Exploration of Culturable Marine Streptomyces from Southern Coastal Region of India with Antibacterial Properties

The Southern coastal region is a geographically positioned area with rich source of microbial diversity, of which producing broad spectrum of bioactive compounds. The marine sediments are collected from the various sites of south east coastal region of Tamil nadu and are processed. Out of 78 isolates, the 4 strains of actinomycetes are showing broad spectrum activity against Klebsiella pneumoniae (MTCC 1687), Proteus vulgaris (MTCC 3160), Salmonella typhi (MTCC 3231), Shigella dysenteriae (MTCC 3642) and Vibrio cholerae (MTCC 3906). The bioactive isolates are proceeded for further morphological features like growth pattern and mycelial coloration, biochemical and polyphasic taxonomical characterisation were documented. Isolates are investigated for abiotic stress condition to study the growth rate of isolates. Finally, the 16S rRNA molecular identification and phylogenetic analysis of the isolates were explored. Further, bioactive isolates may be potential source for discovery of molecules with industrial applications.


INTRODUCTION
The advent of new antibiotics are on rise due to the rapid spread of antibiotic resistant pathogenic organism causing life threatening infections and side effects as most of the current drugs are chemically synthesized [1]. Thus, the drug discovery from natural sources are immense important to overcome the aforementioned problems [2].
Marine environment is the largest resource for taxonomically diverse bacterial groups with potential and novel secondary metabolites, which are unique in nature as they are extremities (Cross, 1981); [3]. The unique properties and compounds that are observed in marine environment microbes are not exhibited in the terrestrial sources [4,5]. So, the research has been focused on the screening of marine sediment derived microorganisms [6], Numerous of antibiotics are isolated from marine source, of which one third of drug are isolated from Actinomycetes [7].
The organism has shown to produce secondary metabolites with novel properties, which can be potentially used as such as antimicrobial, anticancer, and anti inflammatory agents.
Though isolation of marine actinomycetes and cultivating in the laboratory is a complex process, optimizing the culture conditions will provide us efficient source and ample scope for the investigation of the biologically active metabolites. Streptomyces are the most valuable soil inhabiting Gram positive bacteria, with vast source of novel biologically active compounds with unique structure and properties [8,9,10,11]. The new techniques are arrived for isolation, cultivation and screening to increase the diversity of cultivable isolates. Various pre-treatment was employed to enrich the isolation of actinomycetes from other genera. Air dry treatment of soil is a universal method to increase the number of mycelium forming actinomycetes sampling sites, sampling strategy. Geographic and habitat plays are essential role in the isolation of Actinomycetes (Zhang et al, 2006).

Sampling and Processing
Marine sediment was collected from four different sites of Dhanuskodi, Rameswaram, Pamban, and Mandapam of Tamil nadu, India. The samples were collected at maximum depth of 40m. Each 500 g of soil samples were collected in a clean sterile zip lock polythene bag, stored at 4 0 C, and transferred to the laboratory under sterile condition for further analysis. Different pretreatment methods such as mechanical, physical and chemical are generally used for the selective isolation of Actinomycetes. In this study, samples were air dried for 15-20 days to inhibit the growth of other genera and stimulate the growth of actinomycetes. Then the sediments were passed through a 2 mm sieve to remove gravel and debris.

Temperature, pH and Electrical conductivity
The air dried soil sample 10 g was dissolved in 100 mL of distilled water after noting the temperature.
A suspension of 1:10 weight/volume dilution was made and the pH and electrical conductivity were noted. Quantification of Carbon, Nitrogen source [12], Phosphorous, Potassium, Calcium and Sodium (Murugesan and Rajakumari, 2005) were also determined.

Isolation of actinomycetes
One gram of air-dried sample was added to 9 mL of distilled water in a sterile test tube and shaken vigorously, and the sample was further diluted to 10 6 using sterile distilled water. A volume of 100 µL of diluted sample was inoculated by spreading with sterile glass rod in Starch Casein Nitrate Agar medium (Starch -10 g/L, Casien -0.3 g/L, K 2 HPO 4 -2 g/L, KNO 3 -2 g/L, Nacl -2 g/L, MgSo 4 .7H 2 O -0.05 g/L, CaCO 3 -0.02 g/L, FeSo 4 .7H 2 O -0.01 g/L, Agar -15 g/L). Further, the medium was supplemented with nystatin 30 µg/mL and nalidixic acid at 10 µg/mL to inhibit the fungal and bacterial contamination. The inoculated plates were incubated at 30°C for 10 days (Mincer et al, 2002). Individual colonies were streaked on International Streptomyces Project (ISP) agar medium and further incubated for 7 days.

Agar well diffusion method
Antibacterial activity of 78 isolates were screened by Agar well diffusion method, The well (

Effect of temperature and pH
To optimize the best growth conditions of isolates, the culture were grown in ISP2 liquid broth at various pH of acidity and alkalinity, temperature at 28°C for 7 days and agitated at 200 rpm, as described by Xu et al. [14] and Xu et al. [15].

Chemotaxonomy
Cell mass of isolates were obtained by inoculating in ISP2 liquid broth medium at 28°C for 4 days with agitation of 140 rpm. The biomass was centrifuged at 4500 rpm for 10 min and the pellet was washed with methanol and water, and freeze dried at 20°C. Further, the cell mass was hydrolyzed with 5ml of 6M HCl and incubated at 100°C for 18 hours. The filter was evaporated to remove HCl and paper chromatography was preceded with solvent system of methanol, water, 10M HCl and pyridine in the ratio of 80:26:2:5:20. Ninhydrin was sprayed to develop the chromatogram [16].

Extraction of genomic DNA
The culture was grown in yeast extract/malt extract broth at 28 ˚C for 7 days in shaker at 200 rpm. The mycelia was harvested by centrifuging for 10 min at 10,000 rpm in 4˚C. The SET buffer 25 mM Tris HCl, 25 mM EDTA, pH 8, 0.3M sucrose and 3 µL of lysozyme 20 mg/mL was added and vortexed vigorously, followed by incubation at 37˚C for 45 min at 600 µL of 10%, 3%, 5%, 7%, 9% and 11% incubated at 28°C for 5 days. SDS was added and incubated at 56˚C for 15 min. Later, 3 µL of RNase was added and incubated at 37˚C for 15 min. 2ml of phenol: chloroform: isopropanol at the ratio of 25:24:01, and centrifuged at 10,000 rpm in 4˚C for 10 min. The aqueous layer was transferred carefully to the sterile tube and precipitate with ice cold absolute ethanol. Then the mixture was kept in 80˚C for 30 min. The precipitate was transferred to the sterile tube and centrifuge for 10,000 rpm for 10min. The pellet obtained was washed with 70% ethanol and dried in room temperature. Finally, the pellet was dissolved in 200 µL of TE buffer at 10 mM Tris, 1M EDTA and pH 8. On the sequence, it was transferred and the DNA was checked in 1% Agarose.

PCR amplification of 16S rRNA
The extracted DNA molecule was amplified by using two universal primers 27F 5´AGATTTGATCMTGGCTCAG 3´, 1492R 5´GGTTACCTTGTTACGACTT 3´. PCR reaction was performed with the 50 µl reaction mixture containing 0.5 µM of each primer, 1 µl extracted DNA 50 ng, 23 µl sterile distilled H 2 O and 10% (v/v) of DMSO to the final volume of PCR premix Emerald, Takara, Japan. Amplification were performed with the thermo cycler, the polymerase chain reaction for 5 min at 95°C and followed by 35 cycles of 1 min at 94°C, 1 min at 58°C and 3 min at 72°C, followed by 10 min as final extension at 72°C. The amplified products were analyzed by electrophoresis in 0.8% (w/v) agarose gels and purified by DNA extraction kit Quiagen, India. Sequencing was performed on ABI 310 automatic DNA sequencer using the 27f and 1492r promoter primer.

Phylogenetic analysis
The sequences were aligned using BLAST tool. Multiple alignments was preformed using the CLUSTAL_X and the phylogenetic tree was constructed using MEGA 6.0 software.

RESULTS AND DISCUSSION
Soil is one of the valuable property of nature, with essential nutrients andorganic matter that play important role in the distribution of microorganisms [17,18]. Significant amount of potassium was observed, invariably in all the four samples, followed by the nitrogen, calcium and phosphorus. Organic carbon was noted only in P1 and M1 site (Fig. 1).
A sum of 78 actinomycetes were isolated from 4 different soil samples, showing that marine resources are rich inbiologically active actinomycetes, with industrial importantbioactive compound [19] and [14]. Upon the initial incubation, plates showed white chalk powder like dots and turned to thick leathery form after seven days. The culture developed aerial and substrate mycelium and diffused its color around the colony over further period of incubation. Also, the colony exhibited the musty or earthy odour. Actinomycetes isolated from soil samples are reported to exhibit antagonistic activity against pathogenic organisms [20].
The bioassay revealed antibacterial metabolite producing isolate, which enabled to narrow down the best antibacterial metabolite producer. Four isolates from each sampling site was taken for further characterization studies. The isolate D4 from Dhanuskodi sampling site at Arichimunai area spot shows the broad spectrum against pathogens, and P4 isolated from Pamban sampling site exhibits better activity. Isolate M2 from mandapam sampling site has the antibacterial metabolite, and R6 from Rameswaram shows antibacterial activity (Table  1). Marine ecosystem has been reported to be the best source for biologically active metabolite producing Actinomycetes [21], Fiedler et al., 2005. Since the biosynthetic gene cluster seem to play a major role, molecular approach of natural product drug discovery could be explored as a potential tool.  Morphological analysis plays an important role in the characterization of Streptomyces species. Three salient features were observed in the microscopic analysis: vegetative mycelium, aerial mycelium bearing spore chain and mycelium with spore formation only [22,23]. The colour of aerial mycelia, substrate mycelia, spores and the pigment diffuse from spores and around the isolates in the culture was notable and were much useful for the differentiation of species. These growth pattern were recorded by Yeast extract-Malt extract agar (ISP 2), Oat meal agar (ISP3), Inorganic salts-starch agar (ISP 4), Glycerol asparagines agar (ISP 5), Peptone yeast extract iron agar (ISP 6) and Tyrosine agar (ISP 7).
The result of cultural characteristics of the Streptomyces R6, Streptomyces D4, Streptomyces P4, Streptomyces M2, are shown in Figs. 2 and 5, respectively. Different range of colours were observed in the present study, where the isolates shows whitish grey, white, yellowish grey and grey, representing the production of various pigments. The yellow culture of Streptomyces has the ability to produce antitubercular pluramycins [24]. The growth of Streptomyces R6 were observed to be goods in ISP 2, 3, 4, 5, 7 and moderate in ISP 6. The isolate diffused brown pigment in ISP2, ISP3, ISP7 and yellow in ISP4, and showed the moderate sporulation in ISP 4 & 7, and also poor sporulation in rest of the media. The  The morphological analysis of spore is shown in Fig. 6. Streptomyces R6 was found to bear soft spore chains of hexagonal shape. Streptomyces D4 with rectiflexible spore chains, with hairy sheath and Streptomyces P4 with spiny shealths in spore chains. Long, soft spore chains showing ellipsoid structure was observed in Streptomyces M2. Reports on spore surface morphology shows distinct pattern for every species [25,26]. Thus, the present study reports different spore morphology for different isolate.
Biochemical analysis of all the isolates are shown in the Table 2. All the isolates were found to be aerobic, Gram positive and showed positive results for urea hydrolysis and nitrate reductase, whereas, negative result was observed in gelatin.
Citrate production was observed in all isolate except Streptomyces P4. Positive result for starch hydrolysis was observed in Streptomyces D4, Streptomyces P4 and Streptomyces M2. Streptomyces R6 showed positive result for the Catalase production. Nitrate reductase, H 2 S production, gelatin and starch hydrolysis are considered for characterization of Streptomyces sp [27] and [28]. Thus, the study reports different biochemical characteristic for different isolate.
Physiological parameters greatly influence the growth rate of Streptomyces [29]. The morphology of the isolates was dependent on the temperature and incubation time [30]. The pH plays vital role in production of bioactive metabolite in the culture. The optimal temperature and pH for the growth of isolate is 30°C and pH 7 (Fig. 7), whereas culture has grown beyond 40°C. The isolates Streptomyces R6 and Streptomyces D4 utilize the glucose as efficient carbon source to attain maximum growth, whereas Streptomyces P4 and Streptomyces M2 utilize the sucrose and maltose as carbon source to attain maximum growth. Glutamine is the best nitrogen source for Streptomyces R6 and Streptomyces M2. The isolates Streptomyces D4 and Streptomyces P4 utilize Asparagine as nitrogen source (Fig. 8).   NaCl tolerance have been reported in streptomyces taxonomy based on the color and nature of the spore. The yellow or white spore forming colony shows high tolerance to NaCl, whereas the red spotted spore colonies shows lesser tolerance. The spiny spore has much tolerance than the smooth spore [31]. Streptomyces P4 and Streptomyces M2 exhibit tolerance up to 7% of NaCl concentration, whereas the Streptomyces D4 shows high tolerance up to 11%. Streptomyces R6 exhibits low tolerance up to 5% (Fig. 9). Thus, the spiny spores studied in the present investigation exhibited strong tolerance when compared to smooth spores.
The result of cell wall analysis using LL-Diaminopimelic acid (LL-DAP) was demonstrated (Fig. 10), showing it contains LL-Diaminopimelic acid and glycine, thereby indicating that strain exhibits cell wall chemotype I. The actinomycetes have a cell wall composition alike to that of Gram positive bacteria and actinomycetes [32]. DAP is widely distributed as a key amino acid and it has optical isomer. The presence of DAP suggest that, bacteria contains one of the isomers, LL form or mess form, which locates at peptidoglycan layer (Sivakumar et al., 2007). In the present investigation the isolate was found to contain LL-DAP, along with glycine, with the presence of sugar pattern, which indicates characteristic of Streptomyces genus.
The molecular characterization of streptomyces sp was analysed by PCR amplification of 16S rRNA (Fig. 11) and sequenced using 27f and 1492r primers. The resulting sequence was compared with NCBI using BLAST and the obtained sequence was deposited in the GeneBank in the ID of MG198770, KM925137, MG198770, MG198767. The 16s rRNA sequencing is a molecular revolution in bacterial systematic. The phylogenetic analysis was based on 16S rRNA sequence analysis. Streptomyces R6 (Fig. 11) shows that the strain was closely related to the Streptomyces rochi. The distinct branch observed from rooted tree using neighborhood joining method. Streptomyces D4 strain (Fig. 12)

CONCLUSIONS
As per literature studies the marine actinomycete are one of the most bioactive compound producers, so the collected marine soil sediments undergone soil analysis and the micro and macro nutrient are ruled out, which helps to frame the blueprint for metabolites. The specific pretreatment are done to aviod specific growth of other genera and the actinomycetes are isolated.
Out of 78, the 4 isolates exhibits the strong antibacterial activity, so the 4 isolate is proceeded for the polyphasic characterisation, several physiological parameters like salinity, pH, carbon and nitrogen source. These parameters helps to set the essential requirement for the growth of actinomycetes. Then, the isolates are moved to next level study; molecular characterisation are phylogenetic analysis that reveals the similar strains which have highly broad specturm antimicrobial activity. The obtained literature data and experimental studies reveals that the isolates are highly potent, out of which Actinobacterium dagang 5 exhibits the broad spectrum activity among other isolates.

CONSENT
It is not applicable.

ETHICAL APPROVAL
This study does not involve experiments on animals or human subjects.