Abstract---Marine invertebrates such as corals and sponges from deep oceans to shallow coastal area colonize taxonomically diverse bacteria that are rich sources of novel bioactive compounds. The bacterial communities associated with scleractinian corals are highly specific in nature. Thus the present study is aimed to isolate aerobic bacteria from the coral surface for exploring their antimicrobial properties. The crude extracts from the isolates were assessed for its

potential acitivity against Gram positive, Gram negative bacteria and Candida albicans using the agar well diffusion method. About 42 culturable coral surface associated aerobic bacteria were isolated. Among the 42 isolates 5 showed maximum activity (≤ 25 to 33 mm zone of inhibition) and their phylogenetic analysis were done using 16S rRNA sequencing. The phylogenetic analysis of coral associated bacterial belongs to Pseudomonas, Firmicutes and Brevibacterium sp. These bacterial isolates can be exploited for pharmaceutical

application.

Keywords---16S rRNA, antimicrobial activity, coral associated bacteria, scleractinians.

I. INTRODUCTION

EEF-BUILDING corals are comprised of a complex

interaction between the coral host, endo-symbiotic

microalga (Symbiodinium sp.) and close associations with

a broad spectrum of unicellular and multicellular organisms [1], including bacteria [2],[3],[4], archaea [5], [6], viruses and

fungi [7],[8], [9] and hydrozoans[10]. Within individual

corals, there also exist a number of microhabitats (including

the coral surface mucus layer, coral tissues and coral

skeleton), and these each may contain distinct and diverse

microbial communities [4],[11],[1]. Bacteria are known to be

abundant and active in seawater around the corals, in coral

tissues, and within their surface microlayer. Very limited

information regarding the structure, composition and

maintenance of coral associated bacterial communities are

available. [12].

Umagowsalya Gothandapani

1, is with the Department of Marine and

Coastal Studies, School of Energy, Environment and Natural Resources,

Madurai Kamaraj University, Madurai, TamilNadu, India. (Corresponding

author’s e-mail: umagowsalya@gmail.com).

Anithajothi Rajasekaran 2 and Duraikannu Karrupaiah

3 are with the

Department of Marine and Coastal Studies, School of Energy, Environment

and Natural Resources, Madurai Kamaraj University, Madurai, TamilNadu,

India.

Dr. Ramakritinan Chokalingam Muthiah 4, Assistant Professor is with the

Department of Marine and Coastal Studies, School of Energy School of

Energy, Environment and Natural Resources, Madurai Kamaraj University,

Madurai, TamilNadu, India.

Rohwer et al. [13] hypothesized that the microbial community

found on a coral's surface may play a role in the coral's

defense mechanism, possibly by occupying niches or through

the production of antimicrobial metabolites. The coral mucus

layer serves as an ecological niche that is rich in nutrients and

diversified bacterial populations. It is found that 20% of

cultured bacteria from the mucus layer of the coral Acropora

palmata displayed antimicrobial activity. Thus, the presence

of microbial populations on the mucus surface of various

invertebrates may play a part in their defense strategy [14].

Marine microorganisms are of considerable current interest

as a new and promising source of biologically active compounds. Various secondary metabolites produced by these

microbes are used for drug development research [15].

Recently, it was shown that some bioactive compounds

isolated from invertebrates originate from associated

microorganisms [16]. The association between marine

invertebrates and symbiotic bacteria are increasingly

recognized as widespread and of biological importance [17].

Recent molecular methods such as16S rRNA gene

sequencing, RAPD, RFLP, has become the primary tool in

identifying bacteria isolated from various natural

environments. The first use of such methods in coral-associated bacteria characterized the prokaryotic micro-biota

associated with the Caribbean coral Montastraea franksi [2].

The cultured bacteria were found to be closely related to

previously described bacteria and consisted mostly of

representatives of the Gamma-Proteobacteria. However, the

culturable bacteria represent only a small fraction of the coral

micro-biota [12]. The aim of this study was to isolate the coral

surface associated cultivable bacteria and to assess their

antibacterial potential Pathogenic bacterial and fungal test

strains, including Methicillin-resistant S.aureus (MRSA)

which are highly responsible for hospital acquired infections,

i.e., nosocomial infections and Vancomycin-resistant Enterococcus sp.(VRE).

II. MATERIALS AND METHODS

A. Sampling And Isolation Of Coral Associated Bacteria

The coral samples were collected from Palk Bay, South

East Coast of India between Latitude 9° 55' – 10° 45'N and Longitude 78° 58' – 79° 55' E using scuba diving technique

and the samples were identified as Anacropora sp.,

Echinopora sp., Favites sp. according using morphological

The Dynamic Aerobic Culturable Microbiota

from Coral Niches and Their Antimicrobial

Properties

G.Umagowsalya*, R.Anithajothi, K.Duraikannu, and Dr. C.M.Ramakritinan

R

International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)

http://dx.doi.org/10.15242/IICBE.C0115016 62

immediately transferred to the marine shore laboratory of

Madurai Kamaraj University at Pudhumadam coast, Ramnad.

Where the coral fragments were rinsed with sterile seawater

thrice and scrapped off with a sterile knife aseptically. The

resultant tissues were vortexed with 9 ml of sterile water and

serially diluted, spread on Marine Zobell agar 2216E medium (Himedia) and incubated at room temperature for 48 to 96

hours. On the basis of morphological features, colonies were

randomly picked and aseptically sub-cultured in sterile Zobell

marine agar plates by streak plate method to get a pure culture

(Madigan et al., 2000). A slight modification was done in the

media i.e., the seawater which was collected from the 5cm

circumference near to the coral surface was filtered with

Whatmann no.1 filter paper and autoclaved at 121.1°C and

cooled and then used as alternative for distilled water for the

isolation of coral bacteria.

B. Morphological and Biochemical characterization:

The isolated bacterial cultures were subjected to

morphological, biochemical, physiological tests like Grams

staining, motility, oxidase, catalase, urease, esterase, amylase,

protease, lipase, carbohydrate utilization tests, optimum range

of pH and NaCl concentration, were examined.

C. Screening of marine coral associated bacterial isolates

for inhibitory interactions (antagonistic activity).

Bacterium-Bacterium variations may contribute to variation

in micro-scale community structure. In this study the bacteria

isolated from three different coral were pooled together to

study the antagonistic interaction through Burkholder assay. Each of the 42 isolates were examined for their inhibition of

growth against other 41 bacteria. A lawn of a target, isolate

was prepared by mixing 2.5ml of molten (45°C) 0.6% Marine

Zobell agar with 30µl of suspension of the isolate

(OD600,approx.1) , immediately vortexing the preparation for

two seconds and pouring the agar on to a sterile Zobell agar

plate. In a 3x3 matrix, 10µl portions (OD600, approx.0.1) of

the nine isolates were then spotted on the lawn. The plates

were incubated face up for 6 days at room temperature (20 to

24°C) and examined daily for zones of inhibition (areas where

the target lawn culture failed to grow). Potential producers

were considered positive when the diameter of the zone of inhibition was at least 4 mm greater than the diameter of the

colony formed by the potential producer. Potential positive

isolates were tested again to confirm the initial observation. If

ambiguous results were observed in the first two assays, a

third set of assays was performed. In all such cases the third

assay failed to detect inhibition; therefore, the isolates in these

cases were not considered producers.

D. Phylogenetic analysis:

The strains which showed significant activity were

identified genotypically based on 16S rRNA gene sequencing

analysis. The chromosomal DNA was extracted using the

method (Sambrook et al., (1989). The 16S rRNA gene of the

selected marine bacterial isolates were amplified using

primers such as 518F forward primer and 800R reverse

primer. The DNA sequencing reaction was carried out by

using an ABI Prism Bigdye terminator cycle sequencing ready

reaction kit V.3.1 (Applied Biosystems, Foster City, CA,

USA). The PCR cycle-sequencing product was purified by

PCR purification kit according to the manufacturer’s protocol.

The purified PCR products were then subjected to sequencing

with Perkin-Elmer ABI 373S automated sequencer

(Applied Biosystems). The sequences are then submitted to the NCBI nucleotide database for similarity search using

Clustal W package, Version 1.8.1.

E. Antimicrobial activity of coral associated bacteria:

Bacterial strains used:

From the Burkholder assay, those marine bacterial isolate

which showed inhibitory activity to a maximum number of

marine isolate are selected for testing antimicrobial activity. The marine isolates:IS2, IS7, IS14, IS15 and PS1 were used as

producer strain.The test organisms used were as follows:

Bacillus subtilis, Salmonella typhi, staphylococcus aureus,

Staphylococcus epidermidis, Streptococcus mutans, Shigella

flexneri, Vibrio sp., Micrococcus luteus, E. coli, Enterobacter

aerogens, Klebsiella pneumonia, Proteus vulgaris,

Pseudomonas aureginosa, Serratia marcescens, S. aureus

(MRSA), Candida albicans and Aspergillus niger.

Point Inoculation Method:

The antimicrobial activity of coral associated bacteria was

determined using the point inoculation method on sterile pre-

prepared Muller Hinton Agar plates. The overnight culture of

test bacterial isolates in nutrient broth was uniformly swabbed

on the surface of Muller Hinton Agar plates using a sterile

cotton swab, making a lawn culture. The plates were incubated

at 37°C for 24 hours. After the incubation period, the plates

were observed for the zone of inhibition and the results were

recorded as primary screen for antimicrobial activity.

Agar Well Diffusion Method

After screening by point inoculation method, the

antimicrobial substance production of the marine isolates was

tested by agar- well diffusion method. Muller Hinton agar

plate was prepared. The overnight culture of test organisms in

nutrient broth was uniformly swabbed on the surface of Muller

Hinton agar plates using sterile cotton swab. Four wells of 3mm size were made with sterile cork borer on the seeded

plate. Around 30 µl of the overnight broth culture of marine

isolate was added to the well aseptically. The plates were

incubated without inverting for 24hrs at 30°C and the zone of

inhibition was observed and recorded.

III. RESULT

A total of 42 cultivable bacteria were initially isolated from marine coral surfaces. Maximum numbers of bacterial

colonies obtained in Anacropora sp. were 1.3x106 CFU/cm2 of

coral surface and it was 3.6x106 CFU/cm

2 of coral surface in

Favia sp. and in Echinopora sp. it was in 5.6x106CFU/cm2 of

coral surface. The growth period for initial isolation of

bacteria ranges from 24 hrs, 24-36 hrs, 48 hrs, 72 hrs and

some colonies were observed after 4 days.

On morphological observation about 120 isolates were

pooled out from all the coral species for further studies. Based

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http://dx.doi.org/10.15242/IICBE.C0115016 63

on the morphological identifications and reliability of culture

to grow in subculture medium 42 isolates were selected for

further studies. These 42 isolates were then tested for

morphological, physiological and biochemical characteristics.

Fig.1 Percentage of coral associated bacteria isolated according to

morphology

According to morphological characteristics about 55% of

the isolates are rod shaped bacteria and 28% are cocci shaped,

whereas 17% exhibited both rod to cocci or short rod

shape(fig.1). On Gram’s staining 48% of the isolates were

Gram positive and 52% were Gram negative bacteria (fig.2).

Motility test was done with the hanging drop method in which

48% showed non-motility (fig.3). Endospore forming bacteria

were identified with the endospore staining and microscopical

observation. In this study, 24% are endospore forming bacteria

and 76% were non-endospore bacteria of all the isolated

bacteria from coral surface micro-layer (fig.4).

Fig. 2 Percentage of G+ve and G-ve culturable marine coral

associated bacteria

Fig. 3 Percentage of coral associated bacteria showing motility

Fig. 4 Number of endospore forming and non-endospore forming

bacterial isolates

All the 42 isolates were used for studying antagonistic

activity using Burkholder assay. Each of the isolate was

prepared as lawn and the other 41 isolates were spotted by

point culture method for observing the ability of the pointed

culture to inhibit the lawn culture. The zone of inhibition was

calculated from the diameter of clear zone of lawn culture

(sensitive strain) minus the diameter of point culture (producer

strain). The zone of inhibition was ranged as 0 mm; 1-4mm;

above 4-10mm; 11≥15mm and above.

From this assay, the highly potent coral associated bacterial

isolates which inhibited the maximum number of isolates were

selected as potential producer strains for studying antimicrobial activities. Of the 42 isolates, 5 isolates (IS2, IS7,

IS14, IS15, PS1) were selected according to their ability to

inhibit a maximum number of isolates (coral associated

bacteria) and very well acclimatized to laboratory condition.

The biochemical and physiological characteristics observed

were displayed in Table.1.

The 16S rRNA genes of five isolates (producer strains)

were sequenced and compared to the NCBI Genbank

Database. About 60% of the producer strains belonged to

Firmicutes with low G+C content, while 20% of them showed

similarity with gamma-proteobacteria and Actinobacteria

respectively with high G+C content. From the Burkholder assay it is very clear that each of the isolates showed their own

inhibitory activity against each other that is essential for the

competition for their micro-niches in the coral surface micro-

layer. TABLE I

BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERISTICS OF SELECTED

BACTERIA

Biochemical

characteritics

Selected marine coral associated bacterial isolate

IS2 IS7 IS14 IS15 PS1

Amylase - + + + -

Protease + + + + +

Catalase + + - - +

Oxidase + + - - +

Esterase + - - + +

Lipase + -/+ -/+ +

urease - - - - -

Gelatin

hydrolysis + -

+ + -

Starch

hydrolysis - + + + -

H2S - - - - -

Citrate

utilization + (vs) - - + -

Growth at pH

range 7 to 8.5 7 to 12

7.5 to

10.5 6.8 to 11 7to 9.5

Optimum

range of

temperature

15-

35°C 25 -45°C

15-

45°C 25-45°C 25-35°C

Growth at

various % of

NaCl

Growth

occurs

in 1%-

15%

NaCl

Growth

occurs

in3%-

10%

NaCl

Growth

occurs

in3%-

10%

NaCl

Growth

occurs

in3%-

10%

NaCl

Growth

occurs in

1%-10%

NaCl

Carbohydrate

utilization:

Glucose

Sucrose

Fructose

Lactose

Maltose

+/-

+

+

-

-/+

+

+

+/-

-

-

+

+

+

-

+/-

+

+

+

-

-

+

+

++

-

-

Nitrate

reduction -/+

w - + + +

Vs-very slight; -/+w (negative in two test and positive in one trial, so it is considered

as weakly positive.).

This antagonistic activity is essential for studying the

antibacterial activity against various pathogens. The coral

associated bacterial isolate PS1 showed maximum inhibitory

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activity against four pathogenic bacteria Bacillus subtilis,

Micrococcus luteus, E.aerogenes, Enterococcus sp. and both

of the fungal pathogens. The producer isolate IS15 showed

maximum antibacterial activity against S.typhi, S.mutans,

S.flexneri, P.vulgaris, S.marcescens. IS7 showed maximum

antibacterial activity against Vibrio cholera, V.parahaemolyticus and Serratia marcescens. The

antibacterial activity of the selected isolates against different

pathogenic bacteria and fungus are reported in the Figures. 5,

6, 7.

IV. DISCUSSION

In this study, the maximum number of bacterial colonies

were observed from Echinopora sp.(5.6x106CFU/cm2) >Favia

sp.> Anacropora. A similar study was reported by Koren et al.,[27] with different staining method, SYBR gold staining,

the average numbers of bacteria in the coral mucus and tissue

(coral minus mucus) were (6.2± 4.2 x107CFU/cm2) and (8.3±

1.6 x108CFU/cm

2), respectively.

In the present study, 12% of the cultivable coral associated

bacteria showed antimicrobial activity against a maximum

number of microbes which is reliable to the results showed by

Shnit-Orland et al. In their study, they tested for antibacterial

activity among cultivable coral mucus-associated bacteria

from the marine environment of the Gulf of Eilat and

demonstrated that 20-25% of the coral mucus cultivable bacteria displayed antibacterial activity [18]. Species richness

was lower in apparently healthy coral colonies than either the

Healthy and Diseased coral or only diseased coral samples

[19], [20], [21].

Fig.5. Antibacterial activity of coral associated bacterial isolates against Gram Negative Pathogenic test strains

Fig.6.Antibacterial activity of coral associated bacterial isolates

against Gram Positive Pathogenic test strains

Fig. 7.Antibacterial activity of coral associated bacterial isolates

against Fungal Pathogens

In the present study, 48% of the isolates were Gram negative. Similar studies were done in other invertebrate

species. Gnanambal et. al.,[22] observed that out of the 352

bacterial strains isolated from the gorgonoid corals

Subergorgia suberosa and Junceella juncea, 61% were

identified as Gram negative. Chellaram et. al.,[23],[24],[25]

identified that 73.5% of the 632 strains isolated from

Acropora nobilis were Gram-negative. Chelossi et al, [26]

reported similar aspects and their findings showed that 58% of

the aerobic heterotrophic bacterial strains isolated from the

sponge, Petrosia ficiformis were identified as Gram-negative.

Burkholder agar diffusion assay helps to study the antagonistic

bacterium –bacterium interaction within a bacterial consortium. From the Burkholder assay it was found that the

maximum number of isolates showed inhibitory activity to one

or more producers.

The antimicrobial compound producing potential was

expressed as the potency of an isolate to inhibit the maximum

number of bacterial isolates from the same niche. The present

study showed that almost all the 42 coral associated bacteria

expressed inhibitory activity displaying a weak to strong

(1mm to more than 15mm) inhibitory activity. From the assay

it was very clear that least sensitive isolates was the more

potent secondary metabolite producer expressing antimicrobial activity against bacterial and fungal pathogens other than coral

associated cultivable bacterial isolates. Pseudomonas (PS1)

which is a gamma-proteobacteria inhibited maximum

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pathogens. Firmicutes (IS7, IS14, IS15) and Brevibacterium

(IS2) are likely to produce antimicrobial compounds against

pathogens were displayed in the figure.5,6,7. This report was

in agreement with the findings of Long et. al., [28] that

showed the particle attached bacteria are more likely to

produce inhibitory compounds than their free-living counterparts. Antimicrobial potential of microbes from

scleractinian coral, Acropora sp. were reported previously.

Whereas in our study, cultivable bacterial isolates with

antimicrobial potential from selected scleractinian corals of

the Palk Bay helps to further investigate novel bioactive

secondary metabolite for the future prospects of

biotechnology.

V. CONCLUSION

This study aimed to isolate cultivable coral surface

associated microbes which are having more potential to

produce antimicrobial secondary metabolites. Antagonistic

interactions of producer strains were well studied. Dominant

isolate to inhibit a maximum number of pathogens as well as

producer strains were observed. 16S rRNA sequencing and

phylogenetic analysis of the selected potent strains to produce

secondary metabolite were identified as Brevibacterium sp.,

Firmicutes and Pseudomanas sp.

ACKNOWLEDGMENT

The authors wish to thank the Department of Marine and

coastal Studies, School of Energy, Environment and Natural

Resources, Madurai Kamaraj university for providing well

furnished laboratory facilities at Pudhumadam Coast, Ramnad

district, Tamilnadu, India.

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