Microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, ‘reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).

Seaweeds itself secretes secondary metabolites to prevent fouling and grazing. In addition to that epibiotic bacteria on macro algae can also produce antifouling compounds that work in concert with the seaweed derived compounds to protect the seaweed surface. Recent studies have highlighted important roles of epibiotic bacteria colonizing the surface of seaweeds and releasing antifouling compounds. For the past 50 years antibiotics have revolutionized medicine by providing cures for formerly life threatening diseases. However, strains of bacteria have recently emerged that are virtually unresponsive to antibiotics such multidrug resistance, arising mainly through antibiotic misuse, is now recognized as a global health problem. In this situation, it is clear that new classes of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites that display antibacterial properties. The first antibiotic from a marine bacterium was identified and characterized in 1966. In addition, bacteria in biofilm on the surface of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Burgess, et al., 1999). Marine epibiotic bacteria, associated with nutrient-rich algal surfaces have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors. Recently a lot new novel antibiotics such as Phenazine, thiomarinol, phenazine-1-carboxylic acid, 1-hydroxyphenazine 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one pyolipic, loloatins, agrochelin, sesbanimides, pelagiomicins, indomycione and indomycione have been identified from various marine epibiotic bacterial organisms. In particular, some species of the genus Pseudomonas produce both antibiotics and several other bioactive substances. For example, Pseudoalteromonas rubra and Pseudoalteromonas aurantia have been reported to be antibiotic producing bacteria. The phenomenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). A study of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% whereas that from sea water was only a few percent. In addition, some bacteria that previously did not produce any active compounds have been found to be producing such metabolites when they are exposed to other bacterial species or extra cellular chemical from other bacteria. Bacteria may also produce antimicrobial compounds when they sense the presence of competing organisms. However, few attempts have been made to study such chemical communication between different bacterial species or how this might affect. The secretion of antimicrobial compounds (Mearns-Spragg, et al., 1998). Bacterial communication by the chemical signals for specific function is simply known as Quorum sensing. In which a bacterial population receives input from the environment and elicits an appropriate response (Hiroaki and Kristina. 2003). The term “quorum sensing” describes the ability of a microorganism to perceive and response to diffusible signal molecules. Bacterial cells sense their population density through a sophisticated cell to cell communication system and trigger expression of particular genes. Tne first system of density-dependent regulation was studied in detail with the luminescence of Photobacterium fischeri (formerly known as Vibrio fischeri) by Bassler et al., 1997. Eventually, they discovered that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamid or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible the agent in the broth that induced luminescence. Followed by this many researchers have confirmed that in Gram negative bacteria acyl-homoserine lactone is responsible for the cell to cell communication system.

In gram positive bacteria peptide and derivative peptide based signaling molecules seem to be the predominant mode of communication. During high cell density the marine bacteria can produce enzymes, surfactants, toxins, and antibiotics by the chemical signal communication. Marine epibiotic bacteria are also known to produce compounds active against drug resistant hospital pathogen by the cross species induction method. Building on assays described by Austin (Billaud and Austin 1990) a screening procedure has been developed in which marine bacteria are challenged by exposing them to terrestrial bacteria prior to assay of antimicrobial compounds. Hence in this present investigation it is proposed to find out the ability of sea weed epibiotic bacterial organism to produce antibacterial compounds through quorum sensing.

MATERIALS AND METHODS:

SAMPLE COLLECTION:

Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

Table 1. List of Seaweeds species collected for the present study

SPECIES NAME FAMILY

Halimeda gracilis Chlorophyceae

Ulva lactuca Chlorophyceae

Microdictyon tenunis Chlorophyceae

Chondrococcus hornemonii Chlorophyceae

Enteromorpha intestinalis Chlorophyceae

Caulerpa cupressoides Chlorophyceae

Caulerpa racemosa Chlorophyceae

Dictyota dichotoma Phaeophyceae

Turbinaria ornata Phaeophyceae

Padina gymnospora Phaeophyceae

Sargassum cinearifolium Phaeophyceae

Dictyota batryensis Phaeophyceae

Sargassum sps Phaeophyceae

Hypnea musciformis Rhodophyceae

Acanthophora dendroides Rhodophyceae

Jania rubens Rhodophyceae

Hypnea valentiae Rhodophyceae

Hypnea pannose Rhodophyceae

Hypnea esperi Rhodophyceae

Acanthophora spicifera Rhodophyceae

ISOLATION OF EPIPHYTIC BACTERIA

The collected seaweed samples were thoroughly washed with sterile seawater to removes the loosely attached bacteria/particles. Seaweed fronds were scrubbed with sterile cotton swabs to obtain epiphytic bacteria. Epiphytic bacterial organism in the swab were inoculated in sterile peptone broth (50% sea water) and incubated at 28°C in an incubated shaker (220 rpm / min) for overnight. After the incubation period the enriched cultures were serially diluted up to 10-8 concentration and 200 microlitre of each diluted samples were transferred into the nutrient agar plate (50% sea water). The plates were incubated at 28°C for 5 days and the plates with crowded colonies were selected. In the crowded plates those colonies, which showed the sign of inhibition zone around its margin to the neighboring colony, were selected and considered as producer strain. The neighboring sensitive colonies were treated as inducer strain. Both producer and inducer strains were streaked repeatedly until to get pure culture. The pure culture were properly labeled and subjected to the quorum sensing analysis.

QUORUM SENSING

EXPERIMENT NUMBER 1

In this present study, the producer and inducer strains were cross reacted to find out the production of antibiotic compound through quorum sensing. Totally three set of cultures were maintained as follows (along with one as control).

A. Live cells of producer and inducer strains

B. Live cells of producer strain alone

C. Live cells of inducer strain alone

In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

In culture system B 200ul of 16 hours old producer strain alone was inoculated.

In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

All the cultures were incubated at 28°C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

EXPERIMENT NUMBER 2

In this experiment, culture supernatant was obtained as per the procedure given in the experiment 1. 50ml of supernatant was mixed with equal volume of 80% methanol and 1% acetic acid mixture and it was shaked thoroughly in a separating funnel. Finally the methanol and acetic acid fractions were collected and concentrated by evaporation using water bath at 55°C. The viscous colloidal residues were resuspended in 600 microlitre of 50% methanol and it was used for antibacterial assay against different test organism.

TEST ORGANISMS:

1. Epiphytic Vibrio from seaweeds

2. Vibrio from primary film

3. Vibrio from Sediments

4. Pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella sp. and Proteus sp

The test organisms Vibrio species were isolated from seaweed as epiphyles, biofilm, sediment and puffer fish by using TCBS medium (Hi media) The pathogenic bacteria were collected from clinical laboratories.

ANTIBIOTIC ASSAYS

Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37°C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

BACTERIAL IDENTIFICATION

The organisms responded to the quorum sensing process alone were identified by the following biochemical analysis.

Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

Starch hydrolysis test, Protein hydrolysis test, Lipid hydrolysis test, Oxidative / Fermentative test, Salt concentration (0%, 3%, 5%, 7%, 10%), TCBS, Growth in Temperature, 42°C and 47°C

All the above mentioned biochemical tests were performed by following standard methodology given in the Microbiological Laboratory Manual by James 3.Cappuccino (1999).

RESULTS AND DISCUSSION:

QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS

In the present investigation totally 84 isolates were collected out of seaweed species. Among 84 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 50 isolates is normal and not showing any signs of activity (Table.2).

a) Among these 17 producers strain 6 strains were isolated from Hypnea musiformis. 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from Sediment.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

All the 17 strains were named as

PRODUCERS STAINS

BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis

GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

U1+, U2+, U3+, U4+ Ulva lactuca

SA+ Sediment

INDUCER STRAIN

BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis

U1-, U2-, U3-, U4- Ulva lactuca

SA- Sediment

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/Bo-