Indo American Journal of Pharmaceutical Research, 2014
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ISSN NO: 2231-6876
INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH
ANTILEPTOSPIRAL POTENTIAL OF MARINE ACTINOMYCETES (MSU5) AGAINST Leptospira interrogans SEROVAR AUTUMNALIS J.Thirumalairaj1, K. Sivasankari2, K. Natarajaseenivasan2 and R. Balagurunathan1* 1 2
Actinobacterial Research Laboratory, Department of Microbiology, Periyar University Salem - 11 Medical Microbiology Laboratory, Department of Microbiology, Bharathidasan University Tiruchirapalli - 24
ARTICLE INFO Article history Received 26/02/2014 Available online 03/03/2014 Keywords Actinomycetes, Bioactive Compounds. Doxycycline, Autumnalis, Marine Sediment and MIC.
ABSTRACT Leptospirosis is a bacterial zoonotic disease caused by different pathogenic serovars of the spirochaete bacterium Leptospira. It mainly survives in both temperate and tropical regions; the occurrence in the tropics is approximately 10 fold higher than in temperate regions. The present day medicines for the treatment of leptospirosis are only effective during the early phase of the disease and no medicines available after the disease become complicated. In this connection, the present study was carried out for the identification of potential anti-leptospiral compounds from marine actinomycetes. From 18 marine sediment samples nearly 141 isolates of actinobacteria were enumerated. The sediments were collected during the months of March to August 2013, along the coast Bay of Bengal, Palk Strait, Gulf of Mannar and Lakshadweep. Crude compounds were produced from the isolates, and all were screened against the Leptospira interrogans serovar Autumnalis strain N2 (Human isolate) to determine the anti leptospiral activity. In the primary screening, 5 samples were recorded by showing promising activity (strain no MSU5, P1, T10, PGM 15 and TCA6) and determined of having anti-leptospiral activity. In the secondary screening strains MSU5, T10 and P1 were showed significant activity. In both the screening MSU5 isolated from Manakudy mangroves, showed an effective inhibition activity against the serovars Autumnalis with a minimum inhibition concentration (MIC) at 125µg.mL-l. The standard antibiotic doxycycline exhibited the MIC with 200µg.mL-l. This is first report for analyzing marine actinomycetes compounds for antileptospiral activity. The study reveals that strain MSU5 shows promising antileptospiral activity against Leptospira serovar Autumnalis and may be a potential candidate for anti-leptospiral components.
Copy right © 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Please cite this article in press as J.Thirumalairaj et al. Antileptospiral Potential of Marine Actinomycetes (MSU5) Against Leptospira Interrogans Serovar Autumnalis. Indo American Journal of Pharm Research.2014:4(02).
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Corresponding author Dr. R. Balagurunathan Professor and Head,Department of Microbiology, Periyar University Salem – 636011.Tamil Nadu, India. 91+ 94434 46325 0427-2345124.
[email protected]
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INTRODUCTION Leptospirosis is one of the most prevalent emerging zoonotic disease of global importance caused by spirochaete bacterium leptospira that infects human, birds, reptiles and wide range of animal species with protean clinical manifestations from acute subclinical to chronic life threatening diseases [6]. It is estimated that 1 to 10 per million populations have been affected by leptospirosis annually worldwide [12]. But, the exact prevalence of leptospirosis still remains unknown. They are mainly present in water bodies like pond, river, swimming pool, moist soil and mud and transmitted through direct contact to the human beings [9]. Leptospires multiply in blood during 8-10 days after its infection. Then the clinical form of the disease varies between one individual to other. It causes mild to severe form of leptospirosis and often end up with mortality. Any antibiotic used to be effective during the early stage of the disease and in the severe form of the illness none of the antibiotic will be effective [7]. Therefore an efficacious drug for the cure of leptospirosis still remains a unanswered problem and antibiotics like penicillin or doxycycline is the preferred antibiotics for the treatment of leptospirosis [11]. It is imperative to develop a new antibiotic to combat against life threatening diseases. Although significant advancement is made in the field of chemical synthesis and engineered biosynthesis for the production of antimicrobial compounds, nature comprises precious and vital source for the novel antibiotics [14, 2 and 17]. To develop new, effective antibiotics from natural origin to combat the menace of leptospirosis is the need of the hour. Actinobacteria are the most important heterotrophic prokaryotes, with extremely diverse metabolites. They generate copious substances indispensable for health such as antibiotics, enzymes, immuno-modulators, etc. In the past few years actinomycetes became the major thriving source for antibiotics [4, 13]. Marine actinomycetes are the richest source for isolation of distinctive bioactive compounds than the terrestrial ones. Hitherto ~ 13,700 bioactive metabolites from actinomycetal origin was identified in which ~ 10,400 (75%) bioactive metabolites were belonging to Streptomyces sp. and 39% of all microbial products [4]. Currently there was no evidenced report for the treatment of leptospirosis using the actinomycetes. In this connection, the present study was carried out to develop potential anti-leptospiral compounds from marine actinobacteria. MATERIALS AND METHODS Sample collection The sediment samples were collected during the months of March to August 2013, along the coast of Bay of Bengal, Palk Strait, Gulf of Mannar and Lakshadweep. From eighteen different collection sites viz. Parangipettai (Lat: 11° 29' N; Long: 79° 45' E), Killai (Lat: 11° 27' N; Long: 79° 46' E), Pitchavaram (Lat: 11° 25' N: Long: 79° 46' E), Tharangampadi (Lat: 11° 1' N; Long: 79° 51' E), Adiramapattinam (Lat: 10° 18' N; Long: 79° 22' E), Manora (Lat: 10° 15' N; Long: 79° 17' E), S.P. Pattinam (Lat: 9° 49' N; Long: 79° 5' E), Thondi (Lat: 9° 43' N; Long: 79° 1' E), Karankadu (Lat: 9° 41' N; Long: 78° 59' E), Mandapam (Lat: 9° 16' N; Long: 79° 9' E), Pamban (Lat: 9° 16' N; Long: 79° 12' E), Rameswaram (Lat: 9° 17' N; Long: 79° 19' E), Katchatheevu (Lat: 9° 22' N; Long: 79° 31' E),Tiruchendur (Lat: 8° 29' N; Long: 78° 7' E) Manakudy (Lat: 8° 4' N; Long: 77° 29'E), Port Blair (Lat: 11° 34' N; Long: 92° 45'E), Sundarbans (Lat: 22° 8' N; Long: 88° 54' E) and Agatti Island (Lat: 10° 51' N; Long: 72° 11' E). The collected samples were carefully transported to the laboratory for further analysis. Pre treatment of marine sediment The collected sediment samples were air-dried at room temperature for 5days. About 5 g of samples were heated at 55°C for 15 min which favours the isolation of actinobacteria by eliminating most unwanted Gram negative and other spore forming bacteria [1].
Cultivation of Leptospiral strains Leptospiral reference strains were cultivated in the Ellinghausen-McCullough-Johnson-Harris (EMJH-BD, USA) medium containing bovine serum albumin (Sigma). The strains were inoculated in EMJH liquid or semisolid media (with 0.2% agar) at 30ºC for seven days [7]. All Leptospiral cultures were maintained by periodical sub culturing. For primary screening of the extracts, the
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Production and extraction of bioactive metabolites from selected actinobacteria All the selected actinobacterial cultures were inoculated into yeast extract-malt extract (ISP2) agar plates (20 ml/plate) and incubated at 28oC for 10 days. After incubation, mycelial growth was removed aseptically using sterile spatula. The pigment containing agar medium was cut in to small pieces and extracted using methanol for overnight at room temperature [16]. The solvent extracts were concentrated under reduced pressure and quantified.
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Isolation of marine actinobacteria The pretreated marine and mangrove sediment samples were taken separately for serial dilution for the isolation of actinobacteria using starch casein agar medium supplemented with 100 μg/ml of nalidixic acid and 20 μg/ml of nystatin (Himedia, Mumbai) to minimize the bacterial and fungal contaminations respectively. Then the plates were incubated in an inverted position at 28 ± 2 °C for 28 days for isolation of colonies. Of the total isolated colonies, the strains with same size, colour, morphology and reverse side pigments were eliminated and finally 45 strains were selected from 141 isolates for the anti-leptospiral studies. The pure colonies were selected, sub cultured on the yeast extract malt extract medium (ISP2) and preserved both as slant culture in yeast extract malt extract medium (ISP2) [21] and as glycerol stock in 30% glycerol at - 20o C. Apart from this 45 isolates, 50 strains of actinobacteria obtained from culture collection facility maintained at Department of Microbiology, Periyar University, Salem were also included for antileptospiral activity.
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strain from the pathogenic Leptospira interrogans serovar Autumnalis strain N2 (a locally isolated human strain) was used to assess the antileptospiral potency of the actinomycetes extracts. The antimicrobial agents like doxycycline hyclate, doxycycline hydrochloride hemiethanolate hemihydrate (1 mg/ml) (Sigma, USA) were used as standards. Initial antibiotic stocks were prepared as per manufactures instructions, filter sterilized and stored at -20ºC until use [23]. Primary Screening The primary screening was done in 96 well plates, 95 Actinomycetes extracts were processed. Each extract was weighed 5 mg and dissolved in 1 ml of DMSO for the initial stock. Seven days old culture was used as inoculum and the extracts were serially diluted at a concentration of 500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.9 µg. mL -1 with control samples and incubated for 2 hours and the inhibition was determined under the dark field microscope (Optica B500 Italy) [15]. Secondary Screening The minimal inhibitory concentration (MIC) was determined by the broth dilution method, which was performed in 10 ml sterile culture tubes [10]. Seven days old culture was used as inoculums and the concentration were determined under dark field microscope [15]. Inoculum was added to make a final concentration of 1 x 10 8 organisms/ml. The tubes were incubated at 30°C for 7 days in dark condition. For MIC, five concentrations were used (500, 250, 125, 62.5, 31.25 µg. mL-1) as per the observations made in primary screening. The extracts were serially diluted and incubated for seven days at 30°C [23]. All the experiments were performed in triplicates with control samples like culture as control - 1, sterile media as control - 2, and sterile media with the addition of DMSO as control - 3, the pure culture with sterile DMSO also checked for the inhibitory activity of the DMSO. After seven days of incubation the OD (optical density) of the samples were determined using spectrophotometers at 400 nm. RESULTS The marine sediment samples showed the diversity of actinobacteria with maximum 8 X 10 3 CFU.g-1 at Pitchavaram followed by Parangipettai with 6 X 103 CFU.g-1 and minimum was recorded at Tharangampadi with 1 X 103 CFU.g-1 Table 1. In the present study, totally 141 actinobacterial strains were isolated from marine and mangrove ecosystems of eighteen collection sites Table 1. Of which 45 isolates were selected based on the colony characteristics such as growth, consistency, aerial mass colour, reverse side pigment, soluble pigment, aerial mycelium and substrate mycelium. In addition, another 50 isolates were obtained from culture collection facility maintained at Department of Microbiology, Periyar University, Salem. Table 1. Sampling details of various marine sediments S. No
Ecosystem
pH
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Gulf of Mannar (Tamilnadu) Tiruchendur (Tamilnadu) Sundarbans (West Bengal) Thondi (Tamilnadu) Karankadu (Tamilnadu) Mandapam (Tamilnadu) Pamaban (Tamilnadu) Rameswaram (Tamilnadu) Port Blair (Andaman) Agatti Island (Lakshadweep) Parangipettai (Tamilnadu) Pitchavaram (Tamilnadu) Killai (Tamilnadu) Tharangampadi (Tamilnadu) Manora (Tamilnadu) Adiramapattinam (Tamilnadu) Manakudy (Tamilnadu) Katchatheevu (SriLanka)
7.5 7.5 7.4 7.4 7.5 7.5 7.5 7.5 7.5 7.5 7.4 7.4 7.3 7.4 7.4 7.5 7.3 7.5
No. of colonies (10-3XCFU.g-l) 2 3 6 4 3 2 2 3 2 0 6 8 2 1 2 3 5 3
No. of isolates selected 2 3 4 3 2 1 2 2 1 0 6 8 2 1 1 1 4 2
141
45
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Total
Total no. of isolates 5 10 12 11 6 5 5 8 5 0 15 20 5 2 5 7 14 6
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Table 2. Growth and morphological pattern of actinomycetes isolated from different marine ecosystems Characteristics Growth Consistency Aerial Mass colour
Pigment production Micro morphology
Appearance Good Moderate Powdery Rough White Grey Dirty white Chocolate brown Yellow Orange Green Brown Pink Reverse side pigment Soluble pigment Aerial mycelium Substrate mycelium
No. of Isolates (%) 88 (92.6) 7 (7.3) 85 (89.4) 10 (10.5) 41 (43.1) 30 (31.5) 13 (13.6) 3 (3.1) 1 (1) 1 (1) 2 (2.1) 3 (3.1) 1 (1) 61 (64.2) 20 (21) 95 (100) 95 (100)
Table 3. MIC of actinomycetes extracts against pathogenic Leptospira interrogans serovar Autumnalis strain N2 MIC (µg.mL-1) D* E* PGM 15 200 850 TCA6 200 800 T10 200 500 P1 200 250 MSU5 200 125 *D-doxycycline, *E- actinomycetes extract Strain No.
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Totally, 95 crude compounds were screened for anti-leptospiral activity. In the primary screening, 5 samples were recorded by showing promising antileptospiral activity (MSU5, P1, T10, PGM 15 and TCA6). In the secondary screening, strains MSU5, T10 and P1 Table 3. Were showed remarkable activity. In both the screening, MSU5 isolated from Manakudy mangroves, showed an effective inhibition activity against serovars of Leptospira with MIC at 125 µg. mL-1 Table 3 & Fig 1. When compared to the MIC of standard antibiotic doxycycline (200 µg.mL-1), the results evidenced that further purification of the strain MSU5 will definitely shows better activity than standard antibiotics like penicillin and doxycycline used as standards.
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Figure 1. Determination of MIC (Minimum Inhibitory Concentration) of the actinomycetes extracts against pathogenic Leptospira interrogans serovar Autumnalis strain N2
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DISCUSSION The counts of marine actinomycetes were recorded maximum 8 X 103 CFU.g-1 at Pitchavaram similar findings were observed in earlier studies, whereas the maximum 12X 105 CFU.g-1 counts of sediment actinomycetes were recorded in Palk Strait region [24], Andaman Islands [3], and marine actinomycetes were recorded maximum (5X 105 CFU.g-1) at Thondi [19]. Surprisingly, there was no actinomycete found in Agatti Island, like wise reported that, no actinomycetes counts recorded in water sample in Bay of Bengal [18]. This may be actinomycetes can forms only a small fraction of micro flora and in sediment they can survive as spores or resting propagules [8]. In the present study, the lowest MIC of strain MSU5 against pathogenic Leptospira interrogans serovar Autumnalis strain N2 was 125µg. mL-1. Chandan et al [5] reported that the methanolic extract of Phyllanthus amarus and Eclipta Alba shows that significant antileptospiral activity against different serovars of Leptospira at 100 µg. mL-1. Similarly Vedhagiri et al [23] reported that the methanolic extract of seaweed Asparagopsis taxiformis against serovars of Leptospira ranging from 100 ≥ 400µg. mL-1, Seesom et al [20] reported that the crude extract of xanthones from Mangosteen against serovars of Leptospira ranging from 200 ≥ 800µg. mL-1 and Uma et al [22] reported that the extract of nilavembu kudineer and seenthil sarkarai shows that antileptospiral activity against different serovars of Leptospira at 500 µg. mL-1. Even though, the crude extract from actinobacteria showed the MIC of 125µg. mL-1. This has showed significant MIC compare to the medicinal plants extracts reported elsewhere. So Further purification of the crude compound produced by MSU5 will certainly provide efficient activity. Hence the strain MSU5 may be a potent source for isolating novel anti-leptospiral components. Purification, structural characterization and optimization of the bioactive compound from MSU5 to be carried out further for the identification of the active components. CONCLUSION Leptospirosis will definitely stay a considerable public health problem for the forthcoming years, in spite of the extent of climatic changes that are probably to favours raise in its global incidence. Therefore, in this present study, an attempt was made to isolate the potential anti-leptospiral compounds from marine actinomycetes. This is first report for analyzing marine actinomycetes compounds for antileptospiral activity. The present study validates that the marine actinomycete strain MSU5 isolated from Manakudy mangroves will be a potent source for the isolation of bioactive compounds for the antileptospiral activity. Further detailed studies on this strain MSU5 will definitely bring out a potential drug for the treatment of leptospirosis. Conflict of interest statement We declare that we have no conflict of interest.
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ACKNOWLEDGEMENTS Authors are thank to Vice-Chancellor and Registrar Periyar University Salem and Bharathidasan University Tiruchirapalli for providing facilities to conduct research work, and Indian Council of Medical Research New Delhi (Ref no. 58/9/2011- BMS dt. 26.10.2012) for financial assistance in the form of research grant.
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16. Pazhanimurugan R, Gopikrishnan V, Shanmugasundaram T, Radhakrishnan M and Balagurunathan R, Bioactive potential of actinobacteria against drug resistant pathogens. J Appl Pharm Sci. 2012, 02: 167-173. 17. Pelaez F, The historical derive of antibiotic from microbial natural product – can history repeat? J Biochem Pharmacol. 2006, 71: 981-990. 18. Ramesh S, Jayaprakashvel M and Mathivanan N, Microbial status in seawater and coastal sediments during pre and posttsunami periods in the Bay of Bengal, India. Mar Ecol 2006, 27:198-203. 19. Ravikumar S, Thirumalairaj J and Gokulakrishnan R, Assessment of Microbial Diversity In Relation To Biochemical Constituents along Palk Strait. International Journal of Innovative Research in Science, Engineering and Technology, 2013, 2(11): 6431-6437. 20. Seesom W, Jaratrungtawee A, Suksamrarn S, Mekseepralard C, Ratananukul P and Sukhumsirichart W, Antileptospiral activity of xanthones from Garcinia mangostana and synergy of gamma-mangostin with penicillin G. BMC Complement Altern Med. 2013, 13(182): 2-6. 21. Shirling EB and Gottlieb D, Methods for characterization of Streptomyces species. Int J Syst Bacteriol. 1966, 16(3): 313-340. 22. Uma KS, Jacob M, Arumugam G, Kalpana S and Mayilvahanan Natarajan, In vitro antimicrobial activity of the siddha drugs seenthil sarkarai and nilavembu kudineer against leptospira. Int j pharm pharm sci. 2012, 4(2): 75-78. 23. Vedhagiri K, Aseer Manilal, Valliyammai T, Shanmughapriya S, Sujith S, Joseph Selvin et al, Antimicrobial potential of a marine seaweed Asparagopsis taxiformis against Leptospira javanica isolates of rodent reservoirs. Annals of Microbiol. 2009, 9(3): 1- 7. 24. Vijayakumar R, Muthukumar C, Thajuddin N, Pannerselvam A and Saravanamuthu R, Studies on the diversity of actinomycetes in the Palk Strait region of Bay of Bengal, India. Actinomycetologica. 2007, 21(2): 59-65.
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