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Chapter: Basic Concept of Biotechnology - Antibiotics: Microbial Sources, Production and Optimization

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Metabolite production by marine Streptomyces

Actinomycetes comprise about 10% of the bacteria colonizing marine aggregates and can be isolated from various marine sources.

Metabolite production by marine Streptomyces

Actinomycetes comprise about 10% of the bacteria colonizing marine aggregates and can be isolated from various marine sources. Many actinomycete isolates from the depths of the oceans contain non-ribosomal polyketide synthase (NRPS) and polyketide synthase (PKS) pathways, the hallmarks of secondary metabolite production (Li and Piel, 2002; Salmon et al., 2003). Terrestrial soils have hitherto been the predominant and widely exploited source, and investigations on marine Streptomyces are few and inconclusive, though they are the important sources for new bioactive compounds (Okami, 1984). About 23,000 antibiotics have been discovered from microorganisms. It has been estimated that approximately 10,000 of them were isolated from actinomycetes (Okami and Hotta, 1988). Actinomycetes, mainly the genus Streptomyces, have the ability to produce a wide variety of secondary metabolites as bioactive compounds, including antibiotics. The name Streptomyces was introduced in 1943 for the aerial mycelia producing actinomycetes. The genus Streptomyces is represented in nature by the largest number of species among all the genera of actinomycetes and figures over 500 species. The group has an enormous biosynthetic potential that remains unchallenged among other microbial groups. The immense diversity, along with itsunder utilization is the fundamental reason for attracting researchers towards it for discovering novel metabolites. During the last decade, there has been increasing number of novel metabolites possessing potent bioactivity isolated from marine-derived Streptomyces (Lam, 2006; Wu et al., 2007). Many of them are cytotoxic and come from a wide variety of chemical structures such as macrolides, a-pyrones, lactones, indoles, terpenes and quinones.

 

Antagonistic marine actinomycetes in Indian peninsula

Of 9 maritime states of India, only 4 have been extensively covered for the study of marine actinomycetes. Forty years of floristicinventory of marine actinomycetes in Indian Peninsula yielded 41 species belonging to 8 genera, in which the genus Streptomyces was more frequently recorded(Sivakumaret al., 2007). Majority of the surveys have been conducted in the coastal areas, collecting the littoral sediments from the states of Maharastra, Kerala, Tamil Nadu and Andhra Pradesh. Studies covering the Gujarat, Goa, Karnataka, Orissa, West Bengal and Andaman and Nicobar islands are scanty.

 

Ranjithkumar et al., (2014a) isolated 115 actinomycetes from were isolated from the east and west coastal regions of India. Out of 115 actinomycetes strains, 52 isolates (45.2%) had antimicrobial activity, of which 26 isolates (22.6%) showed antibacterial activity (against S.aureus), 17 isolates (14.7%) showed antifungal activity (against T. rubrum), 9 isolates (7.8%) showed both antibacterial and antifungalactivity. Of all 115 actinomycetes strains, BW2-7 showed a broad-spectrum antibiotic activity (Fig. 1) which was isolated from Kannamaly beach had broad spectral antimicrobial activity and was selected for further studies. The physiological and biochemical characteristics and the 16S rRNA sequence analysis confirmed that the strain StreptomycesspBW2-7 was identical to S. fradiae.



Tamil Nadu

 

Laksmanaperumalsamy et al. (1978) isolated 518 Streptomyces strains from the sediments of estuarine, backwater, marine, freshwater and mangrove environment of Porto Novo using Grein and Meyer's agar,

 

Kuster’s agar and Glucose asparagines agar. Majority of the isolates

 

(46.43%) showed combined antibacterial and antifungal activity and 25% showed only antibacterial activity. Balagurunathan et al. (1989) studied the antagonistic behavior of actinomycetes isolated from the littoral sediments of Parangipettai. Among the 51 strains, only 11 strains showed good antibiotic activity and they were identified as Streptomyces spp. and Nocardiaspp. This antibiotic was tested against fish pathogens viz. species ofVibrio, Aeromonas, Pseudomonas, Bacillus and

 

Fusariumand it inhibited all these pathogenic organisms with inhibitionzones ranging from 10-30 mm. The Vibrio sp. and Pseudomonas sp. were more sensitive than the other bacterial species tested.

 

Sivakumar (2001) isolated actinomycetes from the Pitchavaram mangrove environment. The 16S rRNA genes of the isolated two strains were partially sequenced were deposited in the Gen Bank, National Centre for Biotechnological Information, USA under the sequence of the accession numbers AY015427 and AY015428. Patil et al. (2001) reported 133 strains of actinomycetes from 129 marine samples collected from various stations along the Tuticorin coast. Of the 104 strains of actinomycetes screened for the inhibitory activity against bacterial pathogens associated with fish diseases (Aeromonashydrophila,Aeromonassobriaand Edwardsiellatarda), 77 isolates possessedinhibitory activity to at least one of the pathogens. Balagurunathan and Subramanian (2001) isolated 51 strains of Streptomyces from the littoral sediments of Parangipettai coastal waters. Out of these, only eight strains showed very promising antibiotic activity against bacteria and fungi. These strains exhibited higher activity against gram-positive

bacteria than the gram-negative bacteria. Patil et al. (2001) isolated 20 actinomycetes strains from water and sediment samples of mangrove area of Tuticorin. The strains were checked for their antagonistic activity against seven shrimp bacterial pathogens. Among them, 83% showed good antagonistic activity against all the tested pathogens.

Sahuet al. (2004) isolated 40 strains of actinomycetes from the gut contents of three estuarine fishesviz. Chanoschanos,EtroplussuratensisandLatescalcarifer. Among them, only 10 strains ofactinomycetes (30%) showed moderate antagonistic activity against all the tested bacterial pathogens. Kathiresan et al. (2005) isolated 160 strains from the sediments of mangrove, estuary, sand dune and industrially polluted marine environment of Cuddalore. Of the 160 isolates, 10 showed potent activity against all the fungi tested. These isolates produced high antifungal compounds at 120 h of incubation period in the production medium culture. Dhanasekaran et al. (2005) reported 107 strains of actinomycetes from 16 different marine soil samples and studied their antifungal activity against five test fungi. Out of these, only 22 isolate (21.2%) which were grown in starch casein agar produced diffusible antifungal substances in varying quantities. Potency of the culture filtrate was estimated by agar cup assay method usingC.albicans. The antifungal activity was also tested by agar overlay methodusing C. albicansand S. cerevisiaeas test organisms. Six isolates showed strong antifungal action in both agar cup and agar overlay assays.

Sivakumar et al. (2005a) reported 91 strains of actinomycetes from different stations of the Pitchavaram mangrove ecosystem. Out of the 91 strains, only 6 strains showed good activity and they were identified upto species level. Sahu et al. (2005a) studied actinomycetes population density from different samples viz. water, sediments, seaweeds, molluscs and finfishes of the Vellar estuary. The sediment samples harboured higher population density compared to the watersamples. Biological samples viz. seaweeds, molluscs and fin fishes were also analyzed for actinomycetes population. Among them, molluscs recorded higher population density in shell surface region than the gut contents, while the fin fishes recorded higher population in gut contents followed by gills and skin. Seaweed samples also recorded considerable actinomycetes populations. Sahu et al. (2005b) studied the extra-cellular enzyme (amylase, lipase, protease, cellulase and chitinase) activities of actinomycetes isolated from the sediment and molluscan samples of the Vellar estuary. The study indicated that the actinomycetes are the potential sources for extra-cellular enzymes, which play a role in biodegradation of organic matter, thereby enhancing the productivity of the marine environment.

Umamaheswary et al. (2005) isolated 40 strains of actinomycetes from the estuarine fish, Mugilcephalususing Kuster's agar medium. Out of 40 strains tested, only the strain S. galbusshowed good L-glutaminase activity. Various process parameters which influenced L-glutaminase production by the S. galbuswere optimized. Maximal enzyme production (18.93IU/ml) was attained at pH 9.0, 360 C, and glucose and malt-extract as carbon sources after 72 h of incubation. Senthilkumar et al. (2005) isolated 41 halophilic actinomycetes strains from the salt marsh area of the Vellar estuary using four different media. SC agar medium was the best for the isolation of halophilicactinomycetes. Among the isolated strains, the strain SH-9 showed greater resistance towards mercuric chloride in agar diffusion assay. The strain was classified as Actinopolysporasp. by its morphological and chemotaxonomical characters.Sivakumar et al. (2006) isolated actinomycetes strains from skin, gills and gut contents of the estuarine fish,Chanoschanos. Out of 20 strains tested,Streptomyces rimosusshowed L-glutaminase activity. Optimum production of L-glutaminase (18.93IU/ ml) was observed after 96 h at 270 C, pH 9.0 with glucose and malt extract.

Sahu et al. (2006) also reported a total number of 40 strains of actinomycetes from the sediments of the Vellar estuary and checked their antagonistic activity against the human bacterial pathogens (B.substilis, P. vulgaris, S. flexineri, K. pneumoniae V. choleraeand S. aureus). Among them, 9 strains (22.5%) showed activity against thetested pathogens and 5 strains which showed good activity were identified upto species level. Muthurayar et al.(2006) isolated a total of 18 actinomycetes strains from an estuarine fish,Chanoschanosand studied their antagonistic activity against human bacterial pathogens.

 

Saha et al. (2006) isolated four marine actinomycetes from Bay of Bengal and screened against multiple drug resistant (MDR) bacteria. Asha deviet al. (2006) isolated 3 marine actinomycetes from Dhanushkodi coastal region, Tamil Nadu. Vijayakumar et al. (2007) reported 192 actinomycetes colonies from 18 marine sediment samples of Palk Strait region of Bay of Bengal, India. Among them, 68 isolates were morphologically distinct on the basis of color of spore mass, reverse side color, aerial and substrate mycelia formation, production of diffusible pigment and sporophore morphology. From these isolates 39 were assigned to the genus Streptomyces. Gandhimathi et al. (2008) isolated 26 marine endosymbiotic strains, isolated from the Bay of Bengal. In this investigation Streptomyces species showed antimicrobial activity against pathogenic bacteria and fungi. Praveen et al. (2008) isolated two marine actinomycetes and optimized the fermentation conditions.

Selvin et al. (2009) optimized and produced antimicrobial agents from sponge associated marine actinomycetesNocardiopsisdassonvillei MAD08. Suthindhiran and Kannabiran (2009a) isolated Streptomyces VITSDK1 spp from South coast of India which exhibited significant hemolytic activity against rat erythrocytes and human erythrocytes. It also showed moderate antibiosis against fungi and bacterial pathogens. Suthindhiran and Kannabiran (2009b) isolated Saccharopoly

sporasalinaVITSDK4 from Bay of Bengal, which produced an extracellularbioactive metabolite, which inhibited the proliferation of HeLa cells as well as antagonistic to fungal and bacterial pathogens. Deepika and Kannabiran (2009) isolated 100 marine actinomycetes from coastal regions of Tamil Nadu. Out of 100 isolates 3 isolates belonged to genus Streptomyces sp exhibited potential antidermatophytic activity againstthe dermatophyteTrichophytonrubrum. Ramesh and Mathivanan (2009) isolated 209 marine actinomycetes from Bay of Bengal and screened for antimicrobial activity and industrial enzymes.

 

 Kerala

Ranjithkumar et al. (2014b) isolated vancomycin from new source S. fradiaecollected from kannamaly beach, Arabian sea. This vancomycin showed antiquorum sensing activity and wound healing properties again skin pathogens. Dhevendaran et al. (2004) isolated Streptomycetes from Pernaviridis, Grapsusstrigosus, Ulva fasciataandSargassumwightiicollected from Kovalam coast. The distribution patternof the microorganisms with special emphasis on Streptomycetes was carried out using special microbiological media. Streptomycetes isolated from the visceral mass of P. viridisand G. strigosusshowed maximum colonization in Actinomycete agar medium, whereas Streptomycetes associated with the fauna and seaweed showed a high diversity in pigmentation. Streptomyces harboured in the visceral mass of P.viridisexhibited antagonism against Aeromonassp. Remya and Vijayakumar(2008) reported 173 actinomycetes from Kerala, West Coast of India. Out of these isolates 21 had antimicrobial activity.

 

 Andhra Pradesh

Ellaiah and Reddy (1987) isolated 140 strains of actinomycetes from the marine sediments of Visakhapatnam coast and identified them upto genus level. Out of these 140 strains, only 18% exhibited anti-microbial activity against bacteria and fungi. Ellaiah (1996) isolated

actinomycetes from the sediments of Bay of Bengal and the strains which showed good antagonistic activity were identified upto species level. Ellaiah (2002) isolated 80 strains of actinomycetes from the sediments of Bay of Bengal near Machilipatnam by plating on starch casein agar medium. Of these, 7 isolates exhibited broad-spectrum antimicrobial activity, 68 showed proteolytic activity and 62 showed amylolytic activity. Ellaiah (2004) have isolated 60 actinomycetes from Bay of Bengal near Kakinada coast with distinct characteristics, by plating on starch casein agar medium. Among them, 11 isolates exhibited antibacterial (18.3%), 10 isolates showed antifungal (16.6%) while 2 isolates showed both antibacterial and antifungal (3.3%) activities. All 60 isolates were also tested for enzymatic activities on which 49 (81.6%) and 51 isolates (85%) exhibited amylolytic and proteolytic activities, respectively. Sujatha (2005) reported a new marine Streptomycetes BT-408 against methicillin resistant S. aureus.

 

Andaman and Nicobar group of islands

 

Sahu (2007) assessed the population density of actinomycetes from eight different stations of the Little Andaman Island. Mean population density of actinomycetes recorded from the water samples varied from 0.29 to 0.45 x103 CFU/ml with the minimum of 0.29 x103 CFU/ml at Navel Area and the maximum of 0.45 x103 CFU/ml at Chandra Nallah. In the case of sediment samples, population density ranged from 1.21 to 3.29x103 CFU/g with a minimum of 1.21 x103 CFU/g at Navel Area and a maximum of 3.29 x103 CFU/g at Buttler Bay. During the investigation, a total of 41 strains were isolated and tested for their antagonistic activity against the bacteria that are highly pathogenic to shrimps such as V. alginolyticsV. harveyiand V. parahaemolyticus. More than 61% of the strains (26 strains) exhibited varying degree of antagonistic activity. Among them, 6 strains showed good activity and they were tentatively identified. The results suggested that theactinomycetes from the marine environment can be used as bio-control agents in shrimp culture systems to control diseases caused by bacterial pathogens.

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