INTRODUCTION:

Marine environment is largely an untapped source for obtaining actinobacteria, having potential to produce novel, bioactive natural products. Actinobacteria are the prolific producers of pharmaceutically active secondary metabolites, accounting for about 70% of the naturally derived compounds that are currently in clinical use¹. Among the various actinobacterial genera, Actinomadura, Actinoplanes, Amycolatopsis, Marinispora, Micromonospora, Nocardiopsis, Saccharopolyspora, Salinispora, Streptomyces and Verrucosispora are the major potential producers of commercially important bioactive natural products.

In this respect, Streptomyces ranks first with a large number of bioactive natural products. Marine actinobacteria are unique with diverse biological properties including antimicrobial, anticancer, antiviral, insecticidal and enzyme inhibitory activities. They have attracted global attention in the last ten years for their ability to produce pharmaceutically active compounds². The isolation rates of antibiotic producing actinobacteria from marine soil sediments were approximately 27%. Many studies on the antimicrobial activity of the actinobacteria isolated from marine environment have been done, but the results are different. It was reported that the components of the medium, samples differ in types and the exact reason could influence the antimicrobial activities of actinobacteria³.

Since the discovery of antibiotics, bacterial resistance to antibiotic has continued to evolve. Thus, we are witnessing more and more multi-resistant bacteria that pose a serious public health problem⁴ and carbapenem resistance also has been major threat. It is reasonable however to consider that starting from the emergence of carbapenem resistance until its widespread dissemination, some human-related factors play a crucial role⁵. So, the hunt for new production of antibiotics is essential. Due to these reasons the present study aims on the production of antibiotics through the marine soil sediment of Parangipettai and Andaman coast line which has been evinced to have high activity by producing enzymes, antitumour agents and antibiotics⁶.

MATERIALS AND METHODS:

Sample collection and pretreatment

The soil samples were collected from Parangipettai Marshlands (11.5084° N, 79.7568° E) and Andaman Island coastal region (11.7401° N, 92.6586° E). About 15 g of the soil sample was weighed and dried at room temperature for 3 days. The air dried samples was kept in hot air oven at 55°C for 15 minutes⁷.

Isolation of actinobacteria:

Actinobacteria were isolated by adopting standard spread plate method using two different media such as Starch casein agar (SCA) Starch - 10g, Caesin - 0.3g, K₂NO₃- 2.0g, MgSO₄- 0.05g, FeSO₄- 0.01g, CaCO₃ -0.02g, NaCl - 2.0g, distilled water -1000ml, Agar – 22g and Kuster’s agar (KUA) Glycerol-10 g, Caesin - 0.3g, KNO₃ - 0.2g, K₂HPO₄ -2g, Soluble Starch- 0.5g, Asparagine-0.1g, FeSO₄.7H₂O - 0.01g, CaCO₃- 0.02g, MgSO₄ - 0.05g, distilled water - 1000ml, Agar – 22g. All the media used in this study were prepared using 50% sea water⁴. Colonies showing suspected actinobacterial morphology were recovered from the isolation plates using sterile L-shaped needle and inoculated on yeast extract malt extract agar (YEME) Yeast Extract-4g, Malt Extract - 10g, Glucose - 4g, NaCl - 10g, Agar - 22g. After incubation at 28⁰C for 7 days, morphologically different actinobacterial cultures were selected and preserved using YEME agar slants as well as in 30% glycerol⁸.

Production of bioactive compounds:

Bioactive compound from actinobacterial cultures was produced by agar surface fermentation⁹. All the actinobacterial cultures were inoculated into yeast extract malt extract (YEME) agar plates and incubated at 28⁰C for 10 days for the production of secondary metabolites. During incubation, the extracellular metabolites are secreted into the agar medium.

Testing for antimicrobial activity:

Antimicrobial activity of actinobacterial cultures was tested by adopting agar plug method⁹. Actinobacterial cultures grown on YEME agar plates were taken and the mycelial growth was removed from the agar surface using sterile spatula. Test human pathogens such as Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 13439), Klebsiella pneumonia (ATCC 13882), Pseudomonas species (ATCC 27853), Candida albicans (ATCC 90028) and fish pathogens Aeromonas hydrophilla, Vibrio species, Vibrio harveii were used. All bacterial pathogens with 0.5 McFarland standards were inoculated into nutrient agar plates and Candida albicans was inoculated on Sabouraud dextrose agar, using sterile cotton swab. Agar plug with 5 mm diameter were cut from the YEME agar grown with actinobacterial cultures were placed over agar seeded with test pathogens. All the plates were incubated at 37⁰C for 24 hours and observed for zone of inhibition¹⁰. The Actinobacterial strain that showed maximum zone of inhibition against test pathogens was selected as the potential strain for further studies.

Effect of fermentation method on bioactive compound production:

Effect of solid state and submerged fermentation on bioactive compound production by the strain PPA12 was investigated. Spores of the actinobacterial strain PPA12 was inoculated into five YEME agar plates and 100 ml of YEME broth. YEME agar plates were incubated at 28⁰C for 12 days. YEME broth containing flasks were incubated in rotary shaker with 95 rpm for 12 days. For every 24 hours, agar plug from YEME agar plates were taken and tested against Carbapenem resistant Klebsiella pneumonia¹¹. Each 2ml of YEME broth from was taken and centrifuged at 10, 000rpm for 10 minutes. The cell free supernatant also tested against CRKP by adopting well diffusion method¹².

Extraction of bioactive compounds:

The actinobacterial strain produced the bioactive compounds only in solid culture and not in submerged fermentation. Hence, further production of bioactive compound from strain PPA12 was carried out by agar surface fermentation. After incubation, mycelial growth was removed aseptically using sterile spatula. The agar medium was cut into pieces and extracted overnight at room temperature using different solvents (1:2 ratio) such as methanol, chloroform, ethyl acetate and n-hexane. The solvent extracts were concentrated using rotary evaporator and quantified. Antimicrobial activity of different solvent extracts was tested against K. pneumoniae by disc diffusion method¹³. Zone of inhibition was measured after 24 hours of incubation at 37°C and expressed in millimetre in diameter. Solvent extract which showed maximum activity against the test pathogen was used to extract large quantity of bioactive compounds from 1000 ml of YEME agar medium.

Minimum Inhibitory Concentration of crude extract against K. Pneumoniae:

The MIC of the crude extract against K. pneumoniae was determined using microbroth dilution assay as described in the CLSI protocols. The overnight K. pneumoniae culture was diluted to obtain the final concentration of 10⁵ CFU/ml. The compound was dissolved in PBS and was tested at concentrations from 8 to 250 in twofold step intervals. Each well contains 800 μl of nutrient broth, 100μl of culture and 100μl of compound with varying concentrations. Culture control and medium control were maintained. All the tubes were incubated at 37°C for 18-24 hours and then observed for growth inhibition. The lowest antibiotic concentration which showed low turbidity was taken as minimal inhibitory concentration (MIC).

Partial purification of Bioactive Compound:

Analytical Thin Layer Chromatography:

Methanol extract of PPA12 crude extract was spotted on the bottom of TLC sheet with glass capillary tube and marked as solvent front. Chromatography was done with different solvents like chloroform, methanol, ethyl acetate, acetone, dichloromethane and butanol in different ratio. The separated spots were visualized with naked eyes, iodine vapours, UV light, vanillin stain and potassium permanganate strain. Rf value of the separated spots were calculated by standard formula¹⁴.

Bioautogram:

Developed chromatogram sheets were subjected to bioautogram. 1% tetrazolium salt solution was prepared and stored in amber colored bottle. 2ml of the stock tetrazolium salt solution and 100µl of 0.5 Mac Farland’s standard K. pneumoniae were added to molten nutrient agar at hand bearable temperature with constant stirring and poured into petriplates. The developed chromatogram was placed on the agar surface in such a way that the silica faces the medium. The plates were incubated overnight and observed for results¹³.

Characterization and generic identification of potential Actinobacterial strain.

Micromorphology:

Micromorphological characteristics of potential strains were studied by adopting slide culture method. The recorded microscopic characteristics include the presence/absence of aerial mycelium, substrate mycelium, mycelial fragmentation and the details of spore chain morphology. Spore structure and spore surface details were recorded using light microscope¹⁵.

Cultural characteristics were studied by inoculating the growth of actinobacteria strain PPA12 into different ISP (International Streptomyces Project) media such as tryptone agar (ISP1), yeast extract-malt extract agar (ISP2), oatmeal agar (ISP3), inorganic salts-starch agar (ISP4), glycerol-asparagine agar (ISP5), peptone-yeast extract-iron agar (ISP6) and tyrosine agar (ISP7). All the media were prepared by following the guidelines described by Shirling and Gottileb (1966)¹⁶. Plating was done in triplicate and all the plates were incubated for 10-14 days at 28^°C. Cultural characteristics recorded include the nature of growth, consistency, aerial mass colour, presence of reverse side pigment and the details of soluble pigment production, if any.

Physiological Characteristics:

Basal agar medium recommended by Shirling and Gottileb (1966)¹⁶ was used for carbon utilization studies. After sterilization of basal agar medium by autoclaving at 15 lb for 15 minutes, about 1% of various ether sterilized sugars were added and poured into petri plates. About one ml of well grown culture of actinobacterial strain PPA12 was inoculated as a straight line in different sugar containing basal agar plates. Growth was recorded after 14 days of incubation at 28°C. The same procedure was used to study the utilization of nitrogen sources by the potential actinobacteria strains. Sugars used in this study include glucose, fructose, sucrose, mannitol, inositol, xylose, rhamnose, raffinose, and cellulose (Hi media). Nitrogen sources used include aspargine, glutamine and tyrosine. Effect of different pH (5, 7, 9 and 11), temperature (20°C, 30°C, 40°C and 50°C), sodium chloride (0%, 1, 2.5, 5.0, 7.5 and 10%) and sea water (0%-100%) on growth was determined by inoculating the actinobacterial strain PPA12 onto ISP2 agar medium with respective physiological condition⁷. All the plates were observed for growth after 10 days of incubation.

Molecular Characterization:

The molecular characterization of strain PPA12 was carried out at Eurofins Genomics India pvt. Ltd., Bangalore.

Extraction of chromosomal DNA:

The chromosomal DNA of actinobacterial strain PPA12 was extracted using solute ready® genomic DNA kit. The extracted DNA was suspended in TE buffer.

PCR amplification of 16S rRNA:

The PCR amplification of 16S ribosomal RNA gene of strain PPA12 was performed by using two primers 27F 5´-AGAGTTTGATCMTGGCTCAG-3´ (forward) and 1492R 5´-TACGGYTACCTTGTTACGACTT-3´ (reverse)¹⁷ using thermal cycler (Eppendorf Master cycler-nexus). The final volume of reaction mixture of 20 µl contained 50ng of template DNA (1.0µl), 10p mol (each 2µl) of primer (forward and reverse separately), 10µl PCR master mix (2µl Taq DNA polymerase, 10X Taq reaction buffer, 2mM MgCl_2,1µl of 10Mm dNTPs and PCR additives) and 5.0µl of double-distilled water. The amplified 16S rRNA gene was purified using 2% agarose gel prepared in TE buffer.

Sequencing of 16S rRNA:

Sanger dideoxy chain terminator sequencing was done in an automated DNA sequencer. In this fluorescence based method of DNA sequencing, four reaction mixers were prepared. Each containing only one ddNTP, was labeled with four different fluorescent dyes so that only one reaction mix was required. In addition, polymerase enzymes such as dATP, dGTP, dCTP, dTTP were also added. The sequence detection was made by subjecting labeled ddNTP to a single capillary tube. DNA fragments of different colours were separated by their respective sizes in a single electrophoretic gel. The sequences were read by determining the sequence of the colours emitted and the information was fed directly to a computer which determined the sequence.

16S rRNA sequence analysis:

The closely related 16S rRNA gene sequence of strain PPA12 homologs sequence were determined and compared for similarity level with the reference species of Streptomyces contained in genomic database banks, using the ‘NCBI Blast’ (www.ncbi.nlm.nih.gov) web site. After BLAST analysis, a dataset of potential orthologs was prepared by considering those database sequences which had > 99% sequence identity with the query sequence of strain PPA12.

Phylogenetic analysis:

Phylogenetic and molecular evolutionary analyses of potential strains PPA12 was conducted using software included in MEGA version 4¹⁸ package. The 16S rRNA sequence of the strains PPA12 was aligned (http://www.ebi.ac.uk/clustalw)¹⁹ against corresponding nucleotide sequences of representatives of the genus Streptomyces retrieved from GenBank²⁰. Evolutionary distance matrices were generated as described and a phylogenetic tree was inferred by the Neighbor joining method. Tree topologies were evaluated by bootstrap analysis²¹ based on 1000 resembling of the neighbor joining data set.

Nucleotide accession number:

The partial 16S rRNA nucleotide sequence of the potential actinobacteria strain PPA12 were deposited at GenBank database by using Bankit tool (http://www.ncbi.nlm.nih.gov/WebSub/?tool=genbank).

RESULTS:

Isolation and selection of actinobacterial cultures

Colonies with actinobacterial morphology was observed on both the isolation media SCA and KUA in which more number of actinobacterial colonies were observed on KUA than the SCA media. Thirty seven actinobacterial colonies were recovered from the two isolation media and subcultured on ISP2 agar plates. After incubation, about 28 morphologically different actinobacterial cultures were selected for the further study. They showed wide pattern of morphology on YEME agar as well as under microscopic observation.

Screening of selected actinobacterial cultures for antimicrobial activity

In the preliminary antimicrobial screening, 24 out of 28 actinobacterial strains showed activity against atleast one of the four bacterial pathogens tested (Table 1). In particular 13 cultures were found to be active against Carbapenam resistant strains. Based on the results of preliminary antimicrobial screening, actinobacterial strain PPA12, which showed maximum zone of inhibition against six bacteria out of eight bacterial pathogens tested, was selected as potential strain for further studies

Table 1: Antimicrobial activity of isolated actinobacteria by agar plug method

S. No	Strain No.	*S. aureus*	*E. coli*	*K. pneumoniae*	*A. hydrophilia*	*Vibrio sp*	*Pseudomonas sp*	*Vibrio harvei*	*C. albicans*
1	PPA2	-	-	19	18.5	18	-	15	-
2	PPA25	-	-	19	18	21	24	-	13
3	PPA22	-		25	-	-	-	25	25
4	PPA23	-	-	21	13	1	-	-	-
5	PPA20	11	-	18	14	19	25	24	26
6	PPA21	11	-	-	27	24	21	23	-
7	PPA11	11	-	18	23	23	21	22	-
8	PPA28	-	-	19	-	-	-	-	-
9	AIA6	12	-	-	-	-	-	-	-
10	PPA12	11	-	19	25	22	22	23	-
11	PPA19	11	-	-	23	22	17	25	-
12	PPA24	-	-	-	-	-	-	-	-
13	PPA17	19	-	-	21	22	21	22	-
14	PPA16	-	-	19	18	17	-	21	-
15	PPA13	19	-	-	19	22	18	25	-
16	PPA26	13	10	-	-	7	-	22	-
17	PPA7	-	-	12	18	21	34	22	-
18	PPA10	10	-	-	15	19	19	20	-
19	PPA14	-	-	19	16	23	-	24	-
20	PPA13	-	-	19	-	19	-	19	-
21	PPA45		-	-	-	-	-	-	-
22	PPA43		11	19	20	-	10	19	-
23	PPA15		11	-	-	13	-	-	-
24	PPA35		-	-	-	-	-	-	-
25	PPA26		-	-	-	-	-	-	-
26	PPA15		10	-	-	13	-	-	-
27	PPA28		-	-	-	-	-	-	-
28	PPA42		-	12	-	-	-	12	-

Effect of fermentation method on bioactive metabolite production:

Effect of fermentation method on bioactive compound production by the strain PPA12 is given in Table 2. Even after 10 days, the cell free supernatant was not showed activity whereas the agar plug from YEME agar showed activity from 4th day of fermentation. Hence further bioactive compound production was carried out by agar surface fermentation.

Table 2: Effect of fermentation media on bioactive metabolite production

Incubation period	Submerged fermentation In YEME broth		Solid state fermentation YEME agar
Incubation period	Growth	Zone of inhibition (mm) against *K. pneumoniae*	Growth	Zone of inhibition (mm) against *K. pneumoniae*
DAY1	+	-	+	-
DAY2	+	-	+	-
DAY3	++	-	++	-
DAY4	++	-	++	20
DAY5	++	-	++	21
DAY6	++	-	++	21
DAY7	++	-	++	22
DAY8	++	-	++	23
DAY9	+++	-	++	23
DAY10	+++	-	++	24

+++ - good growth; ++ - moderate growth; + - poor growth

Extraction of bioactive compound:

Among the four different solvents tested for extraction, methanol extract showed maximum antimicrobial activity against K. pneumoniae. Hence further large scale extraction was done using methanol.

Determination of MIC:

Minimal Inhibitory Concentration (MIC) of the aqueous methanol fraction against K. pneumonia was found to be 125µg/ml.

Partial purification of bioactive metabolite:

In TLC analysis, among the different solvent systems tested, the methanol extract of strain PPA12 showed three well separated spots viz., PPA12 - A (0.288), PPA12 - B (0.403) and PPA12 - C (0.55) when the Hexane : Ethyl acetate: Methanol used as solvent system at 6:3:1 ratio.

Bioautogram:

The developed chromatogram was run bioautogram with 1% tetrasolium salt. The results showed that the zone of inhibition with PPA12-A spot as well as PPA12-C.

Characterization and taxonomy of potential Actinobacterial strain:

Phenotypic characteristics:

Under bright field microscopic observation, the vegetative substrate mycelium was lengthy and the reproductive aerial mycelium was dark and appeared in spiral (S) arrangement. The aerial and substrate mycelia did not exhibit fragmentation. Smooth, oval shaped spores were borne in long straight chains to spirals with 30-40 spores in a filament.

Strain PPA12 showed good growth on ISP2, ISP4 and ISP7 medium while moderate growth was observed on ISP1, ISP3, ISP5 and ISP6 medium. It produced powdery colonies on all the ISP media. Growth pattern of actinobacterial strain PPA12 is as shown in table 3.

Physiological characteristics:

Strain PPA12 was able to grow well utilizing wide range of sugars except on basal medium supplemented with sucrose, raffinose, cellulose and tyrosine. Growth of strain PPA12 was observed at a wide range of temperature (20⁰C – 40⁰C), pH 7 and 9 and in the presence of 0 – 7.5% NaCl and 100% seawater (Table 4).

Molecular characterization:

The PCR amplification of 16s rRNA gene of actinobacterial strain PPA12 yielded around 1085 base pair sequence. The BLAST analysis showed 99% similarity to Streptomyces fumigatiscleroticus NRRL B-3856. Phylogenetic relationship of the strain PPA12 and related taxa are given in Figure 1. Based on the criteria given by Bosshard et al., (2003), the strain PPA12 was identified to be species of the genus Streptomyces. The 16s rRNA gene sequence of strain PPA12 is submitted to GenBank with the accession number MK573175.

Table 4: Physiological characteristics of actinobacterial strain PPA12

Factors	Variables	Growth
Sugars	Glucose	Moderate
	Arabinose	Good
	Sucrose	Poor
	Xylose	Good
	Inositol	Good
	Mannitol	Good
	Fructose	Good
	Rhamnose	Good
	Raffinose	Poor
	Cellulose	No Growth
Amino acid	Asparagine	Good
	Glutamine	Good
	Tyrosine	Poor
Ph	5	No Growth
	7	Good
	9	Good
	11	Moderate
Temperature (⁰C)	20	Moderate
	30	Good
	40	Moderate
	50	No Growth
NaCl %	0	Good
	1	Good
	2.5	Good
	5	Moderate
	7.5	Poor
	10	No Growth
Seawater (%)	0	Moderate
	10	Good
	25	Good
	50	Good
	100	Moderate

DISCUSSION:

As the burden of MDR K. pneumoniae is growing rapidly, discovery of new drugs is an indispensable process to combat those emerging resistant pathogens²². Actinobacteria are still the promising candidates for the discovery of novel antibiotics which are being isolated from wide range of natural habitats²³. However, research on actinobacteria from parangipetttai mangrove ecosystems is less investigated²⁴. The present study was under taken for bioprospecting of actinobacteria from parangipetttai and Andaman Island for bioactive metabolite production. In the present study, Parangipettai mangrove and Andaman island coastal region sediment were used for isolation of Actinobacteria after dry heat treatment at 55°C for 10 minutes. The use of two different media results in the isolation of more powdery or leathery colonies with substrate and aerial mycelium with the maximum no of actinobacterial colonies in Kusters agar than SCA. Mohana and Radhakrishnan (2014)²⁵ also recommended Kusters agar as a suitable medium for the recovery of actinobacteria from mangrove ecosystem. In the present study majority of the recovered actinobacterial cultures are Streptomyces. Most of the isolated actinobacterial cultures showed powdery growth with aerial and substrate mycelium. These cultures may come under the genus Streptomyces¹⁶. Streptomyces species are reported to produce wide range of pigments with different biological activities²⁶. Further the heat treatment method and antibacterial and antifungal antibiotic supplemented in the isolation medium resulted in the reduction of unwanted bacterial and fungal growth, respectively.

According to Berdy (2012)²² great numbers of antibiotic compounds exhibit exclusive activities against gram positive bacteria while only 1.5% are active against gram negative bacteria. Eco-physiological conditions of particular ecosystem greatly influence the biological and metabolic activity and diversity of actinobacteria²⁷. Salamoni et al., (2012)²⁸ reported that 25 Streptomyces species isolated from compost showed 20 different patterns of activity against 53 bacterial and fungal pathogens. Lalitha et al., (2017)²⁹ isolated 51 morphologically different Actinobacteria isolated from rhizosphere soil of five different medicinal plants. However, only one isolate was shown to have a potential activity against MDR K. pneumoniae. But in the present study nearly 50% of the isolated actinobacteria showed activity against CRKP.

Strain PPA12 showed good growth on ISP2 broth during submerged fermentation by shake flask method. But, in well diffusion test, the cell free supernatant showed no activity against the test organisms. However, in agar surface fermentation, strain PPA12 showed good growth and the methanol extract obtained from the fermented medium showed activity against carbapenam resistant K. pneumoniae. Production of a majority of industrially important secondary metabolites from actinobacteria is carried out using submerged fermentation processes where they exhibit diverse morphological forms³⁰. Morphology is influenced by environmental conditions such as medium composition and shear stress³¹. Further optimization of medium component and concentration may be needed to produce the compound from strain PPA12 by submerged fermentation.

Majority of the actinobacterial metabolites are extracellular in nature and they are extracted using the medium polar solvent ethyl acetate^7,13. In the present the only the extracellular metabolites extracted in methanol showed antimicrobial activity. This result indicates the highly polar nature of the active compound produced by the actinobacterial strain PPA12. The methanol fraction of strain PPA12 showed promising activity against K. pneumoniae. This observation paves the way for the exploration of the actinobacteiral strain PPA12 for clinical applications. The analysis of 16s rRNA gene sequence has also revealed that the strain PPA12 belongs to a species of the genus Streptomyces. The BLAST analysis showed 99% similarity to Streptomyces fumigatiscleroticus NRRL B-3865 deposited in Gen Bank with the accession number MK573175.

Findings of the present study showed that mangrove ecosystem of Parangipettai is a promising source for bioactive actinobacteria and the Streptomyces fumigatiscleroticus PPA12 from this ecosystem is a promising source for the isolation of bioactive compounds for clinical important carbapenam resistant K. pneumoniae.

ACKNOWLEDGEMENT:

Authors thank the authority of Sathyabama Institute of Science and Technology (Deemed to be University). Chennai, Tamil Nadu for the research facilities provided.

REFERENCES:

1. Ibrahim AH, Samar YD, Mostafa AF, Mohamed SK, Tobias AMGand Abdelmohsen UR.Natural Product Potential of the Genus Nocardiopsis. Marine Drugs. 2018; 16(5): 147.

2. Manivasagan P, Kyong-Hwa K, Sivakumar KECY, Li-Chan, Hyun-Myung Oh and Se-Kwon Kim. Marine actinobacteria: An important source of bioactive natural products. Environ. Toxicol. Pharmacol. 2014; 38(1):172-188.

3. Yu J, Liu Z , Qiu L, Xiaohui Q, Ying J and Beom SK. Isolation and characterization of actinobacteria from Yalujiang coastal wetland, North China. Asian Pacific J Tropical Biomed. 2015; 5(7):555-560.

4. Elbendary AA, Hessain AM, El-Hariri MD, Seida AA, Moussa IM, Mubarak AS, Kabli SA, Hemeg HA and El Jakee JK. Isolation of antimicrobial producing Actinobacteria from soil samples. Saudi J. Biol. Sci. 2018; 25(1):44-46.

5. Meletis G. Carbapenem resistance: overview of the problem and future perspectives. Therapeutic adv. Infectious Dis. 2016; 3(1):15-21.

6. Sathiyaseelan K and Stella D. Isolation, identification and antimicrobial activity of marine actinomycetes isolated from parangipettai. Rec. Res. Sci. Tech. 2011; 3(9): 74-77.

7. Radhakrishnan M, Balaji S and Balagurunathan R. Thermotolerant actinomycetes from the Himalayan Mountain-Antagonistic potential, characterization and identification of selected strains. Malaysian Appl. Bio. J. 2007; 36(1): 59-65.

8. Mohamed H, Miloud B, Zohra F, Garcia-Arenzana JM, Veloso A and Rodriguez-Couto S. Isolation and Characterization of actinobacteria from Algerian Sahara soils with antimicrobial activities. Int. J. Mol. Cellular Med. 2017; 6(2):109-120.

9. Radhakrishnan M, Pazhanimurugan R, Gopikrishnan V, Balagurunathan R and Vanaja Kumar. Streptomyces sp D 25 isolated from Thar Desert soil, Rajasthan producing pigmented antituberculosis compound only in solid culture. J. Pure Appl. Microbiol. 2014; 8(1): 333-337.

10. Sathish KS and Kokati VBR. In-vitro antimicrobial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus. Asian Pacific J. Tropical Biomed. 2012; 2(10):787-792.

11. Eccleston GP, Brooks PR and Kurtboke DI. The occurrence of bioactive Micromonosporae in aquatic habitats of the sunshine coast in Australia. Marine Drug. 2008; 6243-6261.

12. Bavya M, Mohanapriya P, Pazhani murugan R and Balagurunathan R. Potential bioactive compounds from marine actinomycetes against biofouling bacteria. Indian J. Geomarine Sci. 2011; 40(4): 578-582.

13. Selvameenal L, Radakrishnan M and Balagurunathan R. Antibiotic pigment from desert soil actinomycetes; biological activity, purification and chemical screening. Indian J. Pharma Sci. 2009; 71: 499 - 504.

14. Augustine SK, Bhavsar SP and Kapadnis BP. A non-polyene antifungal antibiotic from Streptomyces albidoflavus PU23. J. Biosci. 2005; 30: 201 - 211.

15. Balagurunathan R and Radhakrishnan M. Biotechnological, genetic engineering and nanotechnological potential of actinomycetes. 2010; p. 436 In D.K. Maheshwari, R.C. Dubey, and R. Saravanamuthu (ed.), Industrial Exploitation of Microorganisms. I.K. International Publishing House Pvt. Ltd, New Delhi.

16. Shirling EB and Gottileb D. Methods for characterization of Streptomyces species. Int. J. Systematic Bacteriol. 1966; 6(3): 313 -340.

17. Gothwal RK, Nigam VK, Mohan MK, Sasmal D and Ghosh P. Screening of nitrogen fixers from rhizospheric bacterial isolates associated with important desert plants. Appl. Ecol. Environ Res. 2008; 6(2): 101-109.

18. Takahashi YO and Omura S. Isolation of new actinomycete strains for the screening of new bioactive compounds. J Gen Applied Microbiol. 2003; 49: 141-154.

19. Thompson JD, Higgins DG and Gibson TJ. Clustal W – improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22: 4673-4680.

20. Boudjella H, Bouti K, Zitouni A, Mathieu F, Lebrihi A and Sabaou N. Taxonomy and chemical characterization of antibiotics of Streptosporangium Sg 10 isolated from a Saharan soil. Microbiol. Res. 2006; 161: 288-298.

21. Felsenstein J. Phylogenies and the Comparative Method. The American Naturalist. 1985; 125(1): 1-15.

22. Berdy J. Thoughts and facts about antibiotics: where we are now and where we are heading. J. Antibiot. (Tokyo). 2012; 65(8): 385-395.

23. Tiwari, K. and Gupta, R.K. 2012. Rare actinomycetes: a potential storehouse for novel antibiotics. Crit. Rev. Biotechnol. 32(2):108-32.

24. Radhakrishnan M, Vijayalakshmi G, Gopikrishnan V and Jerrine J. Bioactive potential of actinobacteria isolated from certain understudied regions in India. J. Applied Pharma. Sci. 2016; 6(8): 151-155.

25. Mohana S and Radhakrishnan M. Streptomyces sp MA7 isolated from mangrove rhizosphere sediment effective against Gram negative bacterial pathogens. International Journal of PharmTech Research. 2014; 6(4): 1259-1264.

26. Diraviyam T, Radhakrishnan M and Balagurunathan R. Antioxidant activity of melanin pigment from Streptomyces species D5 isolated from Desert soil, Rajasthan, India Drug Invention Today. 2010; 3(3): 12-13.

27. Knight V, Sanglier JJ, DiTullio D, Braccili S, Bonner P, Waters J, Hughes D and Zhang L. Diversifying microbial natural products for drug discovery. Appl. Microbiol. Biotechnol. 2003; 62: 446-458.

28. Salamoni SP, Mann MB, Campos FS, Franco AC, Germani JC and Van der Sand ST. Preliminary characterization of some Streptomyces species isolated from a composting process and their antimicrobial potential. World J. Microbiol. Biotechnol. 2010; 26(10): 1847-1856.

29. Lalitha C, Thiagarajan R, Sudarshan S, Rathore and Jayapradha R. 2010. Bioactive molecule from Streptomyces sp. mitigates MDR Klebsiella pneumoniae in Zebrafish infection model. Front. Microbiol. 2010; 8: 614.

30. Treskatis SK, Orgeldinger V, Wolf H and Gilles ED. Morphological characterization of filamentous microorganisms in submerged cultures by on-line digital image analysis and pattern recognition. Biotechnology and Bioengineering. 1997; 53(2):191-201.

31. Manteca A, Alvarez R, Salazar N, Yague P and Sanchez J. Mycelium differentiation and antibiotic production in submerged cultures of Streptomyces coelicolor. Applied and Environmental Microbiology. 2008; 74(12): 3877-3886.

Received on 10.09.2019 Modified on 15.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3653-3660.

DOI: 10.5958/0974-360X.2020.00646.0

Medium	Growth	Colony consistency	Aerial mass colour	Reverse side pigment	Soluble pigment
Tryptone yeast extract agar (ISP1 medium)	Moderate	Powdery	Gray	-	-
Yeast extract malt extract agar (ISP 2 medium)	Good	Powdery	Gray	Golden Yellow	-
Oat Meal agar (ISP 3 medium)	Moderate	Powdery	Gray	Golden Yellow	-
Inorganic salts starch agar (ISP4 medium)	Good	Powdery	Ash	-	-
Glycerol asparagine agar (ISP 5 medium)	Moderate	Powdery	White	-	-
Peptone yeast extract iron agar (ISP6 medium)	Moderate	Powdery	White	-	-
Tyrosine agar (ISP 7 medium)	Good	Powdery	Gray	-	-