Antimicrobial potential of sponge associated marine actinomycetes

Journal de Mycologie Médicale (2008) 18, 16—22

D i s p o n i b l e e n l i g n e s u r w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m y c m e d

ORIGINAL ARTICLE/ARTICLE ORIGINAL

Antimicrobial potential of sponge associated marine actinomycetes Potentiel antimicrobien d’actinomycètes marins associés aux éponges R. Gandhimathi a, M. Arunkumar a, J. Selvin a,*, T. Thangavelu a, S. Sivaramakrishnan b, G.S. Kiran b, S. Shanmughapriya a, K. Natarajaseenivasan a a b

Department of Microbiology, Bharathidasan University, 620 024 Tiruchirappalli, India Department of Biotechnology, Bharathidasan University, 620 024 Tiruchirappalli, India

Received 5 September 2007; received in revised form 26 October 2007; accepted 3 November 2007 Available online 8 February 2008

KEYWORDS Marine sponge; Marine actinomycetes; Antimicrobial activity; Streptomyces

MOTS CLÉS Éponge marine; Actinomycètes marins;

Summary Objectives. — The sponge—microbial association is a potential chemical ecological phenomenon, which provides sustainable source of supply for developing novel pharmaceutical leads. Material and methods. — A total collection of 26 marine endosymbiotic actinomycete strains, isolated from the Bay of Bengal (coast of India), were screened for antagonistic and antimicrobial activity against pathogenic bacteria and fungi. Marine sponge agar was the most appropriate medium for the isolation of marine endosymbiotic actinomycetes. Results. — No unique actinomycetes association pattern was observed among the sponges. Among the total culturable microbial symbionts associated with Callyspongia diffusa, 38.46% were actinomycetes. The endosymbiotic marine actinomycetes exhibited potent antimicrobial activity against the growth of human pathogens. Particularly, the strains CPI 3, CPI 9, CPI 12 and CPI 13 showed the highest antimicrobial activity. Based on the RDP-II classifier programme and conventional classification system, the potential producer CPI 13 was identified as Streptomyces spp. Conclusion. — The results of the present investigation revealed that, the marine actinomycetes from the sponges are a potential source of novel antibiotic leads. # 2007 Elsevier Masson SAS. All rights reserved. Résumé Objectifs. — L’association éponge—microbe est un phénomène écologique, chimique et potentiel qui constitue une source originale pour le développement d’une nouvelle entité pharmaceutique.

* Corresponding author. E-mail address: [email protected] (J. Selvin). 1156-5233/$ — see front matter # 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2007.11.001

Antimicrobial potential of sponge associated marine actinomycetes Activité antimicrobienne; Streptomyces

17

Mate´riel et me ´thodes. — Vingt-six souches d’actinomycètes symbiotiques marins, isolées de la baie de côte du Bengale, Inde, ont été criblées pour leur activité antimicrobienne contre les bactéries et les champignons pathogènes. L’agar marin d’éponge était le milieu le plus approprié pour l’isolement d’actinomycètes symbiotiques marin. Re ´sultats. — Aucun modèle unique d’association d’actinomycètes avec les éponges n’a été observé. Parmi les microbes symbiotiques associés avec Callyspongia diffusa, 38,46 % étaient des actinomycètes. Les actinomycètes marins symbiotiques ont montré une activité antimicrobienne puissante contre la croissance des germes pathogènes humains. Particulièrement, les souches CPI 3, CPI 9, CPI 12 et CPI 13 ont montré la plus importante activité antimicrobienne. Sur la base de programme de classification RDP et le système de classification conventionnel, le producteur potentiel CPI 13 a été identifié comme Streptomyces sp. Conclusion. — Les résultats de cette investigation ont révélé que les actinomycètes marins associés aux éponges constituent une source potentielle pour le développement d’antibiotiques originaux. # 2007 Elsevier Masson SAS. All rights reserved.

Introduction Sponges (Porifera) have been recognized as a rich source of novel compounds that are of potential interest to mankind [11]. They produce secondary metabolites and other compounds to repel and deter predators [25] and compete for space with other sessile species. Of the investigated marine sponge species, more than 10% have exhibited cytotoxic activity [41] suggesting production of potential medicinals. They were determined as potential source of novel antimicrobial agents [13,31]1. Some sponges seem to produce potentially useful antifouling agents [3]. Although many bioactive compounds have been discovered in sponges [11,28,29,33], only a few of these compounds have been commercialized [11]. A serious obstacle to the ultimate development of most marine natural products that are currently undergoing clinical trails or that are in preclinical trial evaluation is the problem of supply. Therefore, the research has been focused to disclose the mechanism of secondary metabolites synthesis [1,32]. Since marine organisms live in a significantly different environment from those of the terrestrial organisms, it is reasonable to expect that their secondary metabolite will differ considerably. The presence of large amounts of microorganisms within the mesophyl of many demosponges has been well—documented [16,17]. Bacteria can contribute up to 40% of the sponge biomass (equal to about 108—109 bacteria/g of tissue) and are probably permanently associated with the host sponge unless they are disturbed by external stress factors [12,35,40]. A number of bacteria and cyanobacteria associated with sponges were found to be the sources of antibiotics and other bioactive compounds in the marine environment. It was reported that the wider biosynthetic capabilities of sponges were associated with the symbiotic microorganism [1]. The marine bacterium, Pseudomonas isolated from its host sponge Suberea creba collected from the Coral sea of New Caledonia produced strong antibiotic quinines [7]. Albeit, the marine actinomycetes are considered as a rich source of novel antimicrobial agents, these potential resources were

scarcely explored. Though the genomic and metabolomic diversity of marine actinomycetes remains unknown, many promising bioactive compounds including antimicrobial, antitumour, immunosuppressive agents and enzymes are being discovered in marine actinomycetes [21]. Recently, actinomycetes associated with marine sponges have been reported as richest source of potential antagonists [10,26,32]. In the present study, we report the findings of antimicrobial potential of actinomycetes associated with marine sponges collected from the Bay of Bengal coast.

Materials and methods Sample collection Marine sponges were collected from the Bay of Bengal region of the Indian peninsular coast by scuba diving at 10—15 m depth. To avoid cross-contamination, only unbroken samples were used for microbiological analysis. The specimens were kept 2 h in sterilized aged seawater (ASW) to remove loosely associated microorganisms from inner and outer sponge surfaces. It has been hypothesized that this process may eliminate nonassociated bacteria from the host sponge by digestion. Environmental water representing the sponge habitat was taken prior to sponge sampling and filled up in 1 L sterilized glass bottles. Habitat water was used for isolation of bacteria and fungi on ZoBell marine agar and starch casein agar (supplemented with 2% NaCl), respectively. The isolates were screened for antibiotic susceptibility to select highly active antibiotics (data not shown). The highly active antibiotics were used as selective enrichment to prevent cross-contamination of environmental microorganisms remained in the sponge tissue. Samples were surface cleaned with sterile aged seawater and surface sterilized with 70% alcohol to eliminate epiphytic microorganisms. Then, the samples were kept in a sterile incubator oven for 1 h at 40 8C to dry the surface, and frozen ( 20 8C) immediately in sterile sip-lap bags. Identification of samples was carried out by, P.A. Thomas, an eminent sponge taxonomist in India.

Isolation of actinomycetes 1

Touati I, Chaieb K, Bakhrouf A, Gaddour K. Screening of antimicrobial activity of marine sponge extracts collected from Tunisian coast. J Med Mycol; 2007:17;183—187.

Isolation and enumeration of actinomycetes were performed on different media. A measured area of sponge tissue

18 (1-cm2 area) was excised using a sterile scalpel from the internal mesophyl area. The tissue was then homogenized in sterile ASW using a tissue homogenizer. The homogenate was serially diluted and all dilutions were placed on the media. The isolation was performed on the selective media such as marine sponge agar (MSA) (raffinose: 10 g; L-histidine: 1 g; ferrous sulphate: 0.01 g; dipotassium hydrogen phosphate: 1 g; calcium carbonate: 0.5 g; agar: 15 g; sodium chloride: 20 g; aqueous host sponge extract: 100 ml; double distilled water: 900 ml; pH 7.8; autoclaved at 15 lbs for 15 min) and standard media such as Emerson agar (EA) and modified marine agar (MMA). All these media were supplemented with 20 mM each of nalidixic acid and cyclohexamide to inhibit the bacterial and fungal contamination, respectively. Sabouruad dextrose agar (SDA) with 2% NaCl and marine agar were used for the isolation of fungi and other bacteria. The relative occurrence of actinomycetes with marine sponges was determined based on the percentage composition of actinomycetes to the total culturable microorganisms. Classification of actninomycetes were performed as per Lachevelier [20].

R. Gandhimathi et al.

The inoculum (1 ml) obtained from 72 h shake flask culture was used to generate dense growth in marine actinomycetes broth (MAB) and incubated at 30 8C under dark for 12 days. The resultant culture was used as stock culture. During subculture, the isolates were growing on media without NaCl. Therefore, fermentation conditions were optimized using the media without NaCl. To develop fermentation conditions for the production of antibacterial agents, a phosphate-limited minimal medium was developed based on the method described by Bruheim et al. [4]. A 10% inoculum was used in the 500-ml baffled Erlynmeyer flasks for submerged fermentation. The modified mineral medium (actinorhodin mineral medium (AMM)) was prepared as gram per liter of distilled water containing 9.0 NaNO3; 0.3 K2HPO4; 2.0 FeSO4; 1.6 MnSO4; 0.04 ZnSO4; 0.2 CoCl2; 0.1 MgSO4; 0.05 CaCl2; 2.0 NaCl; 1% sponge extract; 0.001 cAMP. The medium was supplemented with 2 ml trace mineral solution containing (gram per liter of distilled water): 13.5 FeCl3
6H2O; 1.5 CuCl2
2H2O; 9 ZnCl2; 3.6 MnCl2
4H2O; 0.6 Na2MoO4
2H2O; 0.4 CoCl2
6H2O and 0.3 H3BO4. Glucose was used as carbon source with a batch concentration of 125 g/L. Triplicates of 12 flasks were incubated at 30 8C at 300 rpm under dark for 12 days. The cell-free supernatant was used for the extraction of active principles. Acetone was added to the cell-free supernatant in the ratio of 1:1 (v/v) and the resultant aliquot was kept overnight at 4 8C. Then, the aliquot was centrifuged at 5400  g for 15 min at 4 8C and the pellet was dissolved in 3 ml of 0.2 M phosphate buffer, 1% between 80 and 3 ml of acetone, respectively. All three aliquots were screened for antibacterial activity and the effective phosphate buffer aliquot was used in further experiments.

buffered saline was used as test solution. The assay was carried out in LB agar for potential human pathogens (clinical and reference strains) such as Micrococcus luteus (ML), haemolytic Streptococcus (HS), Klebseilla pneumoniae (KP), Staphylococcus epidermidis (SE), Proteus mirabilis (PM), Escherichia coli (EC), Enterococccus faecalis (EF), Bacillus spp. (BS), Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA). The concentration of inoculum used for the assay was standardized using 1 ml of different dilutions of 18 h fresh culture per 10 ml of appropriate assay medium, so that clearly defined zones of inhibition were produced up to the minimum test concentration. First, the base layer was prepared with 1.5% agar at a thickness of 1— 2 mm to avoid seepage of test compound through the bottom interface. Six sterile porcelain cylinders with outside diameter of 8 mm and length 10 mm were placed on the base layer about 608 apart before pouring the seed layer. Then, the seed layer was prepared by adding the determined quantity of the test organism to the medium at a temperature of 40 and 50 8C and immediately poured the inoculated medium on the top of base layer. After solidifying the seed layer, the cylinders were carefully removed using a sterile forceps so that the uniform cavities were formed in the seed layer. Fifty microliters each of reference standard (10 mM amphotericin) and test compound were added in each well. Three alternate wells on each plate were filled with a reference standard and the remaining three were filled with test solution. The reference standard was used for the verification of strain sensitivity/resistance pattern. Inhibition area was measured after overnight incubation at 30 8C.Screening for antifungal activity Antifungal activity was determined using the microbroth dilution method as per NCCLS document M27-A [24]. The pathogenic test strains including Candida tropicalis, Aspergillus fumigatus and Aspergillus flavus were grown overnight on SDA at 32 8C. Tests were performed in RPMI 1640 buffered to pH 7.0 with 0.165 M morpholine propane sulphonic acid (MOPS) and supplemented with 2% glucose. Stock inoculums were prepared from 48-h-old cultures grown on SDA at 32 8C. Inoculum was prepared to a turbidity equivalent to that of a 0.5 McFarland standard in 0.9% (w/v) NaCl and desired inoculum levels were obtained based on the number of viable colonies per millilitre on SDA. The dosing range was determined by the factor 1.25 (0.1 log) as to get dose range between 10 and 92.9 mg protein per milliliter of cell-free extract. An aliquot of 0.1 ml inoculum each and 10 ml each of different concentration of test were added to each well of the microdilution titre plat and incubated at 32 8C. The MIC endpoints were read after 72 h of incubation. The minimum fungicidal concentration (MFC) was determined as the lowest test concentration at which there was either no growth or fewer than three colonies. All the tests were conducted in triplicates and averages were expressed as activity in terms of mg protein per milliliter of extract.

Screening for antagonistic potential

Molecular identification of producers

Antagonistic property of the isolates was evaluated using conventional cross-streak method [8]. Modified cylinder plate double layer diffusion method [31] was used for the determination of antibacterial activity of the isolates. Acetone extract of cell-free supernatant aliquoted in phosphate

Genomic DNA of highly active isolates CPI 3, CPI 9, CPI 12 and CPI 13 were extracted by methods as described in earlier reports [9]. Both universal and genus specific primers were used for the amplification of DNA. The reaction mixture was prepared in a total volume of 50 ml including 100 ng of

Extraction of crude bioactives

19

Antimicrobial potential of sponge associated marine actinomycetes template DNA, 5 ml of 10  PCR reaction buffer (100 mM Tris—HCl; pH 9.0; 50 mM KCl; 1.5 mM MgCl2; 0.1% (w/v) Triton X-100; 0.2 ml BSA; 2.5 U of Taq DNA polymerase (Finnzyme); 200 mM of each dNTP; 2 mM forward primer and 2 mM reverse primer). The complete reaction mixture was incubated in a gradient thermocycler (Eppendorf). The PCR temperature profile used was thus 94 8C for 3 min, then 30 cycles consisting of 94 8C for 1 min, 48 8C for 2 min, 72 8C for 2 min and finally an extension step at 72 8C for 6 min. PCR products were analyzed by electrophoresis on 0.8% (w/v) agarose TAE-gels. The PCR product was cloned by the TA cloning method using a TOPO TA Cloning1 kit according to the manufacturer’s instructions (Invitrogen) for sequencing. The 16S ribosomal RNA gene sequence obtained from the isolates was compared with other bacterial sequences by using NCBI BLASTn. Taxonomic affiliation of the sequences was retrieved from classifier program of ribosomal database project (RDP-II) [39]. RDP-II hierarchy is based on the new phylogenetically consistent higher-order bacterial taxonomy proposed by Garrity et al. [14].

Results and discussion The peninsular coast of India is a hotspot of diverse marine floral and faunal assemblages particularly sponges, sea anemones, sea cucumber, sea urchin, soft corals and diverse number of seaweeds. Most of these pristine resources have not been explored for bioprospecting and/or microbial ecological studies. The sponge—microbial association is a potential chemical, ecological phenomenon, which provides sustainable source of supply for developing novel pharmaceutical leads. The collected sponges were extracted in methanol: dicholomethane (1:1) [31] and screened for antibacterial activity against M. luteus and Bacillus subtilis. All the collected sponges including Fasciospongia cavernosa (FC), Spongia offiscinalis (SO), Callyspongia diffusa (CD), Spirastrella inconstans (SI) and Tedania anhelans (TA) showed antibacterial activity. Therefore, microbial isolations were performed from these sponges. Among the different media used, MSA was found to be the most appropriate medium for the isolation of marine endosymbiotic actinomycetes. The predominant morphotypes were white circular colonies (65%) followed by translucent irregular colonies (25%) and translucent circular colonies (10%). White circular and irregular colonies appeared after 96 h of incubation at 26 8C and translucent colonies appeared after eight days of incubation. Out of this colony morphology, only white circular and irregular colonies showed antagonistic property in cross-streak method. Based on the colony morphology, 26 morphotypes were retrieved and designated as CSIR project isolate (CPI). The culturable actinomycetes associated with the sponge C. diffusa was 10  105 cfu/cm2 area of tissue followed by S. offiscinalis (6  105 cfu/cm2), F. cavernosa (5  105 cfu/ cm2), T. anhelans (3  105 cfu/cm2) and S. inconstans (2  105 cfu/cm2). No unique actinomycetes association pattern was observed among the sponges. Among the total culturable microbial symbionts associated with C. diffusa, 38.46% were actinomycetes. The level of actinomycetes association was drastically decreased to 7.69% in S. inconstans (Figure 1). No direct correlation was observed between

Figure 1 Relative composition of actinomycetes in sponges. Figure 1 Composition relative des actinomycètes associés aux éponges marines.

the relative occurrence of actinomycetes and potential producers or antimicrobial activity. The culturable composition of other bacteria and fungi are presented in Table 1. In the case of other bacterial association, the sponge F. cavernosa and S. inconstans represents high degree of association (7  106 cfu/cm2 = 31.82% of total culturable bacteria) whereas 13.64% bacteria associated with the sponge S. offiscinalis and 22.73% bacteria associated with the sponge C. diffusa. The sponge T. anhelans did not possess any bacterial or fungal association. Therefore, the culturable community analysis of the marine sponge T. anhelans requires innovative approaches to validate the present findings. Fungal isolates were obtained only from S. inconstans and all other sponges were free from culturable fungal association. Among the assemblages of eubacteria, the literature evidenced that the actinomycetes group invariably deserved as potential producers [22,30,38]. The endosymbiotic marine actinomycetes showed potent antimicrobial activity against the growth of human pathogens. Among the isolates, 76.9% showed antimicrobial activity in the primary screening. Only actinomycetes were potential producers and no producer was perceived among the bacterial and fungal isolates. It was found that 27% of actinomycetes associated with F. cavernosa and C. diffusa were potential producers (Figure 2). The strains CPI 3, CPI 9, CPI 12 and CPI 13 showed highest antimicrobial activity. All the potential producers invariably showed activity against Gram positive bacteria, but only 60% showed activity against Gram negative bacteria. This trend has already been reported [19] and Gram positive strains particularly M. luteus was Table 1 Culturable microbial composition of marine sponges. Tableau 1 Flore microbienne cultivable d’éponges marines. Sponges Actinomycetes Bacteria Fungi (105 cfu/cm2) (106 cfu/cm2) (102 cfu/cm2) FC SO CD SI TA

5 6 10 2 3

7 3 5 7 —

— — — 5 —

20

R. Gandhimathi et al.

Table 2 Antibacterial activity of sponge associated bacteria. Tableau 2 Activité antibactérienne des bactéries associées aux éponges marines. Sponge isolates

EC

BS

SA

ML

PM

HS

PA

SE

KP

CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI CPI

R R R 31.4 R 37.68 R R R 43.96 R R R R R R R R R R R

R R R R R 47.1 R R R 50.24 34.54 R R R R R R R R R R

R R R R 37.68 R R R R R R 31.4 R R R R R R R R R

R R 34.54 R R R 34.54 34.54 R R R R 31.4 R 37.68 R R 37.68 R 50.24 R

31.4 40.82 34.54 37.68 R R 34.54 R R 50.24 R R R 37.68 R 47.1 34.54 31.4 37.68 34.54 R

R 31.4 R R R R 31.4 R R R R R 34.54 40.82 R R R R 31.4 R R

31.4 R 31.4 R R R 31.4 40.82 37.68 37.68 47.1 47.1 R R R R R 43.96 R R 47.1

R 34.54 31.4 R 34.54 R R R R 34.54 34.54 R R R R R R R R R 40.82

37.68 R 31.4 R R R R R R R 65.94 37.68 31.4 R R R R R 34.54 R R

1 2 3 4 5 7 9 10 11 12 13 14 15 19 20 21 22 23 24 25 26

CPI: CSIR project isolate. Average of inhibition area in mm2, R: resistant. Enterococcus faecalis (EF) is not shown in the Table, since no activity was observed for the sponge isolates (CPI 1 to CPI 26) tested.

used as test strain for standard antibiotic assays. Antibacterial activity of sponge associated actinomycetes is presented in Table 2. The isolate CPI 13 produced a high antibacterial activity against K. pneumoniae to the extent of 65.94 mm2 inhibition area. Based on the RDP-II bacterial taxonomy and convention classification system, the isolates were identified up to genera level. The potential producer CPI 13 was identified as Streptomyces spp. and the producers CPI 3 and CPI 9 were Saccharomonospora spp. and CPI 12 was Micromonospora spp. Association of culturable actinobacteria including Micromonospora and Streptomyces were reported from Haliclona spp.2 Micromonospora have been isolated previously from a marine sponge Hymeniacidon perleve by Zhang et al. [42]. But the association of Saccharomonospora was seldom reported from marine sponges. Maldonado et al. [23] described the ‘‘Micromonospora—Rhodococcus—Streptomyces’’ group seems to be ubiquitous in cultured actinobacteria from marine environments. Among the 26 isolates, seven showed antifungal activity (Table 3). The isolates, CPI 13 was highly active against all three fungi tested. The MIC and MFC against Candida tropicalis was 10 mg protein per milliliter and 12.5 mg protein per milliliter, respectively. Among the isolates which showed no antibacterial activity, the isolate CPI 1 and CPI 2 showed antifungal activity against C. tropicalis and the isolate CPI 26 showed activity against A. flavus. The antimicrobial potential of endosymbiotic bacteria suggests that marine sponges represent an ecological microniche which harbours a largely 2

Jiang S, Sun W, Chen M, Dai S, Zhang L, Liu Y, Lee KJ, Li X. Diversity of culturable actinobacteria isolated from marine sponge Haliclona spp. Antonie Van Leeuwen;2007:92;16.

uncharacterized microbial diversity and unexploited potential resource in the search for new secondary metabolites. Actinomycetes recognized as sources of antimicrobial compounds have been isolated from marine invertebrates [43]. Sponge associated bacteria has already been established as inhibitors of fouling bacteria [18]. But their potential against clinical and human pathogen was scarcely reported. Sponge associated bacteria from the Indian Ocean is being emerged as potential source of antimicrobial agents [2]. Most

Figure 2 Relative composition of potential producers among actinomycetes. Figure 2 Composition relative des éponges marines en actinomycètes producteurs potentiels.

21

Antimicrobial potential of sponge associated marine actinomycetes Table 3 MIC and MFC values of marine sponge bacteria against fungi tested. Tableau 3 Valeurs des CMI et des CMF de bactéries associées aux éponges vis-à-vis des souches fongiques. Test strain

CPI CPI CPI CPI CPI CPI CPI

1 2 3 9 12 13 26

C. tropicalis

A. fumigatus

A. flavus

MIC

MFC

MIC

MFC

MIC

MFC

59.5 30.46 12.5 74.3 74.3 10 R

74.3 38.0 15.6 92.9 92.9 12.5 R

R R 19.5 38.0 74.3 15.6 R

R R 24.3 47.6 92.9 19.5 R

R R 15.6 38.0 74.3 24.3 12.5

R R 19.5 47.6 92.9 30.4 15.6

CPICSIR project isolate. Values are in microgram of protein per millimeter of cell-free extract.

of the available literature envisages the antimicrobial potential of epibiotic and associated bacteria [5,34] but reports on antimicrobial potential of endosymbiotic bacteria are scanty. The findings of present report envisage the antimicrobial potential of endosymbiotic culturable bacteria. New classes of antibiotics are necessary to combat the increased incidence of multiple resistances among pathogenic microorganisms to the available drugs that are currently in clinical use [27]. As observed in the present study, spongeassociated actinomycetes showed high antimicrobial activity against Gram negative bacteria such as Pseudomonas aeruginosa and K. pneumoniae [36]. The present findings are suggestive of a synergistic effect of cell-free extract which may contain different target of more than one antibacterial substance [6,15,37]. The results of present investigation revealed that the marine actinomycetes from the sponges are a potential source of novel antibiotics. In conclusion, development of appropriate fermentation and downstream processing technologies would bring out new classes of antibiotic leads.

Acknowledgements M.A. is thankful to CSIR, New Delhi for the award of Senior Research Fellowship in CSIR funded research project. Authors are thankful to anonymous reviewer, who gave valuable suggestions to improve the MS. This paper is an outcome of CSIR project no. 38(1128)/06/EMR-II.

References [1] Abrell L. Investigations on natural products from marine sponge derived fungi. Diss Abst Int Pt-B Sci Eng 1997;58:3035. [2] Anand TP, Bhat AW, Shouche YS, Roy U, Siddharth J, Sarma SP. Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India. Microbiol Res 2006;161:252—62. [3] Armstrong E, McKenzie JD, Goldsworthy GT. Aquaculture of sponges on scallops for natural products research and antifouling. J Biotechnol 1999;70:163—74. [4] Bruheim P, Sletta H, Bibb MJ, White J, Levine DW. High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain over expressing the pathway-specific activator gene actII-ORF4. J Ind Microbiol Biotech 2002;28: 103—11.

[5] Chelossi E, Milanese M, Milano A, Pronzato R, Riccardi G. Characterisation and antimicrobial activity of epibiotic bacteria from Petrosia ficiformis (Porifera, Demospongiae). J Exp Mar Biol Ecol 2004;309:21—33. [6] Chino M, Nishimura K, Umekita M, Hayashi C, Yumazakin T, Tshchida T, et al. Heliquinomycin, a new inhibitor of DNAhelicase produced by Streptomyces spp. MJ929-SF2. I. Taxonomy, production, isolation, physico-chemical properties and biological activities. J Antibiot 1996;49:752—7. [7] Debitus C, Guella G, Mancini I, Waikedre J, Guemas JP, Nicolas JL, et al. Quinolones from a bacterium and tyrosine metabolites from its host sponge, Suberea creba from the Coral Sea. J Mar Biotechnol 1998;6:136—41. [8] Egorov N. Microorganisms-antagonists and biological methods for evaluation of antibiotic activity. Moscowkva High scool 1965;200 (in Russian). [9] Enticknap JJ, Kelly M, Peraud O, Hill RT. Characterization of a culturable alphaproteobacterial symbiont common to many marine sponges and evidence for vertical transmission via sponge larvae. Appl Environ Microbiol 2006;72: 3724—32. [10] Faulkner DJ. Marine natural products. Nat Prod Rep 2002;19: 1—48. [11] Faulkner DJ. Marine pharmacology. Antonie Van Leeuwen 2000;77:135—45. [12] Friedrich AB, Hacker J, Fischer I, Proksch P, Hentschel U. Temporal variation of the microbial community associated with the Mediterranean sponge Aplysina aerophoba. FEMS Microbiol Ecol 2001;38:105—13. [13] Galeano E, Martínez A. Antimicrobial activity of marine sponges from Urabá Gulf, Colombian Caribbean region. J Med Mycol 2007;17:21—4. [14] Garrity GM, Lilburn TG, Cole JR, Harrison SH, Euzeby J, Tindall BJ. The taxonomic outline of bacteria and archaea. TOBA release 7.7, March 2007. Michigan State University Board of Trustees; 2007. [15] Gonzales I, Niebla A, Lemus M, Gonazales L, Otero I, Ienaga Y, et al. Ecological approach of macrolide-lincosamides-Streptogramin producing Actinomycetes from Cuban soils. Lett Appl Microbiol 1999;29:147—50. [16] Hentschel U, Hopke J, Horn J, Friedrich A, Wagner M, Hacker J, et al. Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 2002;68:4431—40. [17] Imhoff JF, Stöhr R. Sponge-associated bacteria. In: Müller WEG, editor. Marine molecular biotechnology. New York: Springer-Verlag; 2003. p. 35—56. [18] Kanagasabhapathy M, Sasaki H, Nakajima K, Nagata K, Nagata S. Inhibitory activities of surface associated bacteria isolated

22

[19]

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27] [28]

[29]

[30]

R. Gandhimathi et al. from the marine sponge Pseudoceratina purpurea. Microbes Environ 2005;20:178—85. Kokare CR, Mahadik KR, Kadam SS. Isolation of bioactive actinomycetes from marine sediments isolated from Goa and Maharashtra coast lines (West coast of India). J Mar Sci 2004;33:248—56. Lachevelier F H.A. The actinomycetes III. In: A practical guide to genetic identification of actinomycetes. Bergey’s manual of systematic bacteriology. Baltimore: Williams and Wilkins company; 1989. p. 2344—7 Lam KS. Discovery of novel metabolites from marine actinomycetes. Curr Op Microbiol 2006;9:245—51. Locci R. Streptomyces and related Genera. Bergey’s manual of Systematic bacteriology. Baltimore: Williams and Wilkins company; 1989. pp. 2508—41. Maldonado LA, Stach JEM, Pathom-aree W, Ward AC, Bull AT, Goodfellow M. Diversity of culturable actinobacteria in geographically widespread marine sediments. Antonie Van Leeuwen 2005;87:11—8. NCCLS. Reference method for broth dilution antifungal susceptibility testing of yeasts (M27-A); Approved Standard Second Edition. Pennsylvania: Wayne; 2002. Pawlik JR, McFall G, Zea S. Does the odor from sponges of the genus Ircinia protect them from fish predators. J Chem Ecol 2002;28:1103—15. Piel D, Hui G, Wen D, Butzke M, Platzer N, Fusetani N, et al. Antitumour polykedite bio- synthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci (USA) 2004;101:222—7. Pompe S, Simon J, Wiedemann PM, Tannert C. Future trends and challenges in pathogenomics. EMBO Rep 2005;6, 7:600—5. Richelle-Maurer E, Gomez R, Braekman J-C, van de Vyver G, van Soest RWM, Devijver C. Primary cultures from the marine sponge Xestospongia muta (Petrosiidae, Haplosclerida). J Biotechnol 2003;100:169—77. Rifai S, Fassouane A, Pinho PM, Kijjoa A, Nazareth N, Nascimento MSJ, et al. Cytotoxicity and inhibition of lymphocyte proliferation of fasciculatin, a linear furanosesterterpene isolated from Ircinia variabilis collected from the Atlantic coast of Morocco. Mar Drugs 2005;3:15—21. Saadown I, Garaibeh R. The Streptomyces flora of Badia region of Jordan and its potential as a source of antibiotic resistant bacteria. J Arid Environ 2003;53:365—71.

[31] Selvin J, Lipton AP. Biopotentials of secondary metabolites isolated from marine sponges. Hydrobiologia 2004;513: 231—8. [32] Selvin J, Soniya J, Asha KRT, Manjusha WA, Sangeetha VS, Jayaseema DM, et al. Antibacterial potential of antagonistic Streptomyces spp. isolated from the marine sponge Dendrilla nigra. FEMS Microbiol Ecol 2004;50:117—22. [33] Sepcic K. Bioactive alkylpyridinum compounds from marine sponges. J Toxicol Toxin Rev 2000;19:139—60. [34] Thakur AN, Thakur NL, Indap MM, Pandit RA, Datar VV, Muller WEG. Antiangiogenic, antimicrobial, and cytotoxic potential of sponge-associated bacteria. Mar Biotechnol 2005;7:245—52. [35] Thoms C, Horn M, Wagner W, Hentschel U, Proksch P. Monitoring microbial diversity and natural products profiles of the sponge Aplysina cavernicola following transplantation. J Mar Biol 2003;142:685—92. [36] Tortora GJ, Funke BR, Case CL. Microbiology. The Benjamin/ Cummings publishing company Inc; 2000. p. 659—68. [37] Tsvetanova BC, Price NPJ. Liquid chromatography electrospray mass spectrometry of tunicomycin type antibiotics. Anal Biochem 2001;289:147—56. [38] Waksman SA. The Actinomycetes, classification, identification and description of genera and species. Baltimore: The Williams and Wilkins Company; 1961. p. 1—292. [39] Wang Q, Garrity GM, Tiedje JM, Cole JR. Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy. Appl Environ Microbiol 2007;73:5261—7. [40] Webster N, Hill RT. The culturable microbial community of the Great Barrier Reef sponge Rhopaloeides odorabile is dominated by an a-Proteobacterium. J Mar Biol 2001;138:843—51. [41] Zhang W, Zhang X, Cao X, Xu J, Zhao Q, Yu X, et al. Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley). J Biotechnol 2003;100:161—8. [42] Zhang HT, Lee YK, Zhang W, Lee HK. Culturable actinobacteria from the marine sponge Hymeniacidon perleve: isolation and phylogenetic diversity by 16S rRNA gene-RFLP analysis. Antonie Van Leeuwen 2006;90:159—69. [43] Zheng Z, Zeng W, Huang Y, Yang Z, Li J, Cai H, et al. Detection of antitumor and antimicrobial activities in marine organisms associated actinomycetes isolated from the Taiwan Strait, China. FEMS Microbiol Lett 2000;188:87—91.