International Research Journal of Biological Sciences ____________________________________ ISSN 2278-3202Vol. 4(4), 65-68, April (2015) Int. Res. J. Biological Sci. International Science Congress Association 65 Antagonistic effect of marine Actinobacteria on MDR Uropathogens Aruna V and Rajan S2*Research Scholar, Department of Microbiology, R and D Center, Bharathiar University, Coimbatore, Tamil Nadu, INDIA Research Department of Microbiology, M. R. Government Arts College, Mannargudi – 614 001, Thiruvarur, Tamil Nadu, INDIA Available online at: www.isca.in, www.isca.me Received 18th February 2015, revised 1st March 2015, accepted 6th April 2015Abstract Actinobacteria are the antagonistic bacteria able to release antagonistic compounds which inhibits the growth of other microbial species. This study was carried out to identify biologically potential Actinobacteria from natural source. This study also confirms antimicrobial nature of the Actinobacteria. Twenty six marine water samples was collected from different time and points in the Bay of Bengal and isolated thirteen actinobacterial isolates using different culture medium. All actinobacterial isolates were identified by macroscopic and microscopic observation. The isolated actinobacteria were screened for their antimicrobial activity against UTI pathogens. The marine isolate Stretomycetes sp. identified from marine water were found to be more efficient in the production of secondary metabolites. This will pave the way for further investigations to study their potential importance in combating pathogenic bacteria. Keywords: Actinobacteria, streptomyces, antagonism, MDR pathogens.Introduction Actinobacteria are a group of bacteria which shows multiple potentialities as it inhibit the growth of other microbial species through a process called antagonism. They also produce numerous pharmacologically active substances essential for health. During the last few decades multiple numbers of antimicrobial substances are screened from actinobacteria1. Antagonism is one of the best known phenomena exhibited by a group of bacteria called Actinobacteria. Antimicrobial substances secreted by these bacteria are liable for this activity. Over 5000 antimicrobial substances are identified from these groups of microorganisms. Streptomyces, Actinomyces, Micromonospora, Actinomadura, Actinoverticillium and Thermoactinomycetes are the major antagonistic Actinobacteria. Among these, 60% of antimicrobial substances were made from Streptomyces. Current antibiotics were failed due to the development of multidrug resistance among microorganisms. These microbes may cause more vulnerable infections. To overcome these, people are in search of newer antimicrobial substances from the nature. Microbes and plants are the best source of these substances. Among microbes antagonistic actionobacteria is the best choice. Approximately two thirds of all antimicrobial substances are made from these groups of microorganisms. Actinobacteria show a considerable proportion of the population of soil, lakes and river muds. Atinobacteria have been isolated from dry ecosystem since time immemorial. The first report of actinobacteria being recovered from marine sediments appeared several decades ago. Very few reports suggested that marine actinobacteria in sediments of the ocean are the best source for isolation of bioactive compounds. Studies are established that actinobacteria isolated from the marine environment are metabolically active and have adapted to life in the sea. Actinobacteria isolated from marine ecosystem shows different features than the isolates from the dry ecosyetem (Terrestial). They may also produce effective antagonistic compounds. Hence in this study, antagonistic activity of marine actionbacteria is studied to understand its efficiency and the antimicrobial substance production. Material and Methods Sampleprocessing: Seawater samples were collected from Bay of Bengal, India. Sampling area covers from off shore area of Bay of Bengal at Puducherry, UT of India. The collected water samples were stored in sterile polypropylene bottles and preserved in the laboratory for future use. Isolation of marine actinobacteria: Serial dilution along with pourplating was done to isolate Actinobacteria. The serial dilution was done by taking one ml of water sample and mixed with 1ml sterile distilled water in a test tube. The sample was serially diluted up to 1:10000 dilution by transferring 1ml aliquots to a series of test tubes each containing 9ml sterile distilled water and from respective dilution. One ml of diluted sample was transferred to the sterile petri plates and poured starch casein agar (SCA). Different media like actinomycetes isolation agar, ISP2 and ISP6 are used also used for isolation of actinobacteria from marine samples. The isolation media was supplemented with the antibiotics cycloheximide (25mg/ml) and nalidixic acid (25mg/ml) 7. The plates were incubated at 28±1ºC for 7 to 21 days. The colonies were identified by their cultural characters. Characterization of marine actinomycetes: A standard method of Slide culture technique was performed to characterize aerial mycelium, spore arrangements. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(4), 65-68, April (2015) Int. Res. J. Biological Sci. International Science Congress Association 66 Screening of anti microbial activity: Identified actinobacteria were incubated at 30±2ºC on a rotary shaker at 220 rpm for 10 days for complete recovery of antagonistic substances. The culture was collected and centrifuged at 6000 rpm for 15 min. Cell free extract was collected after filtering through millipore filter (0.45m). Well diffusion method was used for assaying the antimicrobial activity against multi drug resistant E. coli and K. pneumoniae.Results and Discussion Twenty six marine water samples was collected from different time and points in the Bay of Bengal and isolated thirteen actinobacterial isolates using different culture medium. All actinobacterial isolates were identified by growth characteristics and Gram staining. Based on these characteristics actinobacteria were identified as Stretomyces sp and Nocadia sp. (table-2). Out of thirteen selected and identified actinobacteria Stretomycetes sp showed significant prevalence and antimicrobial activity against multidrug resistant UTI pathogens. Five pathogenic strains isolated from cases of UTI infection (three Escherichia coli (E-51, E-44, E-71) and two Klebsiella sp.,(K-52 and K-53) were used as a test organism for antagonistic study. Among the actinobacteria tested, test organism RW2-3 and RW2-5 strains produced the best activity against all the test organisms (table-3). When 100µl / disc concentrations of the actinobacteria grown fermentation medium were tested for antibacterial activity against MDR urinaryisolates, the results indicated that all the actinobacterial strains showed good antibacterial activity. All the strains showed a zone of inhibition against urinary pathogens. Best antimicrobial activity was exhibited by RW2-3 and RW2-5 strains and these strains were considered as a Streptomyces sp. In the present study, zone of inhibition ranges from 8mm -15mm with the best activity against E. coli 51 and E. coli 44 by RW2-3 strain (table-4). Similar kind of antagonistic activity also reported by different authors from India but they used different pathogens9-11. Several studies reported that, the actinobacteria are worth mentioning as antibiotic producers making three quarters of all known products and are especially prolific12-17. According to Mohana and Radhakrishnan18 actinomycetes isolates are often encounter activity against gram positive bacteria than gram negative bacteria, which is due to secondary metabolites, whereas our results shows different report. In the present study all the test urinary MDR pathogens were belonging to gram negative group E.coli and K. pneumoniae). Marine Steptomycetes sp exhibited the highest antibacterial activity against E .coli and K. pneumoniae. Conclusion The present study concluded that the marine actinobacterial isolate, Streptomyces sp is dominant in marine water and is considered as a budding resource of novel bioactive compounds. A further study on the molecular characterization of the isolates and purification of bioactive compounds is in progress. Table-1 Isolation of Actinobacteria from Marine for the Production of Antimicrobial CompoundS. No Strain No. Nature of isolate 1. SW02-001 Nil 2. RW02-002 7 types of colonies were observed (3 white, 3 gray, 1green spot colour colonies) 3. MHT05-001 - 4. MLT05-002 White colour 5 MLT5-003 - 6 MLT5-004 - 7 MLT5-004 - 8 MLT-001 - 9 MLT-002 White colour 10 MLT-003 - 11 MLT-004 - 12 MLT-005 - 13 M10F05-003 - 14 ELT05-004 - 15 EHT-005 White colour 16 E10F05-006 - 17 E20F -007 White colour 18 MLT01-009 - 19 MLT02-009 - 20 MHT01-010 White colour 21 M10F01-011 White colour 22 M10F01-012 - 23 ELT01-012 - 24 EHT01-013 - 25 EHT02-014 - 26 EHT03-015 - References 1.Kassahun S.B., Thangavel S. and Tariku H., In vitro Evaluation of Actinobacteria against Tomato Bacterial Wilt (Ralstonia solanacearum EF Smith) in West Showa, Ethiopia, J. Plant Pathol. Microbiol., 4, 1 (2013)2.Nakade K., Antimicrobials in Microbial Diversity and Bioprospecting, Edited by Bull, T. ASM Press., 336-355 (2012)3.Sunil L.A., Gaviraj N.E., Asif A.K., Ravindra K., Nagesh C. and Chandrashekara S., Screening, isolation and purification of antibacterial agents from marine actinomycetes, Int. Cur. Pharma. J.,1(12), 394-402 (2012)4.Sajad A.B., Ruqeya N., Tauseef A.M. and Fayaz A.S., Secondary metabolites of actinomycetes as potential source of antibiotics, Stem Cell., 4(2), 44-48 (2013) Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(4), 65-68, April (2015) Int. Res. J. Biological Sci. International Science Congress Association 67 Table-2 Characteristic features of actinobacterial isolatesS. No Test isolate Aerial mycelium Substrate mycelium pigment Spore chain morphology Gram staining Glucose Fructose Lactose Maltose Mannital identification 1 RW2-1 gray yellow no spirals Gram positive rod + + _ _ _ Streptomyces sp., 2 RW2-2 gray yellowish brown no spirals Gram positive rod + _ _ + Streptomyces sp. 3 RW2-3 light brown yellowish brown no Simple spirals Gram positive rod - - + + Streptomyces sp. 4 RW2-4 white white no retinaculiaperti Gram positive rod _ + _ _ Streptomyces sp. 5 RW2-5 yellowish brown yellowish brown no Flexible Gram positive rod + _ + + Streptomyces sp. 6 RW2-6 Yellowish brown yellowish brown no spirals Gram positive rod _ + _ _ Streptomyces sp. 7 RW2-7 yellow yellow no spirals Gram positive rod _ + _ + Streptomyces sp. 8 E20F-007 gray Ash-yellow no spirals Gram positive rod _ _ Streptomyces sp. 9 MLT-002 light yellow no spirals Gram positive rod _ _ _ _ Streptomyces sp. 10 EHT-005 cream light no Spirals Gram positive rod _ _ + + Streptomyces sp. 11 MHT01-011 gray brown no spirals Gram positive rod + _ _ + Nocardia sp 12 M10F01-010 gray Yellow-ash no Spirals Gram positive rod _ _ _ _ Nocardia sp 13 EHTOI gray Yellow green no flaxibile Gram positive rod _ _ _ _ Streptomyces sp., Table-3 Antagonistic activity of actinobacteria multidrug resistance pathogens S. No Test organism E.coli 51 E.coli 44 E. coli 71 K.pneumoniae 52 K.pneumoniae 53 1 RW2-1 + - - - + 2 RW2-2 + - - - - 3 RW2-3 + + + + + 4 RW2-4 + - + - + 5 RW2-5 + + + + + 6 RW2-6 + - + - - 7 RW2-7 + - + - + 8 E20F-007 + - + + - 9 MLT-002 - - + - - 10 EHT-005 + - + + - 11 MHT01-011 - - + + + 12 M10F01-010 - - + - - 13 EHTOI - - + + - Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(4), 65-68, April (2015) Int. Res. J. Biological Sci. International Science Congress Association 68 Table-4 Antimicrobial activity of the product obtain from marine sample against Uropathogens S. No Isolate number Organisms Used / Zone of inhibition in mm E-51 E-44 E-71 K-52 K- 53 1 RW2-1 08 - 09 10 11 2 RW2-2 - - 09 10 12 3 RW2-3 15 15 12 11 12 4 RW2-4 - - 11 12 11 5 RW2-5 14 15 13 12 12 6 RW2-6 10 12 12 11 10 7 RW2-7 13 11 13 14 11 8 E20F-007 09 11 12 12 11 9 MLT-002 13 12 13 11 - 10 EHT-005 13 12 11 13 09 11 MHT01-011 - 09 09 08 - 12 M 10F01-010 10 10 09 11 - 13 EHTOI - 09 09 11 - 5.Valli S., Suvathi S.S., Aysha O.S., Nirmala P., Vinoth Kumar P. and Reena A. Antimicrobial potential of Actinomycetes species isolated from marine environment, Asian Pacific J. Tropical Biomed.,, 469-473 (2012)6.Reddy N.G, Ramakrishna D.P.N. and Raja G., A Morphological, Physiological and Biochemical Studies of Marine Streptomyces rochei (MTCC10109) Showing Antagonistic Activity against Selective Human Pathogenic Microorganisms, Asian J. Biol. Sci., 4 (1), 1-14 (2011)7.Kumar S. and Kannabiran K., Diversity and optimization of process parameters for the growth of Streptomyces VITSVK9 Spp. Isolated from Bay of Bengal, India, J. Nat. Env. Sci.,1(2), 56-65 (2010) 8.Rajan S and Selvichristy R, Essentials of Lifesciences, In: Characterization of Actinobacteria, Anjana Publishers, Chennai, 1, 255-274 (2014)9.Sathish K.S.R., Kokati V. and Bhaskara R., In-vitro antimicrobial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus, Asian Pacific J. Tropical Biomed.,5., S1802-S1807., (2012)10.Karthikeyan P., Senthilkumar G. and Panneerselvam A. Isolation, characterization and identification of actinobacteria of Mangrove ecosystem Ennoor, east coast of Tamil Nadu, India, Advances in Appl.Sci. Res.,4(5).,296-301(2013)11.Gulve R.M. and Deshmukh A.M., Antimicrobial activity of the marine actinomycetes, Int. Multidiscip. Res. J.,2(3)., 16-22 (2012)12.Astalakshmi A., Thangapandian V. and Lingakumar K. Isolation and Characterization of Actinomycetes from the Soil of Devathanam: A Foot-hill of Western Ghats, Int. J. Pharma Res. and Rev., 3(1).,15-20 (2014) 13.Deepa S., Panneerselvam A., Dhanasekaran D., Thajuddin N. and Vijayakumar R., Diversity and Antimicrobial Potential of ctinobacteria from Salt Pan Environment, Global Adv. Res. J. Microbiol.,1(8)., 140-148 (2012)14.Gunasekaran M. and Thangavel S., Antagonistic activity of marine Streptomyces sp LCJ94 against the shrimp pathogens, Annals of Biol. Res., 4 (4)., 224-227 (2013)15.Gunasekaran M. and Thangavel S., Isolation and Screening of Actinomycetes from marine sediments for their potential to produce antimicrobials, Int. J. Life Sc. Bt and Pharm. Res., 2.,4-7(2013) 16.Mukesh S., Actinomycetes: Source, Identification, and their Applications, Int. J. Curr. Microbiol. App. Sci.,3(2), 801-832(2014) 17.Radhakrishnan M., Pazhanimurugan R., Gopikrishnan V., Balagurunathan R. and Vanaja K., Streptomyces sp D25 isolated from Thar Desert soil, Rajasthan producing pigmented antituberculosis compound only in solid culture, J. Pure and App. Microbiol.,8(1), 333-337 (2014) 18.Mohana S. and Radhakrishnan M., Streptomyces sp MA7 isolated from mangrove rhizosphere sediment effective against Gram negative bacterial pathogens, Int. J. Pharm Tech Res.,6(4), 1259-1264 (2014)