Bioactive potential of actinobacteria isolated from certain under-studied regions in India

Article history: Received on: 25/05/2016 Revised on: 13/06/2016 Accepted on: 01/07/2016 Available online: 30/08/2016 The present study reports the bioactive potential of actinobacteria isolated from certain under-studied regions in India. Soil and sediment samples and mangrove leaves were collected from 16 different under-studied regions in India. Actinobacteria was isolated by adopting selective isolation methods. Totally 158 actinobaterial cultures were selected from the collected terrestrial, marine and plant samples. More number of colonies was isolated from magnesite area, Kolli Hills and forest area in Himachal Pradesh. Majority of the isolates produced powdery (40%) and leathery (25%) colonies with white (38%) or Gray (37%) colour aerial mycelium. Bioactive compound from all the isolates were produced by agar surface fermentation and its activity was tested by agar plug method against S. aureus, E. coli and C. albicans. About 64 out of 158 cultures showed antibacterial activity in which 62 cultures was active against S. aureus whereas 26 were active against E. coli. Twenty three actinobacterial cultures were exhibited antifungal activity. About 11 actinobacterial cultures were active against both S. aureus and E. coli. In antifungal testing whereas fourteen actinobacterial cultures were found to be active against S. aureus, E. coli and C. albicans. Maximum of 21 antibacterial cultures and antifungal cultures were obtained from Magnesite soil followed by 12 antibacterial and 7 antifungal cultures were obtained from the soil sample collected from Himachal Pradesh. This evidenced that the under-studied ecosystems in India are the promising source for bioactive actinobacteria with broad spectrum antibacterial and antifungal activity. Further studies on the potential actinobacterial strains result in the isolation of broad spectrum antimicrobial metabolites.


INTRODUCTION
The discovery of antibiotics to treat infectious diseases has revolutionized the field of medicine in the mid-twentieth century.However, due to overuse or misuse of antibiotics over a prolonged period, most of the pathogens have become resistant to the antibiotic therapy.Thus, there is a dire need for the discovery and development of new antibiotics to effectively target the life threatening disease causing pathogens (Sharma et al., 2016).Microbial resources are reported as promising source for novel metabolites.Actinobacteria are Gram positive bacteria that .constitute one of the largest bacterial phyla and they are ubiquitously distributed in both aquatic and terrestrial ecosystems (Balagurunathan and Radhakrishnan, 2010).Actinobacteria are of great importance in terms of secondary metabolite producers with promising biological activities.They produce two-thirds of all naturally derived antibiotics in current clinical use as well as many anticancer, antifungal and antiviral compounds (Barka et al., 2016).
But, in the past two decades, there has been a decline in the discovery of novel metabolites from actinobacteria including from Streptomyces which yield disappointingly high number of previously described molecules (Berdy, 2012).To solve this issue, bioprospecting of un/less explored ecosystems like marine, desert, forests, caves, and hills has been proved as useful approach for tapping innumerable number of bioactive compounds from novel bioactive actinobacteria (Radhakrishnan et al., 2014).
In addition, new sources of bioactive metabolites from rare actinobacteria from different ecological niches have promoted recent advances in the discovery of new antibiotic molecules (Nimaichand et al., 2015).With this view the present study has been initiated for bioprospecting actinobacteria from under-studied regions in India to determine their antimicrobial potential.

Sample collection and pre-treatment
Soil samples were collected from 11 different terrestrial regions and sediment samples were collected from three marine and a fresh water region.Leaves sample from the mangrove plant Rhizophora apiculata was also collected from Parangipettai coastal area, Tamil Nadu (Table 1).All the soil and sediment samples were air-dried at room temperature for 3-5 days and the sieved samples were kept at 55°C for 10 minutes in a glass container (Radhakrishnan et al., 2007).Plant leaves were pretreated by adopting three step process described by Coombs and Franco (2003).

Isolation of actinobacteria
Actinobacteria were isolated by adopting standard spread plate method using different media such as Starch Casein agar, Kuster's agar and Oat meal agar.All the media used in this study was prepared using distilled water whereas 50% sea water was used for the isolation of marine actinobacteria.About 10 gram of pre-treated sediment sample was added into 90 ml of sterile distilled water in 500 ml conical flask.The flask was kept in rotary shaker for 30 minutes for mixing of sample.The particulate matter was allowed to settle down and the suspension was serially diluted up to 10 5 dilutions using sterile distilled water blank.Hundred microliter of aliquot from 10 2 , 10 3 , 10 4 dilutions was taken and spreaded over agar plate using sterile L-rod.The same procedure was followed for all the remaining samples.All the plates were incubated at 28°C for one month (Radhakrishnan et al., 2007).
Colonies showing suspected actinobacterial morphology were picked up from the isolation agar plates using sterile Lshaped loop and inoculated on yeast extract-malt extract agar (ISP2 medium).The plates were incubated at 28 0 C for 7 days.Morphologically different actinobacterial cultures were selected and preserved using ISP2 agar slants as well as in 30% glycerol broth.

Characterization of actinobacteria
Cultural characterization was done by inoculating all the actinobacterial cultures into ISP2 agar medium.All the plates were incubated for 10 days at 28 0 C. Cultural characteristics recorded include growth, consistency, aerial mass colour, presence of reverse side pigment and soluble pigment production (Shirling and Gottileb, 1966).Micromorphological characteristics were studied by adopting slide culture method (Balagurunathan et al., 2010).About 2 ml of ISP2 agar medium inoculated with actinobacterial spores were poured as a thin layer over the surface of sterile microscopic slides.The slides were kept in sterile petriplates and incubated at 28 °C for 10 days.Then the slides were observed under bright field microscope at 40X magnification.The recorded microscopic characteristics include presence of aerial mycelium, substrate mycelium, mycelial fragmentation and spore chain morphology.
Based on the results of growth pattern of actinobacteria on ISP2 agar medium and microscopic appearance, similar actinobacterial isolates were discarded and different isolates were selected for further investigations.The selected isolates were grouped into Streptomycetes and non-Streptomycetes/rare actinobacteria.

In vitro screening of actinobacteria for antimicrobial activity
Bioactive compounds from actinobacterial cultures were produced by agar surface fermentation (Radhakrishnan et al., 2014).All the actinobacterial cultures were inoculated into ISP2 agar plates and incubated at 28 0 C for 10 days for the production of secondary metabolites.During incubation, the extracellular metabolites are secreted into the agar medium.Test pathogens used in this study includes Staphylococcus aureus MTCC96, Escherichia coli MTCC739 and Candida albicans MTCC227.All the cultures were maintained as slant as well as stab culture using nutrient agar.
Antimicrobial activity of actinobacterial cultures was tested by adopting agar plug method (Radhakrishnan et al., 2014).Actinobacterial cultures grown on IS2 agar plates for bioactive compound production were taken and the mycelial growth was removed from the agar surface using sterile spatula.Test pathogens were inoculated into nutrient agar plates using sterile cotton swab.Agar plug with 5 mm diameter were cut from the ISP agar grown with actinobacterial cultures were placed over nutrient agar seeded with test pathogens.All the plates were incubated at 37 °C for 24 hours.Zone of inhibition was expressed in millimetre in diameter.

Isolation of actinobacteria
Colonies with actinobacterial morphology were observed and recovered from all the samples collected from 16 different regions.More number of actinobacterial colonies was isolated from terrestrial soil and fresh water sediment samples plated on starch casein agar where are Kusters agar yielded more number of actinobacterial colonies from marine sediments and mangrove plant sample.
Totally about 400 actinobacterial colonies were recovered from the samples collected from 16 different places from which 158 morphologically different colonies were selected for further study.More number of colonies was selected from the soil sample collected from Kolli Hills, and Magnesite area, Salem, Tamil Nadu, forest area of Himachal Pradesh and Utharakand (Table 1).

Characterization of actinobacteria
During recovery and preservation, about 88% of the actinobacterial cultures showed good growth on ISP2 agar.Majority of the isolates produced powdery (40%) and leathery (25%) colonies with white (38%) or Gray (37%) colour aerial mycelium.
Under microscopic observation, all the actinobacterial cultures showed the presence of substrate mycelium whereas 86% of the cultures showed the presence of both aerial and substrate mycelium in which majority of them are Streptomyces.
The remaining 14 % of the cultures which showed only the presence of substrate mycelium are considered as non-Streptomyces/rare actinobacteria (Table 2).

Screening for antimicrobial activity
In agar plug method, 64 out of 158 cultures showed antibacterial activity in which 62 cultures was active against S. aureus whereas 26 were active against E. coli.About 11 actinobacterial cultures were active against both S. aureus and E. coli (Table 3).The results are presented as mean ± SD (n = 3) ; RA -Rare Actinobacteria; + -present; --absent.

CONCLUSION
Findings of the present study evidenced that the understudied ecosystems in India are the promising source for bioactive actinobacteria with broad spectrum antibacterial and antifungal activity.Further studies on the potential actinobacterial strains result in the isolation of broad spectrum antimicrobial metabolites.

Table 1 :
Details of samples collected for the isolation of actinobacteria.

Table 2 :
Morphological pattern of actinobacteria isolated from different regions in India.

Table 3 :
Actinobacterial cultures showing broad spectrum antibacterial activity against Gram positive and Gram negative bacterial pathogens.