Introduction

The marine environment is a complex ecosystem with an enormous diversity of different life forms often existing in close associations. Among these, microorganisms- eukaryote associations have gained significant attention in the past decade7. The surface of all marine eukaryotes are covered with microbes that live attached in diverse communities often embedded in a matrix, forming a bio-film11. Moreover, host specificity has also been illustrated by studies that have shown the presence of unique stable communities living on geographically distant individuals belonging to the same species16.

Marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactivities because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surface of marine eukaryotes1.

The number of natural products, discovered from various living organisms including plants, animals and microbes, to date exceeds one million2, with the majority (40-60%) derived from terrestrial plants. Of these natural products, 20-25% possess various bioactive properties including antibacterial, antifungal, antiprotozoal, ant nematode, anticancer, antiviral and anti-inflammatory activities, Plants and plant extracts have been used for the treatment of human diseases for millennia, and their use has been recorded in the most ancient archaeological sources. In contrast, the exploration of microorganisms as producers of therapeutically agents only began in the 20th century10.

The bacteria are found associated with all organisms. Out standing examples from the sea are the 50% bacterial biomass in sponges of the order Verongia sp., abundant Cyanobacteria sp. and oxychlorobacteria in many sponges. Many eukaryotic microorganisms are also known as symbionts, for example dinoflagellates in sponges fungi in a variety of invertebrates, algae and sea-grass14. Thew polychaeta or polychaetes are a class of Annelid worms. Each body segment has a pair of fleshy protrusions called parapodia that bear many bristles called chaeta, which are made of chitin.

The microbes associated with polychaetes were not studied much. There are several advantages for using microbes as source of bioactive compounds. Hence, in the present study an attempt has been made to screen the antimocrobial activity of bacxteria associated with polychates. A study of this kind will provide more details on the bioactivity of bacteria associated with marine invertebrates.

Materials and Methods

Isolation and identification of Bacteria

Polychaetes were collected from the Kanyakumari coast of west coast of India. For the collection of the polychaetes, a portion of the seaweeds Sargasum sp was detached from the surface and kept in polythene bags with seawater and brought to the laboratory.

In the laboratory, the polychaetes were removed from the seaweed by gentle agitation and collected in a jar with seawater. The polychaetes were rinsed with sterile seawater to remove the loosely attached organism and the surface was swabbed with a sterile nylon brush. The bacterial film swabbed using the brush was dispersed in 1ml filter -sterilized seawater (Millipore, 0.45µm). This bacterial suspension was serially diluted and appropriate dilutions were poured on zobell marine agar. The plates were incubated at room temperature for 48 hours and the developed colonies were purified by repeatedly streaking on zobell marine agar plates. The permanent cultures were maintained in zobell marine agar slants at 4°C. The bacterial colonies were chrecterized based on Bergey’s manual of determinative bacteriology.

Isolation of Extracellular Polymeric Substance (EPS) of the Bacteria associated with Polychaetes

The amount of EPS produced by the cultures was determined by estimating total carbohydrates. 100ml culture broth was centrifuged at 5000 rpm for 15 minutes at 4oC. The cell pellets were discarded and the supernatant was mixed with equal amount of cold absolute ethanol. After one day incubation the precipitated EPS was collected and stored at 4oC.

ANTIBACTERIAL ACTIVITY OF EPS

Disc diffusion (Kirby-Bauer) method

In this method sterile filter paper discs were used 50μl of the EPS was loaded on discs and placed on the sterile Zobell marine agar plates seeded with the test organisms. The test organisms used in the present study were Galionella sp., Alteromonas sp., S.aureus and Klebsiella sp. After firm placement of the discs the plates were incubated at 37oC in inverted position for 48 hours. After the period of incubation the inhibition zone if any around the discs were measured.

Characterization of Extracellular Polymers by Thin Layer Chromatography

The bioactive compounds present in the EPS were partially characterized by Thin-layer chromatography. Methanol, acetic acid and benzene (3:1:1) were used as solvents for the Thin layer chromatography. The samples were loaded on silica gel plates and kept in the TLC chamber for the mobility of the compounds were detected by iodine crystals.

Results

Biochemical Analysis

The biochemical and physiological characteristics of the isolated strains are given Table-I.

Table-1

Biochemical and physiological characteristics of the Bacterial strains
S.No Biochemical and physiological Test Organisms
Strain A Strain B Strain C
1 Gram Staining - - +
2 Morphology cocci cocci cocci
3 Motility + - -
4 Indole production + + +
5 Methyl Red + - +
6 Voges-Prousker + + +
7 Citrate utilization + + +
8 Starch Hydrolysis - + -
9 Urea Hydrolysis - - -
10 TSI Agar test
(i)Acid		 + + +
(ii)Alkaline + + +
(iii)Gas Production + + +
(iv)H2S Production - - -
11 Catalase + + +
12 Oxidase + + +
13 Nitrate Reduction + - +
14 Casein Hydrolysis + + -
15 Gelatin Hydrolysis - + +

Most of the strains are gram-negative, cocci and non-motile. Strain A isolated from the polychaete was gram-negative, cocci and showed motility. It was indole positive, methyl red positive and voges-proskauer positive. It also utilized citrate and showed negative results for starch hydrolysis and urea hydrolysis. In triple sugar iron test, strain A showed positive result for acid, and alkali. It also showed negative result for gas production and hydrogen sulphide production. The strain A positive for catalase, oxidase and nitrate reduction test. It also hydrolysed casein and showed negative result for gelatin hydrolysis.

Strain B isolated from surface of the polychaetes was gram-negative, cocci and non motile. It was indole positive, methyl negative and voges-proskauer positive. It also utilized citrate and positive for starch hydrolysis and negative result for urea. In triple sugar iron test, strain B showed positive result for acid, alkali. It showed positive result for gas production and negative for hydrogen sulphide production. Strain B also showed positive result for catalase, oxdiase and negative for nitrate reduction test. It hydrolysed casein and gelatin.

Strain C isolated from the surface region of polychaete was gram-positive, cocci and showed motility. It was indole positive, voges-proskauer positive. It also utilise citrate and showed negative result for starch hydrolysis. In triple sugar iron test, strain C showed positive result for acid, alkali and gas production and negative for hydrogen sulphide production. Strain C also showed positive result for catalase, oxdiase and positive for nitrate reduction test. It showed negative for casein hydrolysis and positive for gelatin hydrolysis.

Antibacterial Activity of EPS

The extracellular polymeric substance isolated from all the three bacterial strain was tested for their antimicrobial activity against the four bacterial strains (Galionella sp., Alteromonas sp., S.aureus and Klebsiella sp.). and the results are given in (Table -2).

Table- 2

Antibacterial activity of EPS against target Bacteria
S.No Target Bacteria Diameter of zone of inhibition(mm)
Strain A Strain B Strain C
1 Galionella Sp. 10 - 9
2 Alteromonas Sp. 9 15 13
3 Staphylococcus aureus 8 - 9
4 Klebsiella Sp. 9 10 10

The EPS isolated from the strain A showed inhibitory activity against all the four target bacteria and showed a maximum inhibition zone against Galionella sp. 10 mm and minimum of 8 mm against S.aureus. The zone of inhibition against Alteromonas sp. was 9 mm (Fig-1) and the zone of inhibition against Klebsiella sp. was 9 mm.

Fig. 1: Antibacterial activity of EPS of strain A against Alteromonas sp.

The EPS isolated from the strain B showed inhibitory activity against Alteromonas sp. and klebsiella sp. It showed a maximum inhibition zone against Alteromonas sp. 15 mm and minimum of 10mm against Klebsiella sp. The EPS of strain B did not show inhibitory activity against Galionella sp and S.aureus.

The EPS isolated from the strain C showed inhibitory activity against all the four target bacteria and showed a maximum inhibition zone against Alteromonas sp. and the diameter of zone was 13 mm. The minimum inhibition zone of 9 mm was observed against Galionella sp. and S. aureus. The zone of inhibition against Klebsiella sp. was 10 mm.

Thinlayer Chromatography Analysis of EPS

The EPS was loaded on silica gel glass plates for the characterization of active compounds. Thinlayer chromatogram showed the presence of a single spot in all the strains. The EPS isolated from the strain A showed a single spot with the Rf value of 0.75 cm. The Rf value of the compound present in the EPS isolated from the strain B was 0.72 cm. The EPS isolated from the strain C showed a single spot with the Rf value of 0.53 cm (Fig-3).

Fig. 2: Antibacterial activity of TLC purified compounds of strain C against Staphlococcus aureus.

Fig. 3: Thin-layer chromatiogram of EPS isolated from the bacterial strains.

Discussion

The present study strongly revealed the ecological rationale for polychaetes and its associated microorganisms for the maintenance of antimicrobial defenses. Seawater typically contains 10-7 viruses, 10-6 bacteria, 10-3 fungi and 10-2 microalgae/ml6, including those which have been identified as causative agents in marine infectious diseases5. Given that marine invertebrates and their symbionts are continuously exposed to a broad array of potentially deleterious microorganisms, it is reasonable that the production of bioactive secondary metabolites could act as fundamental mechanisms of antimicrobial defense.

Chemical interaction between different species of bacteria can affect the production and secretion of antimicrobial secondary metabolites12. The enhancement of antimicrobial compound by bacteria when they are exposed to a different strain of bacteria suggest that competition for space between epibiotic bacteria may provide further antifouling protection to the basibiont. In addition, some bacteria that previously did not produce such metabolites when they are exposed to other bacterial species or extracellular products from other bacteria. As surface-associated bacteria would be exposed to similar pressures in the surface biofilm, this occurrence further increase the number of strains of bacteria isolated from seaweed surface that that are producing active compounds.

In the present study, of the three strains isolated from the polychaetes, two were Gram-negative. Various studies have confirmed the predominance of Gram-negative producers in the marine environment8. In a study on antibiotic production in marine bacteria3, have reported that 36% of the strains were Gram-negative rod. In this study, Gram-positive as well as Gram-negative bacteria were isolated from the polychaetes. Since, only three strains were isolated from a polychaetes species, more rigourous culture methods may reveal the actual diversity of bacteria associated with polychaetes.

The diversity of antibiotic producing marine bacteria isolated in the present study suggests that polychaetes are rich of bacteria9. The bacteria isolated in the present study may be only a fraction of the total diversity of associated bacteria, given that only a small percentage (1%) of the bacteria can be cultured using the currently available medium and fermentation techniques13.

Serious attempts to tap the vast potential of marine organisms as source of bioactive metabolites that may be directly utilized as drug or serve as lead structures for drug development started in the late 1960s. The discovery of sizeable quantities of prostaglandins, which had just been discovered as important mediators involved in inflammatory disease, fever and pain , from the gorgonian plexaura homomalla by Weinheimer and Spraggins in 196915 is usually considered as the “take –off point” of any serious search for “drug from the sea”16.

The discovery of new classes of antibiotics is necessary due to the increased incidence of multiple resistance among pathogenic microorganisms to drug that are currently in clinical use4. Peninsular India enjoys a large coastline with diverse marine ecosystems. The microbial diversity was not much studied from Indian marine ecosystem in respect to bioactive compounds search. This true in the case of polychaetes, in which only a few studies are available on the bioactive potential of associated bacteria.

In general, results of the present study suggest that bacteria associated with polychaetes are having strong antimicrobial activity and could be used as a potential source for the development of marine drugs. The extracellular polymeric substance produced by the marine bacteria are reported to have various application. Hence, more studies on the characterization of the isolated strains may improve our understanding on the chemical ecology of bacteria associated with polychaetes.

Table -3

Rf value of compounds observed in the TLC
Rf values (cm) Strain A Strain B Strain C
0.75 0.72 0.53