |
| ECRST7 | - | - | - | - | - |
| ECRST8 | - | - | - | - | - |
| ECRST9 | - | - | - | - | - |
| ECRST10 | - | - | - | ||
| ECRMM1 | - | - | - | - | - |
| ECRMM2 | - | - | - | ||
| ECRMM3 | - | - | - | - | - |
| ECRMM4 | - | - | - | ||
| ECRMM5 | - | - | - | - | - |
| ECRMM6 | - | - | - | ||
| ECRMM7 | - | - | - | - | - |
| ECRMM8 | - | - | - | ||
| ECRMP1 | - | - | - | - | - |
| ECRMP2 | - | - | - | ||
| ECRMP3 | - | - | - | - | - |
| ECRMP4 | 18±0.68mm | 20±0.1mm | 16±0.2mm | 8±0.24mm | 62 |
| ECRSV1 | - | - | - | - | - |
| ECRSV2 | - | - | - | ||
| ECRSV3 | - | - | - | - | - |
PC: Positive control Streptomycin, NC: ethyl acetate
In the antimycobacterial study of these 25 isolates, Microployspora (ECRMP 4) is a rare actinobacteria showed potent antimycobacterial activity against both Mycobacterium. The maximum inhibition zone was 20 ± 0.1mm against M. tuberculosis activity followed by 18 ± 0.68 mm zone of inhibition against M. smegmatis (table 4). Similarly the percentage of relative inhibition zone was 66% against M.tuberculosis at 100µg. However the Microployspora (ECRMP 4) grown in ISP1 and ISP2 did not show any antimicrobial activity. Rare actinomycetes are become an increasingly attractive source in the search for new antibiotics to fight drug resistance [19]. From the discovery of streptomycin from Streptomyces griseus, numerous anti TB antibiotics such as kanamycin and rifampicin have been reported from actinomycetes isolated only from terrestrial origin but not from marine source [20]. The bioassay of TLC fraction prevails that the Rf value of active compound is 0.16. Consequently further investigations like IR, NMR and MS are needed in order to determine the active metabolite.
Fig. 1: Growth rate of isolated actinomycetes on different ISP medium at 200 rpm.
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