ORIGINAL ARTICLE

Antimicrobial potential of metabolites extracted frombacterial symbionts associated with marine sponges incoastal area of Gulf of Mannar Biosphere, IndiaS. Skariyachan, A. G. Rao, M.R. Patil, B. Saikia, V. Bharadwaj KN and J. Rao GS

Department of Biotechnology, R & D Centre, Dayananda Sagar College of Engineering, Bangalore, Karnataka, India

Significance and Impact of the Study: This is the first study demonstrating antimicrobial potential offlurophoric and chromophoric metabolites extracted from bacterial biosymbionts associated with mar-ine sponges. Our study has significant scope as Indian coastal area especially harbours vast varieties ofsponges with novel secondary metabolites-producing organisms. The natural metabolites extracted fromsponge-derived bacteria pave novel therapeutic remedy against various pathogens when most of themare emerged as extreme drug resistant superbugs.

Keywords

chromophoric, endosymbiotic bacteria,

fluorophoric, Gulf of Mannar, marine

sponges, secondary metabolites, therapeutic

potential.

Correspondence

Sinosh Skariyachan, Department of Biotech-

nology, R & D Centre, Dayananda Sagar

College of Engineering, Bangalore 560 078

Karnataka, India.

E-mail: sinoshskariya@gmail.com

2013/1529: received 30 July 2013, revised 14

October 2013 and accepted 15 October 2013

doi:10.1111/lam.12178

Abstract

Marine coastal areas of India have vast diversity of sponges which harbours

many endosymbiotic bacteria which are the source of many potential

antimicrobial metabolites. This study focuses the screening and

characterization of drug-producing bacteria symbiotically which are associated

with marine sponges collected from Gulf of Mannar, South Coast India. Six

different sponges were collected and they were identified on the basis of their

morphology. The drug-producing isolates were screened by agar overlay

method towards various clinical strains. The secondary metabolites were

characterized and were found to be quinones, alkaloids, flavanoids and flavonyl

glycosides. The metabolites showed significant inhibitory properties against

clinical strains that were further identified as chromophoric and fluorophoric

in nature. Ethyl acetate extracts of chromophore and floureophore substances

showed significant inhibitory properties against Methicillin resistant

Staphylococcus aureus (MRSA) and Salmonella typhi respectively. 16S rRNA

gene sequencing of theses isolates revealed that chomophore-producing strain

were closely related to Pseudomonas spp. RHLB12, isolated from Callyspongia

spp. and floureophore-producing bacteria was related to Bacillus licheniformis

T6-1 which was isolated from Haliclona spp. Hence, our study demonstrated

that antimicrobial metabolites extracted from symbiotic bacteria associated

with marine sponges have high therapeutic potential against many bacterial

pathogens including multidrug-resistant strains.

Introduction

Marine sponges are one of the richest sources of biologi-

cally active secondary metabolites with vast chemical

diversity. Various metabolites obtained from such sponges

have yielded pharmaceutical products such as novel anti-

inflammatory agents, anticancer agents and antibiotics

(Boopathy and Kathiresan 2010). The marine coastal

areas in India have vast diversity of marine sponges in

which Gulf of Mannar region is a bed for valuable

sponges. The Gulf of Mannar is located in the state of

Tamil Nadu on the South-eastern tip of India. Around

3600 species of marine fauna and flora have been identi-

fied in the Gulf of Mannar area consisting of 108 species

of sponges (Mol et al. 2010). Until now, more than 5000

different compounds have been isolated and identified

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology 231

Letters in Applied Microbiology ISSN 0266-8254

from sponges with nearly 800 of them exhibiting antimi-

crobial properties. These compounds belonged to differ-

ent class of compounds such as terpenoids, alkaloids,

macrolides, polyethers, nucleoside derivatives and pep-

tides (Ravichandran et al. 2011; Dhinakaran et al. 2012).

Marine sponges harbour micro-organisms on their sur-

faces, in their canal systems and intercellular spaces, and

these contribute up to 40% of the total cellular content of

a sponge. Most of these micro-organisms are endosymbi-

otic bacteria and are probably major source of novel

secondary metabolites and other valuable compounds

(Petchi et al. 2013). These symbiotic bacteria may play

roles in digestion, waste removal and help in the nutri-

tional process, either by intracellular digestion or by

translocation of metabolites including photosynthesis,

nitrification and nitrogen fixation. They also stabilize the

sponge skeleton and participate in the host’s chemical

defence. This has greatly improved our knowledge of

sponge–microbe interactions. Symbiotic bacteria associ-

ated with sponges produce potential antimicrobial

compounds and bioactive compounds. Many bioactive

compounds extracted from Dendrilla nigra (Ivanova et al.

1999), Axinella donnani and Clathria gorgonoides

(Aishwarya et al. 2013) have significant antimicrobial

activities against various Gram-positive pathogenic bacte-

ria. Similarly, dichloromethane-methanol (1 : 1) extract

of the sponge Phycopsis sp. collected from Tuticorin coast

of India exhibited antibacterial activity (Venkateswarlu

and Biabani 1995). Many bacterial metabolites have been

isolated from the marine sponges, Cryptophycin I and

Chondramide isolated from Dysidea spp. and Jaspis spp.

respectively (Sabdono and Radjasa 2008). In addition to

therapeutic applications of bioactive compounds from

marine sponges against various bacteria, there are many

reports which reveal the antitumor drugs such as Kendar-

imide A, isolated from sponge Haliclona sp. has been

shown to reverse multidrug resistance in tumour cells

(Roser et al. 2005).

Recent studies revealed that endosymbionts associated

with sponge Amphimedon ochracea produced cytotoxic

compounds against HepG2 (hepatocellular carcinoma),

HCT (colon carcinoma) and MCF-7 (breast carcinoma)

cancer cell lines (Aboul-Ela et al. 2012), Proteobacteria

and novel Pseudomonas sp. associated with the sponge

Suberites domuncula produced cytotoxic compounds

against HeLa and PC12 cells (Thakur et al. 2005), Bacillus

megaterium and Pseudoalteromonas aurantia isolated from

Eupexaura curvata, Pseudoalteromonas piscicida isolated

from Hymeniacidon perleve and Psedoalteromonas rubra

isolated from sea water produce bioactive compounds

with wide range of antimicrobial spectrum (Zheng et al.

2005a,b). Similarly, Bacillus atrophaeus was isolated from

the marine sponge Dysidea avara in the South China Sea,

which can produce Bacillamide C and a new compound

of Neobacillamide A (Liu et al. 2012).

The use of molecular approaches for describing the

microbial diversity has greatly enhanced the knowledge of

these natural microbial communities associated with mar-

ine sponges. Cloning and sequencing of 16S rRNA genes

gives data which can be used to describe complete micro-

bial community, composition and phylogenetic relatives,

and they can indicate possible nutritional requirements

and physiological riches of many endosymbiotic microbes

(Webster et al. 2001) present in various sponges. This

study mainly focuses on the screening and characteriza-

tion of drug-producing bacteria that are symbiotically

associated with marine sponges found in South Coast of

India, Gulf of Mannar. The identification and character-

ization of secondary metabolites or similar bioactive com-

pounds isolated from symbiotically associated bacteria is

also studied. Further, antimicrobial properties of metabo-

lites isolated from endosymbiotic bacteria from sponges

against various bacterial pathogens, especially multidrug-

resistant strains have been performed in this study.

Results and discussion

Six different types of marine sponges were collected from

Olaikuda in Gulf of Mannar Bioshphere region, Ramesh-

warm, Tamil Nadu, India. Based on morphological fea-

tures of spicules and other specialized structures, the

collected marine sponges were identified as Cacaspongia

spp., Sigmadocia spp., Callyspongia spp., Dendrila spp.,

Gorgonid spp. and Haliclona spp. There are many reports

which reveal that these kinds of marine sponges are very

common in Indian coastal areas including Tuticorin coast

(Singla et al. 2013), Kavaratti Island, Lakshadweep archi-

pelago (Gopi et al. 2012) and Mandapam bay and Gulf of

Mannar (Velho-Pereira and Furtado 2012). Other reports

reveal that these marine sponges harbour various types of

symbiotically associated micro-organisms they are unique

sources of natural bioactive secondary metabolites with

pronounced pharmacological activities (Thomas et al.

2010; Hardoim et al. 2012; Velho-Pereira and Furtado

2012). For high microbial abundance (HMA) sponges,

around 38% of sponge wet weight is composed of bacte-

rial cells, and that such bacterial abundance surpasses that

of the seawater by 2–4 orders of magnitude (Taylor et al.

2007).

The bacteria which are symbiotically associated with the

collected sponge samples were isolated by Marine Zobell

agar. The cultural characteristics of the isolates were found

to be pale yellow coloured, medium sized, opaque and con-

vex colonies. The viable bacterial count in terms of colony

forming units (CFU g�1) ranged from 1�10 9 103 to

6�4 9 103, with an average of 3�85 9 103 CFU g�1. After

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology232

Antimicrobial potential of metabolites extracted S. Skariyachan et al.

analysing the bacterial count from the sponge samples,

Sigmodocia spp. was found to have the highest CFU g�1

(Table 1).

The bacteria isolated from the sponges were character-

ized by standard microbiology techniques. Most of the

bacteria isolated from the sponges were found to be

Gram-positive bacilli and cocci. Few bacteria were found

to be Gram-negative rods (Fig. S1). Various studies on

antibiotic production in marine environment have

reported that around 36% of the isolates are Gram-nega-

tive rods (Beveridge 2001). To determine the characteris-

tics of the isolated bacteria, the sponge homogenate was

plated on differential and selective media. When plated

on MacConkey’s agar, the isolates from tissues were

found to be Gram-negative bacteria. MacConkey’s agar

indicates the presence of Gram-negative coliforms in the

sponges which are probably due to the presence of faecal

products from marine environments (Boopathy and

Kathiresan 2010). When the sponge homogenate was pla-

ted on Thiosulfate citrate bile salts sucrose (TCBS) agar,

it showed small, circular, golden yellow coloured, opaque

colonies indicating the presence of Vibrio spp. All sponges

showed the presence of Vibrio spp. Marine sediments and

sponges often harbours Vibrio spp. and optimum growth

parameter for Vibrio spp. is high salinity (West and

Colwell 1984). Studies have reported that Vibrio spp. are

predominant bacterial symbionts associated with marine

environment (Anand et al. 2006). When the sponge

homogenates were plated on Bromo thymol blue (BTB)

Lactose agar, the plates showed deep yellow coloured,

medium sized, circular colonies indicating the presence of

Staphylococcus spp. Such kinds of bacterial isolates were

observed from the tissue homogenates of Gorgonid spp.,

Haliclona spp., Cacaspongia spp., Callyspongia spp., Sigm-

odocia spp. and Dendrila spp. Similarly, circular, med-

ium-sized, good luxuriant, blue–green coloured colonies

were observed when the sponge homogenates were plated

on milk agar with cetrimide, which are the characteristic

features of Pseudomonas spp. Such types of colonies were

isolated from the marine sponge Callyspongia spp. Other

reports reveal that many species of Pseudomonas found as

biosymbiont with marine sponges and they are the poten-

tial sources of antimicrobial agents against drug resistant

pathogens (Marinho et al. 2009; Thomas et al. 2010).

Various types of symbiotically associated bacteria isolated

from the collected sponges are shown in Table 1. Maxi-

mum types of endosymbiotic bacteria were isolated from

the sponge Callyspongia spp. (Table 1).

The antimicrobial properties of marine isolates were

tested against MRSA, Proteus mirabilis, Klebsiella pneumo-

niae and Salmonella typhi by agar overlay method. A clear

zone of inhibition was observed on the soft agar mixed

with two test organisms (Fig. 1) indicating that marine

isolates have significant antimicrobial activities, which are

probably due to the production of bioactive secondary

metabolites. The drug-producing bacteria identified by

preliminary screening were further characterized by bio-

chemical tests. The microbiology characterizations of the

bacterial isolates from the marine sponges are shown in

Table 2. From our results, it is evident that the bioactive

metabolites produced by biosymbionts were extracellular

in nature. Most of the bacteria showed zones of inhibi-

tion against the two test organisms were isolated from the

sponges; Callyspongia spp. and Haliclona spp. Out of all,

the symbiotic isolates from Callyspongia spp. have signifi-

cant antimicrobial properties against MRSA. Similarly, the

bacteria isolated from Haliclona spp. showed significant

antimicrobial properties against clinical strain of Salm.

typhi. Studies have revealed that members of the Cally-

spongia spp. and Haliclona spp. are potential drug-bearers

and they have potential antimicrobial activities against

various pathogens (Ely et al. 2004; Aishwarya et al. 2013).

There are other reports where sponge-derived isolate of

Vibrio spp. have shown to be rich source of novel biologi-

cally active bacterial metabolites (Anand et al. 2006).

Similar studies demonstrated that marine derived Pseudo-

monas and Bacillus are sources of potential antimicrobial

substances (Anand et al. 2006; Devi et al. 2010).

The secondary metabolite-producing isolates were cul-

tured in Marine Zobel broth and minimum essential

media for extraction and purification of antimicrobial

compounds. The supernatant from appropriately incu-

bated culture was used for purification. The metabolite

from the supernatant was further purified by solvent

extraction method. The purified extract showed inhibitory

properties, zone of inhibition in the agar medium (well

Table 1 Total bacterial count (CFU g�1) and major types of symbiotic

bacteria isolated from selected marine sponges

Sponge

sample

Number of

bacteria

(CFU g�1) Major types of bacteria isolated

Dendrilla

spp.

5�7 9 103 Staphylococcus spp., Vibrio spp.

Cacaspongia

spp.

5 9 103 Staphylococcus spp., Bacillus spp.,

Vibrio spp.

Callyspongia

spp.

3�2 9 103 Staphylococcus spp., Pseudomonas spp.,

Klebsiella spp., Bacillus spp., Vibrio

spp.

Haliclona

spp.

9�1 9 103 Staphylococcus spp., Bacillus spp.,

Vibrio spp.

Sigmodocia

spp.

6�4 9 103 Staphylococcus spp., Bacillus spp.,

Vibrio spp.

Gorgonoid

spp.

1�8 9 103 Staphylococcus spp., Bacillus spp.,

Vibrio spp.

CFU, colony forming unit.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology 233

S. Skariyachan et al. Antimicrobial potential of metabolites extracted

diffusion method) against selected pathogenic bacteria as

observed in the preliminary screening. The effect of vari-

ous concentrations of the purified metabolites against the

selected strains is shown in Table 3. Among the tested

clinical isolates, the secondary metabolite extracted from

the bacterial isolate from the marine sponges Callyspongia

spp. showed better antimicrobial activities against MRSA

which indicate a zone of clearance around the well

(Fig. 1a), where various concentration of metabolites have

been incorporated. Similarly, the bioactive substance

extracted from the bacterial isolate associated with the

sponge Haliclona spp. showed significant inhibitory prop-

erties against the lawn culture of Salm. typhi (Fig. 1a).

These are significant finding of study as other reports

reveal that both Staphylococcus aureus and Salm. typhi are

resistant to many conventional antibiotics and have

emerged as multidrug-resistant strains (Frye and Jackson

2013; K€ock et al. 2013; Lee et al. 2013; Torpdahl et al.

2013). From our studies, it is evident that the drugs pro-

duced by the biosymbionts from marine sponges have sig-

nificant inhibitory properties against such MDR strains.

Bacteria such as Pseudomonas spp. (Thakur et al. 2005)

and Bacillus spp. (Gebhardt et al. 2002) is common

inhabitant of marine sponges that produce powerful anti-

microbial substance against multidrug-resistant bacteria

(Ivanova et al. 1999; Zheng et al. 2005a,b; Marinho et al.

2009). Present data are one time observation and can be

reproduced to achieve significant results. Further, we

compared our data with experimental error range in

inhibitory zone assays (Schwalbe et al. 2007). From the

comparative analysis, it is evident that our result is within

the experimental error ranges. Currently, only the zone of

inhibition method is used to assess the inhibitory effect of

extracted compounds against selected clinical strains. The

inhibitory effect using a liquid culture method and

growth curve studies at OD600 may give more clarity to

the nature of the antimicrobial action. If the antimicrobial

substance inhibits only the multiplication, removal of the

extract after specific period of time would revive the

growth curve. If the antimicrobial agent is responsible for

lysing the cell membrane or permanent inhibitory mecha-

nism, the growth curve will not be revived.

The drug-producing efficiency of marine isolates was

compared both in Marine Zobell agar and minimum

essential medium. From our study, it is evident that the

drug-producing efficiency of isolated bacteria against the

clinical isolates of Staph. aureus and Salm. typhi was high

in the minimum essential medium than marine Zobell

agar (Fig. 1b) A comparative study of suitable solvent sys-

tem for the drug is also studied which showed ethyl

acetate extract of the drug produced maximum zone of

inhibition compared to other solvents such as methanol,

chloroform and water. It has been reported that ethyl ace-

tate extracts of many metabolites exhibited more antimi-

crobial activity than methanol extracts, solubility of the

drug increases in presence of ethyl acetate (Chairman

et al. 2012).

Phytochemical characterization of the metabolites asso-

ciated with Pseudomonas spp. and Bacillus spp. was

carried out. The extract from Pseudomonas spp. showed

(a) (c) (d)

(b) (f)(e)

Figure 1 Preliminary screening of drug producing bacteria associated with marine sponges by agar overlay method against (a) clinical strains of

Staphyloccus areus (b) Salmonella typhi. Clear zones of inhibition (highlighted in the figure) represent the bacterial isolate have antimicrobial activ-

ity of against the test organism. Antimicrobial activity of bacterial metabolite isolated from the Callyspongia spp. against Staph. areus (c) in

Marine Zobel agar medium and (d) Minimum essential medium. Antimicrobial activity of bacterial metabolite isolated from Haliclona spp. against

Salm. typhi (e) in Marine Zobel agar medium and (f) Minimum essential medium.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology234

Antimicrobial potential of metabolites extracted S. Skariyachan et al.

the presence of alkaloids, quinones, flavonyl glycosides

and flavanoids in marine Zobell broth. The extract from

Bacillus spp. showed the presence of carbohydrates,

proteins, lipids, alkaloids, quinones and flavonyl glyco-

sides. Bioactive compounds such as Micacocidin A, B, C

(antimycoplasmal) and C-I4, a cyclic dipeptide (chitinase

inhibitor) have been reported from marine Pseudomonas

spp. (Kobayashi et al. 1998). Many antibiotics including

cyclic peptides, cyclic lipopepides and novel thiopeptides

have been reported from marine Bacillus spp. (Nagai

et al. 2003).

The ethyl acetate extracts was spotted on the thin layer

chromatography (TLC) plates. On separation, a purple-

coloured compound was observed on the plate which

Table 2 Microbiology characterization of various symbiotic bacteria isolated from selected group of marine sponges

Sponge

sample CNa SMb Mc Mtd Ie Mrf Vpg Ch Cti Uj Ok Sl Sfm An Hso Mfp Nrq Cor Organism

Cacaspongia

spp.

CN1 MZAs Gram +ve

bacilli

� � � � � + + � + + + + + � � Bacillus spp.

CN2 TCBSt Gram �ve

bacilli

+ � � + + + + + � + � � + + � Vibrio spp.

CN3 BTBLu Gram +ve

bacilli

� � + + � + � � � + + � + + + Staphylococcus

spp.

Callyspongia

spp.

CN1 Macv Gram �ve

bacilli

� � � + + + + � + + � � � � � Klebsiella spp.

CN2 MZAs Gram +ve

bacilli

� � � � � + + + � + � � � � � Bacillus spp.

CN3 MCX Gram �ve

bacilli

+ � � � + + + + + + + � � + � Pseudomonas

spp.

CN4 BTBLu Gram +ve

cocci

� � + + � + � � � + + � + + + Staphylococcus

spp.

CN5 TCBSt Gram �ve

curved

bacilli

+ + + � + + � + � + + � + + � Vibrio spp

Haliclona

spp.

CN1 BTBLu Gram +ve

cocci

� � + + � + � � � + + � + + + Staphylococcus

spp.

CN2 MZAs Gram +ve

bacilli

+ � � � + + + � + + + � � + � Bacillus spp.

CN3 TCBSt Gram �ve

curvedbacilli

+ + + � + + � + � + + � + + � Vibrio spp.

Dendrilla

spp.

CN1 BTBLu Gram +ve

cocci

� � + + � + � � � + + � + + + Staphylococcus

spp.

CN2 TCBSt Gram �ve

curved

bacilli

+ + + � + + � + � + + � + + � Vibrio spp.

Sigmodocia

spp.

CN1 BTBLu Gram +ve

cocci

� � + + � + � � � + + � + + + Staphylococcus

spp.

CN2 TCBSt Gram �ve

curved

bacilli

+ + + � + + � + � + + � + + � Vibrio spp.

CN3 MZAs Gram +ve

bacilli

+ � � � + + + � + + + � � + � Bacillus spp.

Gorgonoid

spp.

CN1 BTBLu Gram +ve

cocci

� � + + � + � � � + + � + + + Staphylococcus

spp.

CN2 TCBSt Gram �ve

curved

bacilli

+ + + � + + � + � + + � + + � Vibrio spp.

CN3 MZAs Gram +ve

bacilli

+ � � � + + + � + + + � � + � Bacillus spp.

aColony Number, bSelective media used, cMorphology, dMotility, eIndole, fMethyl red, gVoges Proskauer, hCitrate, iCatalase, jUrease, kOxidase,lStarch hydrolysis, mSugar fermentation (Glucose, Lactose and Sucrose), nAcid and gas, oHydrogen sulphide production, pMannitol fermentation,qNitrate reduction. rCoagulase, sMarine Zobel agar, tThiosulfate-citrate-bile salts-sucrose agar, uBromo Thymol Blue Lactose Agar, vMacConkeys’s

agar, xMilk agar with Cetrimide.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology 235

S. Skariyachan et al. Antimicrobial potential of metabolites extracted

indicates the presence of a chromophore and the remain-

ing compounds of interest were colourless. The colourless

fractions are further visualized under UV lamp which

indicate that the extract also contains few fluorophoric

substances (Fig. 2). The UV spectrum of the chromo-

phoric compound showed a peak at 600 nm, whereas the

flourophoric compound produced peaks at 280 and

320 nm (Fig. 2c,d). The chromophoric and flourophoric

substances were extracted from marine Pseudomonas spp.

and Bacillus spp. respectively. Many studies showed that

the secondary metabolites from marine sponges have the

capability to absorb the ultraviolet rays and emit fluores-

cence (Ahamed 2012). The ethyl acetate extract of

chromophoric substance showed better antimicrobial

properties than the flourophoric substances against

selected clinical strains. Hence, we focused the

chromophoric substance for understanding of structural

features of the metabolites. Fourier transform infrared

(FT-IR) spectroscopic analysis of the chromophoric com-

pound was further carried out to determine the func-

tional groups. The spectrum showed strong peaks at the

following frequencies 3429�55, 2924�18, 2860�53, 2360�96,1741�78 and 1641�48 Hz (Fig. 2e). This was compared

with the standard chart (Chu et al. 1999) and thus

confirmed the presence of functional groups such as

-OH, -CN, -CH and -CO. However, further biophysical

characterization is required to elucidate the structure of

identified metabolite.

The drug- (chromophoric substance) producing

biosymbiont isolated from the sponge Callyspongia spp.,

showed antibacterial activity against MRSA, further

confirmed by 16S rRNA gene sequencing (Clarridge 2004;

Table 3 Effect of various concentrations of ethyl acetate (50% v/v) extract of secondary metabolites extract from biosymbionts on various clinical

strains by well diffusion method. Present data are one time observation and it is compared with experimental error range in inhibitory zone assays

Sponge Media

Nature of main

secondary metabolite Producer strain

Test

organism Phase

Concentration

(lmol l�1)

Diameter ofc ZOI

(mm)

Callyspongia spp. MZAa Chromophore

substance

Pseudomonas

spp.

MRSAb Organic 25 2�550 3�0100 4�0

Haliclona

occulata

MZAa Fluorophore compound Bacillus spp. Salmonella

typhi

Organic 25 No zone

50 2�0100 8�0

Callyspongia spp. MEMd Chromophore

substance

Pseudomonas

spp.

MRSAb Organic 25 3�050 4�0100 7�0

Haliclona

occulata

MEMd Fluorophore compound Bacillus spp. Salm. typhi Organic 25 3�050 6�0100 8�0

aMarine Zobel agar, bMethicillin resistant Staphylococcus aureus, cZone of Inhibition, dMinimum essential media.

(a) (b)

(c) (d)

(e)Figure 2 Thin Layer chromatography plate

showing visual spots of (a) chromophoric (b)

fluorophoric metabolites under UV spectrum.

UV spectrum of (c) chromophoric (peak at

600 nm) and (d) flourophoric substance (peak

at 350 nm). (e) FTIR spectrum of the

chromophoric substance, the data compared

with standard chart indicate the presence of

functional groups -OH, -CN, -CH and -CO.

The peaks referring to the functional groups

were specifically marked in the figure.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology236

Antimicrobial potential of metabolites extracted S. Skariyachan et al.

Janda and Abbott 2007). The sequence obtained was

compared with known sequences in the GenBank data-

base, identified by BLAST search; indicate closest phyloge-

netic relationship with 99% sequence identity (Hentschel

et al. 2001) towards Pseudomonas spp. RHLB12. Similarly,

the drug- (flourophoric substance) producing biosymbi-

ont isolated from the sponge Haliclona spp., showed anti-

bacterial activity against clinical strain of Salm. typhi, was

also characterized by 16S rRNA gene sequencing. From

the BLAST result we noticed that the sequence showed 96%

sequence identity with Bacillus licheniformis T6-1. The

16S rRNA sequences of Pseudomonas spp. RHLB12 were

deposited to GenBank database and can be accessed by

the accession number KF225558.1.

Materials and methods

Description of the sampling spot

The area under study was a beach by name Olaikuda in

Gulf of Mannar, Marine National Park, Rameshwaram,

Tamil Nadu, India. Gulf of Mannar is a Bio-sphere

Reserve which is developed for preserving aquatic life. It

comprises 21 islands running almost parallel to the coast-

line between latitude 8°47′ N and 9°15′N and longitude

78°12′E and 79°14′E with three distinct marine ecosys-

tems namely corals, sea grass and mangroves.

Collection, transportation and storage of sponge samples

The sponge samples were collected from bottom set crab

nets, for the harvest of edible crabs at a depth of 7 feet.

The net were deployed in the sea at 4 PM and was

retrieved at 6 AM the following morning on January 12,

2013. The sponge samples were carefully removed from

the wires of the crab net and washed with sea water to

remove the sand and adhered debris. The samples were

kept in different zip lock bags in an ice box. The samples

were transported to the laboratory with seawater under

required aseptic precautions (Dhinakaran et al. 2012).

The samples were aseptically stored at �4°C for 12 h. All

the samples were processed on the very next day and the

duration of this study was 6 months.

Isolation of symbiotic bacteria associated with marine

sponges

The sponges were washed thoroughly with sterile sea water

and homogenized in sponge dissociation medium (Anand

et al. 2006). The homogenate was serially diluted and pla-

ted on marine Zobell agar 2216 (Anand et al. 2006) by

pour plate technique (Geldreich et al. 1972). All the plates

were incubated at 37°C for 24 h. Subsequently, the

symbiotic bacteria that are associated the sponges were

selectively isolated using TCBS agar, Milk agar with cetri-

mide, BTB lactose agar and MacConkey’s agar (Hi-media,

Mumbai, India). The total number of endosymbiotic bac-

teria (CFU g�1) associated with each sponge sample was

determined by digital colony counter (Labtronics, India).

Preliminary screening of drug-producing bacteria

Preliminary screenings of drug-producing bacterial iso-

lates were carried out by agar overlay method (Anand

et al. 2006). The test strains were gently overlaid using

3�5 mol l�1 soft agar over the marine isolates. The clinical

isolate of MRSA, Pr. mirabilis, Kl. pneumoniae and

Salm. typhi were used as the test organisms. The test

organisms were collected from Sagar Hospitals, Bangalore,

India. The soft agar was prepared by inoculating 1 mL

fresh cultures of test strain in 100 mol l�1 of soft agar

and mixed thoroughly. For marine strains, 1�5% NaCl

was added to the soft agar. The overlaid plates were incu-

bated at 37°C for 48 h. The zones of inhibition produced

by the marine isolates against the test organisms were

thereafter interpreted.

Secondary screening of drug-producing bacteria

Marine Zobell broth 2216 (Anand et al. 2006) and minimal

essential media (30 g l�1 sucrose, vitamin tablet and sea

water) were used for the extraction of drugs from marine

isolates. Ten ml culture of marine isolate was inoculated to

100 ml of marine Zobell broth and minimal essential media

and kept in a shaker incubator (Eppendorf) at 35°C for

7 days. The culture was centrifuged at 10 000 g for 20 min

and the supernatant was separated and stored at �4°C. Thesupernatant was further screened for antimicrobial activity

against the test organisms by well diffusion method at con-

centration ranges of 25–100 lmol l�1. The plates were

incubated at 37°C for 48 h and screened for antimicrobial

activities, presence of zone of inhibition.

Phytochemical analysis of secondary metabolites

produced by marine isolates

The supernatant from the marine isolates which showed

significant antimicrobial activities against the test organ-

ism were selected and transferred to separating funnels.

An equal mixture of supernatant and ethyl acetate, suit-

able solvent was poured to a separating funnel and the

separation process was carried out at room temperature

(Gokulkrishnan et al. 2011). The organic phase, contains

the antimicrobial metabolite, was collected and stored at

�4°C for further studies. The analysis for major classes of

antimicrobial substance present in the collected phase was

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology 237

S. Skariyachan et al. Antimicrobial potential of metabolites extracted

carried out by standard phytochemical analysis. Meyer’s

and Wagner’s test (Miller et al. 2010) were used for the

detection of alkaloids; Molisch’s test (Usman et al. 2009)

was used for the detection of carbohydrate; Libermann

Burchard test (Aryantha et al. 2002) was used for the

detection of steroids; sulphuric acid test (Firdouse and

Alam 2011) was used for the detection of quinines; ferric

chloride test (Firdouse and Alam 2011) was used for the

detection of phenols; biuret, ninhydrin and Bradford

assays (Singh et al. 2012) were used for the detection of

proteins. Similarly, standard phytochemical screening was

performed for the detection of flavonyl glycosides, lipids

and flavanoids (Singh et al. 2012).

Purification and characterization of bioactive metabolites

Purification of the secondary metabolites, probable drugs,

was carried out by TLC. The organic extract was spotted

on silica gel plates (Merck Millipore, Mumbai, India)

using 30 : 70 ethyl acetate-hexane as solvent system. A

one-dimensional ascending technique was used to sepa-

rate the components present in the extract (Chairman

et al. 2012). The plates visualized under UV radiation by

short, middle and long wavelengths of 254 nm, 302 nm

and 600 nm respectively. The separated compounds were

eluted and subjected to Ultra Violet-Visible (UV-Vis)

spectral analysis using methanol as standard. A FT-IR

spectroscopic analysis was carried out to determine the

functional groups of bioactive compounds.

Antimicrobial testing of the identified compounds

An antimicrobial assay was further performed with the

identified compound to confirm the inhibitory properties.

Fractions of the identified compounds were diluted with

sterile distilled water. The antimicrobial solutions at vari-

ous concentrations were subjected to selected clinical iso-

lates using agar well diffusion method. All the plates were

incubated at 37°C for 48 h and the plates were screened

for zone of inhibition.

Microbial characterization of drug-producing isolates

The potent isolates selected from the preliminary screen-

ing were characterized by standard microbiology methods.

The morphological (Boobathy et al. 2009), physiological

and biochemical characteristics of each isolate were also

studied based on Bergey’s manual of systematic bacteriol-

ogy (Krieg et al. 2010). The biochemical tests performed

for the identification of the isolates include indole pro-

duction, methyl red, vogues prauskauer and citrate utili-

zation (Krieg, 2010), triple sugar iron test (Sulkin and

Willett 1940), starch hydrolysis (Colonna et al. 1992),

nitrate reduction (Moreno-Vivi�an et al. 1999), urease test

(MacFaddin 1980), catalase test (Miller et al. 2007),

oxidase test (Gordon and McLeod 1928) and hydrogen

sulphide production (Singh et al. 2012) tests.

Molecular characterization of drug-producing bacteria

The marine strain that produce antimicrobial secondary

metabolites was further characterized by 16S rRNA gene

sequencing (BioAxis DNA Research Centre Private Limited,

Hyderabad, India). The 16S rRNA gene from the selected

isolate was amplified using universal primers 16F (5′-AGA-GTTTGATCCTGGCTCAG-3′) and 16R (5′-AGA-GTTTGATCCTGGCTCAG-3′). A PCR was performed with

25 ml volume using 10 ng of genomic DNA, 19 reaction

buffer (0�01 mol l�1 Tris-HCl, pH 8�8 at 25°C, 0�0015 mol

l�1 MgCl2, 0�05 mol l�1 KCl and 0�1% Triton X-100), 0.

0�0004 mol l�1 (each) deoxynucleoside triphosphates and

0�5 U of DNA polymerase (New England Labs, Hitchin,

UK). The PCR was performed in an automated PCR system

9700 thermal cycler (Applied Biosystems, Foster City, CA,

USA) under the following conditions. The amplification

conditions were as follows 94°C for 1 min (denaturation),

55°C for 1 min (annealing), 72°C for 90 min (elongation)

and 72°C for 10 min final elongation. Expected PCR prod-

uct of around 1�5 kb was checked by electrophoresis of

5 ml of the PCR product on 1% agarose gel in 19 TBE buf-

fer and stained with 0�005 mol l�1 ethidium bromide. The

PCR product was precipitated by PEG-NaCl (20% PEG in

2�5 mol l�1 NaCl) precipitation at 37°C for 30 min. One

microlitre of purified PCR product was sequenced by auto-

mated sequence analyser (Applied Biosystems-3500, Capil-

lary sequencer). Further analysis of sequences was

performed by similarly searching tool, NCBI BLAST server

(http://www.ncbi.nlm.nih.gov/BLAST).

Acknowledgements

The authors acknowledge The Principal Chief Conserva-

tor of Forest, Govt of Tamil Nadu, India for grant per-

mission (C.No.WL5/11399/2013) for collecting the marine

sponges from Gulf of Mannar Biosphere, Ramesharam.

The authors are very much grateful to Dr. V. Deepak

Samuel, Programme Specialist, Energy and Environment

Unit, United Nations Development Programme, UNDP-

GEF, Gulf of Mannar Biosphere Reserve Trust, for his

immense support towards the collection and identifica-

tion of marine sponges.

Conflict of Interest

The authors are disclosing that there are no potential

sources of conflict.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology238

Antimicrobial potential of metabolites extracted S. Skariyachan et al.

References

Aboul-Ela, H.M., Shreadah, M.A., Abdel-Monem, N.M.,

Yakout, G.A. and van Soest, R.W.M. (2012) Isolation,

cytotoxic activity and phylogenetic analysis of Bacillus spp.

bacteria associated with the red sea sponge Amphimedon

ochracea. Adv Biosci Biotechnol 3, 815–823.

Ahamed, N. (2012) Isolation and identification of secondary

metabolites producing organisms from marine sponge.

Discovery 1, 14–17.

Aishwarya, M.S., Lipton, A.P. and Sarika, A.R. (2013)

Phylogenetic appraisal of the drug bearing marine sponge

Callyspongia subarmigera (Ridley, 1884) from South India.

Indian J Geo-Mar Sci 42, 139–145.

Anand, T.P., Bhat, A.W., Shouche, Y.S., Roy, U., Siddharth, J.

and Sarma, S.P. (2006) Antimicrobial activity of marine

bacteria associated with sponges from the waters off the

coast of South East India. Microbiol Res 161, 252–262.

Aryantha, N.P., Adinda, A. and Kusmaningat, S. (2002)

Occurrence of triterpenoids and polysaccharides on

Ganoderma tropicum with Ganoderma lucidum as

reference. Aust Mycol 20, 123–129.

Beveridge, T.J. (2001) Use of the Gram stain in microbiology.

Biotech Histochem 76, 111–118.

Boobathy, S., Soundarapandian, P., Subasri, V., Vembu, N.

and Gunasundari, V. (2009) Bioactivities of protein

isolated from marine sponge, Sigmadocia fibulatus. Curr

Res J Biol Sci 1, 160–162.

Boopathy, N.S. and Kathiresan, K. (2010) Anticancer drugs

from marine flora: an overview. J Oncol 2010, 214186.

Chairman, K., RanjitSingh, A.J.A. and Ramesh, M. (2012)

Screening twelve species of sponges for biomedical activity

in Gulf of Mannar Tuticorin Coast. Int J Mar Sci 2, 43–50.

Chu, P.M., Guenther, F.R., Rhoderick, G.C. and Lafferty, W.J.

(1999) The NIST quantitative infrared database. J Res Natl

Inst Stand Technol 104, 59.

Clarridge, J. (2004) Impact of 16S rRNA Gene sequence analysis

for identification of bacteria on clinical microbiology and

infectious diseases. Clin Microbiol Rev 17, 840–862.

Colonna, P., Leloup, V. and Bul�eon, A. (1992) Limiting factors

of starch hydrolysis. Eur J Clin Nutr 46, S17–S32.

Devi, P., Wahidullah, S., Rodrigues, C. and Souza, L.D. (2010)

The sponge-associated bacterium Bacillus licheniformis

SAB1: a source of antimicrobial compounds. Mar Drugs 8,

1203–1212.

Dhinakaran, D.I., Manohari, V., Atchya, B., Tamilselvi, K. and

Lipton, A.P. (2012) Antifungal and cytotoxic activities of

some marine sponges collected from the South East Coast

of India. J Appl Pharm Sci 2, 52–55.

Ely, R., Supriya, T. and Naik, C.G. (2004) Antimicrobial

activity of marine organisms collected off the coast of

South East India. J Exp Mar Biol Ecol 309, 121–127.

Firdouse, S. and Alam, P. (2011) Phytochemical investigation

of extract of Amorphophallus campanulatus tubers. Int J

Phytomed 3, 32–35.

Frye, J.G. and Jackson, C.R. (2013) Genetic mechanisms of

antimicrobial resistance identified in Salmonella enterica,

Escherichia coli, and Enteroccocus spp. isolated from U.S.

food animals. Front Microbiol 4, 135.

Gebhardt, K., Schimana, J., Muller, J., Fielder, H.P.,

Kallenborn, H.G., Holzenk€ampfer, M., Krastel, P., Zeeck,

A. et al. (2002) Screening for biologically active

metabolites with endosymbiotic bacilli isolated from

arthropods. FEMS Microbiol Lett 217, 199–205.

Geldreich, E.E., Nash, H.D., Reasoner, D.J. and Taylor, R.H.

(1972) The necessity of controlling bacterial populations in

potable waters; community water supply. J Am Water

Works Assoc 64, 596–602.

Gokulkrishnan, K., Kusuma, S. and Boopalan, K. (2011)

Antimicrobial activity of marine bacteria isolated from the

Mangalore coast, West Coast of India. Recent Res Sci

Technol 3, 15–17.

Gopi, M., Kumaran, S., Kumar, T.T., Deivasigamani, B.,

Alagappan, K. and Prasad, S.G. (2012) Antibacterial

potential of sponge endosymbiont marine Enterobacter

spp. at Kavaratti Island, Lakshadweep archipelago. Asian

Pac J Trop Med 5, 142–146.

Gordon, J. and McLeod, J.W. (1928) Practical application of

the direct oxidase reaction in bacteriology. J Pathol

Bacteriol 31, 185–190.

Hardoim, C.C.P., Esteves, A.I.S., Pires, F.R., Goncalves, J.M.S.,

Cox, C.J., Xavier, R.J. and Costa, R. (2012) Phylogenetically

and spatially close marine sponges harbour divergent

bacterial communities. PLoS ONE 7, e53029.

Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Gernert,

C. and Hacker, J. (2001) Isolation and phylogenetic

analysis of bacteria with antimicrobial activities from the

Mediterranean sponges Aplysina aerophoba and Aplysina

cavernicola. FEMS Microbiol Ecol 35, 305–312.

Ivanova, E.P., Vysotskii, M.V., Svetashev, V.I.,

Nedashkovskayal, O.I., Gorshkoval, N.M., Mikhailovl,

V.V., Yumoto, N., Shigeri, Y., et al. (1999)

Characterization of Bacillus strains of marine origin. Int

Microbiol 2, 267–271.

Janda, J.M. and Abbott, S.L. (2007) 16S rRNA gene sequencing

for bacterial identification in the diagnostic laboratory:

pluses, perils, and pitfalls. J Clin Microbiol 45, 2761–2764.

Kobayashi, S., Hodaka, S., Kawamura, Y., Ozaki, M. and

Hayase, Y. (1998) Micacocidin A, B and C, novel

antimycoplasma agents from Pseudomonas spp. J Antibiot

(Tokyo) 51, 323–332.

K€ock, R., Schaumburg, F., Mellmann, A., K€oksal, M., Jurke, A.,

Becker, K. and Friedrich, A.W. (2013) Livestock-associated

methicillin-resistant Staphylococcus aureus (MRSA) as

causes of human infection and colonization in Germany.

PLoS ONE 8, e55040.

Krieg, N.R., Ludwig, W., Whitman, W.B., Hedlund, B.P.,

Paster, B.J., Staley, J.T., Ward, N., Brown, D. et al. (2010)

Bergey’s Manual of Systematic Bacteriology. New York, NY:

Springer.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology 239

S. Skariyachan et al. Antimicrobial potential of metabolites extracted

Lee, Y.J., Kim, K.S., Kwon, Y.K. and Tak, R.B. (2003)

Biochemical characteristics and antimicrobials

susceptibility of Salmonella gallinarum isolated in Korea.

J Vet Sci 4, 161–166.

Liu, F., Sun, W., Su, F., Zhou, K. and Li, Z. (2012) Draft

genome sequence of the sponge-associated strain Bacillus

atrophaeus C89, a potential producer of marine drugs.

J Bacteriol 194, 4454.

MacFaddin, J.F. (1980) Biochemical Tests for Identification of

Medical Bacteria, pp. 173–183. Baltimore, MD: Williams &

Wilkins.

Marinho, P.R., Moreira, A.P., Pellegrino, F.L., Muricy, G.,

Bastos Mdo, C., Santos, K.R., Giambiagi-de Marval, M.

and Laport, M.S. (2009) Marine Pseudomonas putida: a

potential source of antimicrobial substances against

antibiotic-resistant bacteria. Mem Inst Oswaldo Cruz 104,

678–682.

Miller, J.M., Krisher, K., Holmes, H.T. (2007) General

principles of specimen collection and handling. In

Manual of Clinical Microbiology, 9th edn ed. Murray,

P.R., Baron, E.J., Jorgensen, J.H., Pfaller M.A., et al.,

p. 43. Washington, DC: American Society for

Microbiology.

Miller, J.H., Singh, A.J. and Northcote, P.T. (2010)

Microtubule-stabilizing drugs from marine sponges: focus

on peloruside A and zampanolide. Mar Drugs 8,

1059–1079.

Mol, V.P.L., Raveendran, T.V., Abhilash, K.R. and

Parameswaran, P.S. (2010) Inhibitory effect of Indian

sponge extracts on bacterial strains and larval settlement of

the barnacle, Balanus amphitrite. Int Biodeterior Biodegrad

64, 506–510.

Moreno-Vivi�an, C., Cabello, P., Mart�ınez-Luque, M., Blasco,

R. and Castillo, F. (1999) Prokaryotic nitrate reduction:

molecular properties and functional distinction

among bacterial nitrate reductases. J Bacteriol 181,

6573–6584.

Nagai, K., Kamigiri, K., Arao, N., Suzumura, K., Kawano, Y.,

Yamaoka, M., Zhang, H. and Watanabe, M. (2003) YM-

266183 and YM-266184, novel thiopeptide antibiotics

produced by Bacillus cereus isolated from marine sponge.

J Antibiot (Tokyo) 56, 123–128.

Petchi, R.R., Vijaya, C. and Parasuraman, S. (2013) Anti-

arthritic activity of ethanolic extract of Tridax procumbens

(Linn.) in Sprague Dawley rats. Pharmacognosy Res 5,

113–117.

Ravichandran, S., Wahidullah, S., D’Souza, L. and

Anbuchezhian, R.M. (2011) Antimicrobial activity of

marine sponge Clathria indica (Dendy, 1889). Bioorg Khim

37, 483–489.

Roser, D.J., Ashbolt, N., Ho, G., Mathew, K., Nair, J., Ryken-

Rapp, D. and Toze, S. (2005) Hydrogen sulphide

production tests and the detection of groundwater faecal

contamination by septic seepage. Water Sci Technol 51,

291–300.

Sabdono, A. and Radjasa, O.R. (2008) Microbial symbionts in

marine sponges: marine natural product factory. J Coast

Dev 11, 57–62.

Schwalbe, R., Steele-Moore, L. and Goodwin, A.C. (2007)

Antimicrobial Susceptibility Testing Protocols, pp. 91–109.

Boca Raton, FL: CRC Press.

Singh, D., Singh, P., Gupta, A., Solanki, S., Sharma, E. and

Nema, R. (2012) Qualitative estimation of the presence of

bioactive compound in Centella Asiatica: an important

medicinal plant. Int J Life Sci Med Res 2, 5–7.

Singla, N., Bansal, N., Gupta, V. and Chander, J. (2013)

Outbreak of Salmonella typhi enteric fever in sub-urban

area of North India: a public health perspective. Asian Pac

J Trop Med 6, 167–168.

Sulkin, S.E. and Willett, J.C. (1940) A triple sugar-ferrous

sulfate medium for use in identification of enteric

organisms. J Lab Clin Med 25, 649–653.

Taylor, M.W., Radax, R., Steger, D. and Wagner, M. (2007)

Sponge-associated microorganisms: evolution, ecology, and

biotechnological potential. Microbiol Mol Biol Rev 71, 295–

347.

Thakur, A.N., Thakur, N.L., Indap, M.M., Pandit, R.A., Datar,

V.V. and Muller, W.E.G. (2005) Antiangiogenic,

antimicrobial, and cytotoxic potential of sponge-associated

bacteria. Mar Biotechnol 7, 245–252.

Thomas, T.R., Kavlekar, D.P. and LokaBharathi, P.A. (2010)

Marine drugs from sponge-microbe association-a review.

Mar Drugs 8, 1417–1468.

Torpdahl, M., Lauderdale, T.L., Liang, S.Y., Li, I., Wei, S.H.

and Chiou, C.S. (2013) Human isolates of Salmonella

enterica serovar typhimurium from Taiwan displayed

significantly higher levels of antimicrobial resistance than

those from Denmark. Int J Food Microbiol 161,

69–75.

Usman, H., Abdulrahman, F. and Usman, A. (2009)

Qualitative phytochemical screening and in vitro

antimicrobial effects of methanol stem bark extract of

Ficus thonningii (Moraceae). Afr J Tradit Complement

Altern Med 6, 289–295.

Velho-Pereira, S. and Furtado, I. (2012) Antibacterial

activity of halophilic bacterial bionts from marine

invertebrates of Mandapam, India. Indian J Pharm Sci

74, 331–338.

Venkateswarlu, Y. and Biabani, M.A.F. (1995) Phycopsisenone

A new phenolic secondary metabolic from the sponge

Physopsis spp. J Nat Prod 58, 269–270.

Webster, N.S., Wilson, K.J., Blackall, L.L. and Hill, R.T. (2001)

Phylogenetic diversity of bacteria associated with the

marine sponge Rhopaloeides odorabil. Appl Environ

Microbiol 67, 434–444.

West, P.A. and Colwell, R.R. (1984) Identification and

Classification of Vibrionaceae – An Overview, pp. 285–363.

New York, NY: Wiley & Sons.

Zheng, L., Chen, H., Han, X., Lin, W. and Yan, X. (2005a)

Antimicrobial screening and active compound isolation

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology240

Antimicrobial potential of metabolites extracted S. Skariyachan et al.

from marine bacterium NJ6-3-1 associated with the

sponge Hymeniacidon perleve. World J Microbiol Biotechnol

21, 201–206.

Zheng, L., Yan, X., Xu, J., Chen, H. and Lin, W. (2005b)

Hymeniacidon perleve, associated bioactive Pseudomonas

spp. NJ-6-3-1. Prikl Biokhim Mikrobiol 41, 35–39.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1 Microscopic view of drug producing bacte-

rial isolates from marine sponges after Gram Staining.

Letters in Applied Microbiology 58, 231--241 © 2013 The Society for Applied Microbiology 241

S. Skariyachan et al. Antimicrobial potential of metabolites extracted