Antimicrobial Attributes Of Rare Actinobacteria DetectedFrom Limestone Quarries

Rare actinobacterial isolates belonging to the genera Micromonospora, Nonomuraea, Kribbella, Lechevalieria, Saccharotherix and commonly occurring Streptomyces were isolated from Limestone quarry a unique and geographical area and harsh habitat. pH – 9.0, temperature – 45 C and sodium chloride concentration – 3% were found to be optimum for the maximum growth and physiological ability of the test isolates indicating them as thermo-alkaliphilic in nature. It was found that, the test isolate DRQ 10 belonging to the genus Streptomyces and DRQ 72 belonging to the genus Micromonospora were very effective showing maximum zone of inhibition against the bacterial pathogen B. subtilis (37 mm and 35 mm) and fungal pathogen Fusarium solani (33 mm and 31 mm) respectively. The similar test isolates exhibited their higher efficacy with the MIC value of 2 μg/ml and 4 μg/ml for B. subtilis and 4 μg/ml and 8 μg/ml for C. albicans, and also showed more potency then the commercially available antibacterial drug, Ampicillin and antifungal drug, Nystatin. KEYWORDS; Limestone Quarry, Soil sample, Rare actinobacteria, Antibacterial activity, Antifungal activity, Minimum Inhibitory Concentration *Corresponding author DAYANAND AGSAR A-DBT Research Laboratory, Department of Microbiology, Gulbarga University, Gulbarga – 585106, Karnataka, India Int J Pharm Bio Sci 2012 July; 3(3): (P) 137 147 This article can be downloaded from www.ijpbs. net P 138 INTRODUCTION The Actinobacteria are Gram-positive organisms with high G+C composition. It is an important class within Bacterial domain with 56 families and over 280 genera. They can be found in a variety of habitats and are particularly abundant in soil and aquatic bodies. These are filamentous, branching bacteria with a fungal type of morphology among which they were originally classified under the older name actinomycetes. Streptomyces is a familiar name to all microbiologists, but few will recognize actinobacteria, the group to which it belongs. Identified from 1870s onwards, these organisms confused their discoverers by sharing characteristics of both bacteria and fungi. Different type of rocks like Cinder, China clay, Coquina, Blue rock, Granite, Grit stone, Gypsum a mineral, Limestone, Marble, Sandstone, and Slate occur naturally in the Deccan Plateau. Limestone is one of the chief available sedimentary rocks of the Deccan Traps and makes up about 10% of the total volume of all sedimentary rocks. Limestone is a sedimentary rock composed largely of the minerals calcite and aragonite, which are different crystal forms of calcium carbonate (CaCO3) . These are mainly being used as raw materials for the production of cement. Their excavation for the said purpose from the earth leaves behind huge quarries with typical habitat. The harsh climatic conditions in the lime stone quarries supposed to be a good niche for detection, which includes isolation and screening of potential isolates as well as novel bioactive molecules. The screening of microbial natural products, especially antibiotics, continues to represent an important route to the discovery of novel bioactive molecules, for development of new therapeutic agents and for evaluation of the potential of established as well as new bacterial taxa. Antibiotics have been used in many fields including agriculture, veterinary and pharmaceutical industries. Actinobacteria have the capability to synthesize many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, antiparasitic, and several enzymes. Of these compounds, antibiotics predominate in therapeutic and commercial importance. It has been estimated that approximately two-third of the thousands of naturally occurring antibiotics have been isolated from actinobacteria. Indeed, the Streptomyces species produce about 75% of commercially and medically useful antibiotics. The search for novel metabolites especially from actinobacteria requires a large number of isolates in order to discover a novel compound of pharmaceutical interest. The aim of this study was to isolate, screen and investigate the antimicrobial activities of the rare actinobacteria obtained from limestone quarries of Shahabad town of Gulbarga district, Karnataka, India. MATERIALS AND METHODS Sampling and Isolation Soil samples were collected from limestone quarries around Gulbarga, Karnataka, India. Actinobacteria were isolated from the collected soil samples by following serial dilution plate culture technique employing starch casein agar and ISP – 2, 3, 6 and 7 media. The inoculated plates were incubated at 35 C for 1 2 weeks. After the completion of incubation period, typical actinobacterial colonies were picked up from mixed colonies and subcultured on fresh medium to obtain pure cultures. The pure cultures were stored at 4 C. Identification and Characterization Actinobacterial colonies were identified and characterized morphologically, biochemically and physiologically following the standard features described in International Streptomyces Project (ISP) 18 and Bergey’s Manual of Systematic Bacteriology . Cultural characteristics of pure isolates in various media were recorded after incubation Int J Pharm Bio Sci 2012 July; 3(3): (P) 137 147 This article can be downloaded from www.ijpbs. net P 139 for 7 to 14 days at 35 C. Morphological observations were made with a light microscope (Nikon, SE) by using the method of Shirling and Gottlieb. Essential biochemical reactions were carried out following the standard procedures of Gottlieb. Carbon and nitrogen utilization was determined on plates containing ISP basal medium 9 as described by Gottlieb. Antimicrobial Activity Antimicrobial activities were examined in vitro against standard strains of bacterial and fungal pathogens that includes, Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 29998), Klebsialla pneumonia (ATCC 15380), Pseudomonas aeruginosa (ATCC 25619), Salmonella typhi (ATCC 6539),Staphylococcus aureus (ATCC 6538), Aspergillus fumigatus (ATCC 46645), Aspergillus niger (ATCC 16404), Fusarium solani (ATCC 36031), Candida albicans (ATCC 90028), Cryptococcus gatti (ATCC 32609) and Mucor indicus (ATCC 4855) obtained from the culture depository unit, Department of Microbiology, Gulbarga University, Gulbarga. Determination of antimicrobial activity of pure actinobacterial cultures was performed by well diffusion method. The test organisms were grown on Muller Hinton medium (bacteria) and Sabouraud Dextrose agar (fungi and yeast). Wells were bored and the culture filtrate of the selected isolates of actinobacteria were dropped in the wells and kept for incubation at 37 C for bacterial and 28 C for fungal strains. The antimicrobial activity was determined by measuring the size of the inhibition zone. The activity from 7 to 15 mm inhibition zone was recorded as low, 16 to 24 mm as medium and more than 25 mm as high activity. Minimum Inhibitory Concentration The minimum inhibitory concentrations (MIC) of the crude extracts of all the test isolates were evaluated against the previously described bacterial and fungal pathogens by serial dilution technique according to standard methods of Noble and Sykes . Dilutions of the antibiotic were made in nutrient medium containing phenol red indicator. The trays were incubated at 37 C for 24 hours. The MIC (μg/ml) was taken as the smallest concentration of antibiotic at which the indicator remained red and yellow color indicated acid production caused by growth of the organism. Antibiotic activity against the fungal cultures was determined in tubes by serial dilution method. Tubes containing fungal and yeast pathogens were incubated at 28 C for 1, 2 or 5 days. The MIC (μg/ml) was taken as the smallest concentration of antibiotic preventing growth of the test organism in the presence of a standard Ampicillin for bacterial and Nystatin for fungal pathogens.