Introduction

It is well established fact that microbial natural products remain one of the most important sources of lead compounds for the pharmaceutical industry. Among microorganisms, the actinomycetes are a fascinating group that they are sources of most of the antibiotics used in medicine today. They also produce metabolites that are used as anticancer drugs, antihelminthics and drugs that suppress the immune system in patients who have received organ transplants. This becomes apparent in 1940, following Selman Waksman’s discovery of actinomycin from S. gresous¹ and was fully realized by the 1980s, when actinomycetes accounted for almost 70% of the world’s naturally occurring antibiotics².

In addition to terrestrial sources actinomycetes have been isolated from marine water, sediments, plants and animals. Actinomycetes comprise about 10% of bacteria colonizing marine aggregates³. Despite their abundance, however, reports in marine actinomycetes are yet not explored. As a result in recent years marine environment be comes a major focus in the search for the next generation of pharmaceutical agents^4. This is because; organisms in marine environment have developed unique adaptations that enable them to survive in dark, cold and highly pressurized environments. These novel adaptations can offer a wealth of opportunities for the discovery of new drugs for the treatment of infectious diseases ^{5, 6, 7}.

The goal of this study were to screen and isolate antibiotic producing actinomycetes that are found in different samples of marine environment in the East Coast of Bay of Bengal using refampicin or naldxic acid as a selective inhibitor of bacterial contaminant.

 Materials and Methods

Sample collection and processing

A total of 37 different samples were taken from marine environment  (10 sea water, 20 sediments, 1sample each from sponges, hard corals and snails, and 4 algae and sea weeds) at a various depth using  sterile screw capped bottle and sterile plastic bags.

Samples were processed as soon as collected and some of the samples collected from distant site were processed in the following days of collection by preserving the samples with a sterile plastic bag in an ice box. Algae and sea weeds samples were dried overnight in a laminar flow hood and crushed with an alcohol sterilized mortar and pestle. Other samples like sponges, corals and shell of snail samples were processed as described by Jensen et. al. ⁸. These samples were subjected overnight in a laminar flow hood and the samples were scrapped with a sterile spatula generating a powder.

About 1gm of the above samples (sediments, dried powdered samples) was suspended aseptically and were homogenized with sterile sea water and kept in 250 ml flask having 50 ml of sterile sea water and incubated in an orbital shaker at 26 ⁰C with shaking at 140 rpm for 30 minutes. Mixtures were allowed to settle then ten fold serial dilutions were prepared. Depending on the condition water samples were either directly or serially diluted.

One millilitre of each of these dilutions was added to 50ml of starch casein agar (g/l: starch 10, casein 0.3, KNO₃ 2, NaCl 2, K₂HPO₄ 2, MgSO₄.7H₂O 0.05, CaCO₃ 0.02, FeSO₄.7H₂O 0.01, agar 20) and oat meal agar (g/l: oat meal 20, agar 20, trace salt solution 1ml) when the temperature is about 40-45 ⁰C. Samples were thoroughly mixed and poured in to a sterile Petri-plate (6 diam). One copy of the samples was plated following spread plating technique using sterile L-shaped glass rod. Inoculated samples were incubated at 28⁰C for 21 days. To inhibit growth of non-filamentous bacteria and fungi medium was supplemented with 5µg/ml rifampicin⁸ or 20µg/ml naldixic acid and 50µg/ml cyclohexamide ⁹ respectively.

Identification of actinomycetes

Actinomycetes colonies were recognized by their characteristics tough leathery colonies that adhered to the agar surface, branched vegetative mycelia, and when present, aerial mycelia and spore formation ^8,10.  Actinomycetes colonies were counted on different plates after 10-21 days incubation at 28 ⁰C. Data are expressed as colony forming unit (CFU/ml) for water or CFU/gm for dry weight samples. Actinomycetes colonies were characterized morphologically and physiologically following the methods given in the International Streptomyces Project (ISP) ¹¹. Species were identified by the morphological characteristics of colonies, substrate and aerial mycelium, structure of spore chains and pigment production on different ISP media. Detailed physiological and biochemical characterization of promising isolates were performed following the standard procedure¹².

Determination of antimicrobial activity

Primary screening of antimicrobial activities of actinomycetes isolated from different samples were screened following the streak plating technique. The test organisms employed in this study were supplied by the National Chemical Laboratory (NCIM), Pune and Microbial Type Culture Collection and Gene bank (MTCC), Chandigarh. The following test organisms were used for antimicrobial activities. Bacillus pimilus (NCIM-2327), Bacillus subtilis  (NCIM-2063),  Staphylococcus aureus (NCIM-2079), Escherichia coli (NCIM-2065),  Shigella flexinari (MTCC-1457), Salmonella typhimerium (MTCC-98),  Aspergillus niger (NCIM-548), Aspergillus oryzae (NCIM-643), Penicillium cryseogenum (NCIM-738),  Candidia albicans, and Saccharomyces cervisae.

Further screening of activities were made using an agar overlay methods following methods of Willims et.al. 1983a ¹³. That is, spot inoculated 5 days old plated colonies were killed by inverting them over 1.5ml chloroform for 40 minutes. The dead colonies were overlaid with 5ml of nutrient agar (bacteria) and 5 ml of potato dextrose agar (yeast and fungi) that has been inoculated with the test organism. Zone of inhibition around the colonies was recorded after 24 hours at 37 ⁰C for bacteria and 48 hours at 28 ⁰C for yeast and filamentous fungi.

Result and Discussion

The total number of actinomycetes recovered from different samples showed that the maximum population density was recorded in samples of sea weeds and algae, 3.5 X 10⁵ CFU/gm followed by samples of sponges, corals and snails, 4.7 X10⁴ CFU/ml; sediments 2.6X10³ CFU/ml and water with least population counts, 6.2X10²CFU/ml. (Table -1). Sahu et.al¹⁴ observed similar patterns of population density of marine actinomycetes from sediment and water samples collected in the East coast of India. The maximum count of actinomycetes in algae and sea weeds samples may be because of nutrient availability. That is, nutrient availability in plant and animal residues is higher and this may create a fertile ground for spores of actinomycetes to germinate. However, the population of actinomycetes decrease in water samples than other samples, this is also because dormancy of spore imposed by lack of nutrients in water and a spore population will decline¹⁵ and results low number of actinomycetes in water sample than other samples.

Tables 1:  Total microbial count obtained in different samples using Rifampcin.

To avoid bacterial contaminant from different samples, 5μg/ml rifampicin or 20μg/ml naldixic acid have been used for comparison.  Comparing to naldixic acid, the rate of recovery of antibiotic producing actinoomycetes using rifampicin is relatively high (Table-2). Similar report by Pisano et.al.¹⁶ showed that, in pre-treated samples 16 % of the actinomycetes produced bioactivity while in rifampicin treated marine samples 46% of actinomycetes showed bioactivity¹⁷.

As indicated in table-2, four water samples were taken for comparison of rifampicin and naldixic acid. It was found that actinomycetes colonies were recovered only in one sample using rifampicin (1.5X10¹CFU/ml). Out of 10 sediment samples taken for comparison, actinomycetes were recovered in 5 samples for rifampicin (1.1X10³ CFU/gm) and 3 samples for naldixic acid (1.3X10² CFU/gm). One sample each from corals, sponges and shells were also compared, both rifampicin (4.7 X10⁴ CFU/gm) and nalixic acid (2.8X10³ CFU/gm) were showing similar recovery with high population density in rifampicin. Four samples from algae and sea weeds were also compared, 3 of the samples were showing high density count using rifampicin (3.6X10⁵ CFU/gm) and 2 of the samples showing also good count using naldixic acid (3.0X10⁴ CFU/gm). As naldixic acid, rifampicin is also effective against some gram-positive and gram-negative bacteria. However, rifampicin is mostly effective when there is combination of other drugs (Examples, treatment of tuberculosis, leprosy); this might be the reason that using rifampicin alone may favour recovery of more actinomycetes populations than using the broad spectrum antibiotic, naldixic acid.

Table 2: Comparison of actinomycetes recovery from same samples using rifampicin and naldixic acid as inhibitors of bacterial contaminants. (Only 21 samples were compared)

During screening of 37 different marine samples, 420 suspected actinomycetes colonies were recovered. Among these, on the basis of their colony morphology, colour of their aerial mycelium 150 colonies suspected to be different were selected and looping out. Although rifampicin can suppress the growth of some of the strains of actinomycetes, 5μg/ml concentration added to the sample during primary screening found to be effective to protect rapidly growing members of non-filamentous bacteria.

After detail study of their morphology and characteristics of aerial and substrate mycelium, a total of 132 isolates were confirmed to be actinomycetes (Table-3). Among these, 76 (57.6%)  exhibit antimicrobial activity to one and/ or more test organisms of bacterial, and filamentous fungi or  yeast strains while the other 56 (42.4%) isolates don’t show and/or poor  activities. This result was lower than reported by Pisano et.al.¹⁸, 73% of chitinolytic actinomyctes were showing bioactivity. The reason may be due to correlation between chitinolysis and bioactivity of actinomycetes ¹⁸.

Most of the inhibitory activity was directed against bacteria than filamentous fungi and yeast. As shown in table-3, 64 (48.9%) of the total number isolated, proved to be active to one or more bacteria, while 50 (37.9%) were active to filamentous fungi and yeast.

Table 3: Distribution of actinomycets exhibiting antimicrobial activities.

Among bacterial group B. subtilis (39.4%) was the most susceptible organism followed by S. aureus (37.1%), B. pimilus (28.8%), E. coli (25%); P. vulgaris (11.4%) was the least susceptible. Among fungal group Penicillium cryseogenum (34.1%) was the most susceptible followed by Aspergillus niger (18.2%), Saccharomyces cervisae (17.4%), Candidia albicans (14.4%); Aspergillus oryzae (10.6%) was the least susceptible (Figure-1). Total number of actinomycetes exhibiting broad spectrum activity (bacteria and fungi) were 35(26.5%).

Figure 1:  Total number of actinomycetes exhibiting broad spectrum activity (bacteria and fungi) were 35(26.5%).    Click here to View figure

SA= S. aureus (NCIM 2079); BS=B. subtilis  (NCIM 2063); BP=B. pimilus (NCIM2327);  EC= E. coli (NCIM 2065); PV= P. vulgaris; AN= A. niger (NCIM-548); AO=A.  oryzae (NCIM 643);  PC= P. cryseogenum(NCIM-738); CA= C. albicans; SC= S. cervisae 

Fifteen promising isolate were selected and their detailed biochemical and physiological study showed that 10 of them were under the Sterptomyces species and 5 of them were under the group Micromonospora species. This result was in agreement with reports of Das et.al., ¹⁹ and Peela  et.al., ²⁰  as the major genera isolated in the bay of Bengal were the Streptomyces and Micromonospora species. However, this result is different from reports by Bredholt et.al.²¹ that Micromonospora species are dominant. The reason may be because of difference in depth of the sample collected. The deep water sediments contained a higher relative amount of Micromonospora compared to the shallow water sediments²².  Although, the other isolate showed strong antagonistic activities against different test organisms two of the Streptomyces species were also strongly active against methicillin resistant S. aureus (MRSA (Table-4). The antimicrobial spectrums of these species were also evaluated by submerged fermentation and their activities are still very promising against the test organism of bacterial and fungal strains. The diversity of these promising isolates and their content of biological activities are understudy.

Table 4: Activity of promising isolates on different test organisms using an agar overlay method.

 AN= A. niger (NCIM-548); AO=A.  oryzae (NCIM 643);  PC= P. cryseogenum; SC= S. cervisae;  CA= C. albicans; SA= S. aureus (NCIM 2079); BP=B. pimilus (NCIM2327); BS=B. subtilis  (NCIM 2063);  EC= E. coli (NCIM 2065); PV= P. vulgaris; SF= S. flexinari (MTCC1457); ST=S. typhimerium (MTCC 98); MRSA= Methicillin Resistant Staphylococcus aureus.

Conclusion

As it  is well documented drug resistance pathogens such as methicillin resistant S. aureus become  the most problematic gram positive bacterium in public health not only because it is highly prevalent but also it has reported that, the organism shows reduced susceptibility to the last resort antibiotics of vancomycin and teicoplanin. Therefore, despite the challenges of identifying new natural product antibiotics; searching and screening of antibiotic producing actinomycetes from new unexplored area using different pre-treatment of marine samples such as rifampicin as selective inhibitors for non filamentous bacterial contaminants during primary screening may help to identify new novel bioactive molecules.

Acknowledgment

The authors are thankful to DBT, New Delhi for the financial support to carry out this work. We are also grateful to Dr. B. Narasinga Rao (King George Hospital) for providing methicillin-resistant S. aureus.

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