In- Vitro Antibacterial Activity of Stereum Ostrea a Wood Decaying Macro Fungus

Transcription

1 Journal of Microbiology Research and Reviews Vol. 2(2): 12-18, February, 2014 ISSN: In- Vitro Antibacterial Activity of Stereum Ostrea a Wood Decaying Macro Fungus Amiya Kumar Prusty 1 *, Laxmikumari Samad 2, Abhijita Rout 2 and Apramita Patra 2 1 Institute of Pharmacy and Technology, Salipur, Cuttack, Odisha Department of Botany, College of Basic Science and Humanities, OUAT, Bhubaneswar for Correspondence: amiyaprusty@gmail.com Abstract The increasing prevalence of multidrug resistant bacterial strains has prompted the need for antibacterial controls other than the existing antibiotics. In the present study, a wood decaying macro fungi Stereum ostrea was assessed in-vitro for its ability to inhibit the growth of different bacterial strains. The antibacterial activities of acetone, ethanol and aqueous extracts of fruiting body of Stereum ostrea were evaluated against the bacterial strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentration of acetone extract against the test microorganisms was lesser than that of ethanol and aqueous extracts. From the minimum inhibitory concentration data it could be concluded that acetone extract was most active as antimicrobial against the test microorganisms followed by ethanol and aqueous extracts. The acetone extract, ethanol extract and aqueous extract of Stereum ostrea were having maximum zone of inhibition of 19.17mm, 12.67mm and 10.17mm respectively against Bacillus subtilis, and minimum zone of inhibition of 11.33mm, 8.50mm and 7.33mm respectively against Pseudomonas aeruginosa. The study concluded that the different extracts of fruiting body of Stereum ostrea contain potential compounds that inhibit growth of both gram positive and gram negative bacteria. Keywords: macro fungi, Stereum ostrea, In-vitro antimicrobial activity, Minimum inhibitory concentration. INTRODUCTION Infectious diseases remain one of the major threats to human health. With the emergence and increase of microbial organisms resistant to multiple antibiotics, and the continuing emphasis on health care costs, more and more research are carried out to develop new, effective antimicrobial reagents free of microbial resistance and of low cost. Such problems and needs have led to resurgence in the use of natural products that may be linked to broad spectrum activity and far lower propensity to induce microbial resistance than the existing antibiotics and also cheaper (Jun et al., 2007). It had been known since Greek and Roman period that macro fungi were used as food and medicine. Therefore macro fungi may be explored as a source of new and useful bioactive compounds (Anke et al., 1980). Macro fungi need antibacterial and antifungal compounds to survive in their natural environment (Kim et al., 1999). Therefore, antimicrobial compounds could be isolated from many macro fungi species and could be of benefit for humans. As a matter of fact, macro fungi produce a large number of metabolites that show antibacterial, antifungal, antiviral, antitumor, hypoglycemic, antiallergic, immunomodulating,anti-inflammatory, hypolipidemic, and hepatoprotective activities (Hatvani, 2001). This was succeeded by the isolation and identification of pleuromutilin. That compound had served for the development of first commercial antibiotic of Basidiomycete origin (Mustafa and Fatma, 2006). Stereum ostrea a macrofungi belongs to family Stereaceae, and division Basidiomycota. It is inedible due to its

2 13 tough, leathery texture and is often called as False turkey tail as it mimics Trametes versicolor. Like the True turkey tail, Stereum ostrea has somewhat fuzzy cap that displays zones of brown and reddish brown colors. The Stereum ostrea is distinguished by its relatively large size and it tends to develop individual, sliced funnel-shaped fruit bodies, rather than laterally fused flat ones. It is saprophytic as it is found on the dead hard woods, growing in dense overlapping clusters and widely distributed in various parts of the world. This fungus has long been used in folk remedies even without any knowledge of which compounds are responsible for its activity. The ethnobotanical uses of this mushroom to heal both plant and human diseases have been accumulated but scientific evidences are not yet well known (Praveen et al., 2012). At present there is little information available on the antimicrobial activity of these species but some study is here focused:-recently, some new compounds such as a sesquiterpene, three aromatic compounds and a known compound methyl 2, 4-dihydroxy-6-methylbenzoate was isolated from a culture broth of the fungus Stereum sp. The novel sesquiterpene was determined to be stereumone and the three new aromatic compounds were elucidated together with the known compound. The combination of these compounds showed evident nematocidal activity against nematode Panagrellus redivivus (Li et al., 2006). The main focus of present study lies in the investigation of a white-rot fungus Stereum ostrea isolated from wood logs and their inhibitory activity against selected Gram-positive and Gram-negative bacteria. MATERIALS AND METHODS Chemicals used The chemicals used like ethanol, methanol, agar, peptone, beef extract, sodium chloride, sodium hydroxide, gentamycin sulphate, etc were of analytical grade. Microbial strains used Following four microbial strains were used for the antimicrobial activity studies. Gram + ve: Bacillus subtilis (MTCC 1789) and Staphylococcus aureus (MTCC 187) Gram ve: Escherichia coli(mtcc 1591) and Pseudomonas aeruginosa (MTCC 779) [MTCC: Microbial type culture collection] The microbial Strains were obtained from Institute Of microbial technology, Chandigarh, India in lyophilized form. The lyophilized microorganisms were cultured in suitable medium at 37ºC overnight for rejuvenation and preserved in slant culture under refrigeration for future use. METHODS Collection of Stereum ostrea The mushroom Stereum ostrea was collected from the Bateswar village of Salipur, district Cuttack, Odisha during the morning hour of First week of April The mushroom was authenticated by the taxonomist of Department of Botany, College of basic science and humanities, OUAT, Bhubaneswar and a specimen sample was preserved in the museum of Institute of Pharmacy and Technology, Salipur, Cuttack. After separation from the wood log, the mushroom was washed properly in sterile water to remove any dirt or other unwanted materials adhered to it, shade dried for fifteen days followed by oven dry at 50ºC for 24hours. After complete drying it was preserved in a dry container for future use. Preparation of different extracts of the fruit body of Stereum ostrea The dried fruit body of collected mushroom was grounded to powder by a mechanical grinder. The powdered materials (10 gm) were taken in the thimble of soxhlet extractor packed properly and covered with glass wool. 200ml of acetone and few porcelain pieces were added to the extraction chamber. The condenser was attached with a water source at the lower end and removed from the upper out let; water supply to the condenser was continuous till completion of extraction process. The soxhlet apparatus was fixed properly and heated using a heating mental at 50ºC till liquid present in the siphon was colourless. The soxhlet apparatus was dismantled. The extract present in the boiling flask was separated. The powder was removed from the thimble; air dried and once again packed in the thimble for

3 14 Table 1. Zone of inhibition of Gentamycin sulphate (50 µg /ml) against different bacteria. Zone of inhibition in mm Mean (mm)+/-sd / / / /-0.89 extraction using 200ml ethanol as solvent. After completion of extraction, the procedure was repeated using 200ml distilled water. The extracts were filtered, and the solvents were completely evaporated to dryness at 40ºC using a rotary vacuum evaporator and stored in freezer for future use with proper labeling (Prusty et al., 2008). Determination of Minimum Inhibitory Concentration An agar dilution assay was performed for determining minimum inhibitory concentration (MIC) of the three extracts. Tubes of 15 ml molten agar were prepared and maintained at 50ºC. A single concentration of extract was added to the agar in each test tube to obtain a range of final concentrations of 10, 20, 30, 40 and 50µg/ml. Different concentration of extracts were added to molten agar and poured to pre-sterilized petriplates and allowed to solidify. The test microorganisms were diluted in 0.1% (w/v) peptone water to get a concentration of 10 6 CFU/ml determined by UV-VIS spectrophotometer. The test microbial culture was added to the extract added petriplates and spreaded by a spreader. A control plate, without added extract, was prepared and inoculated to ensure adequate growth of the test microorganism. The plates were incubated at 37ºC for overnight. The MIC was determined as the lowest concentration that completely inhibited growth of microorganism after incubation (Ahmed et al., 2007). Assay for antibacterial activity The antibacterial activity of the extracts was determined by paper disc method (Norrel and Messley, 1997). The extracts were diluted to 50µg/ml with sterilized distilled water. The sterile filter paper (Whatman filter paper) discs of 6mm diameter were soaked with 50μl of the extract dilution and placed on bacteria seeded petirplates (10 6 CFU/ml) of solid nutrient agar. For positive control, Gentamycin sulphate in same concentration was used. The plates were first incubated at 4 o C for 12 hours to allow proper diffusion of the extract into the solid agar medium and then incubated at 37 o C for 24 hours. After incubation, the inhibition zone was observed and measured after marking at six different positions in the periphery of the zone of inhibition. Statistical Analysis The mean and standard deviation of six zone of inhibition reading were calculated by using Smith s statistical package (SSP) software. The diameters of zone of inhibition at different points were put in the first variable column. Then by going to statistical inference followed by one simple test of mean, the value of mean and standard deviation were determined. RESULTS AND DISCUSSION From the Zone of inhibition data for Gentamycin sulphate ( Table 1) it was observed that Escherichia coli was showing maximum zone of inhibition of 28.67mm+/-1.03, followed by Pseudomonas aeruginosa with 25.00mm +/-

4 15 Table 2. Zone of inhibition of acetone extract(50 µg /ml) against different bacteria. Zone of inhibition (mm) Mean (mm)+/-sd / / / /-1.51 Table 3. Zone of inhibition of ethanol extract(50 µg /ml) against different bacteria. Zone of inhibition in mm Mean (mm)+/-sd / / / /-1.05 Table 4. Zone of inhibition of aqueous extract (50 µg /ml) against different bacteria. Zone of inhibition in mm Mean (mm)+/-sd / / / / , Bacillus subtilis with mm+/ and Staphylococcus aureus with mm+/ In addition from the Zone of inhibition data for acetone extract (Table 2) it was observed that Bacillus subtilis was showing maximum zone of inhibition of 19.17mm+/-1.94, followed by Staphylococcus aureus with 16.83mm +/-1.94, Escherichia coli with 12.83mm +/-1.47 and Pseudomonas aeruginosa with 11.33mm +/ In contrary from the Zone of inhibition data for ethanol extract (Table 3) it was observed that Bacillus subtilis was having maximum zone of inhibition of mm+/- 1.75, followed by Staphylococcus aureus with 11.50mm+/-1.52, Escherichia coli with 9.67mm+/-1.03and Pseudomonas aeruginosa with 8.50mm+/ From the Zone of inhibition data for aqueous extract ( Table 4) it was observed that Bacillus subtilis was having maximum zone of inhibition of mm+/-0.75, followed by Staphylococcus aureus with 8.50 mm+/-1.05, Escherichia coli with 7.83mm+/-0.75 and Pseudomonas aeruginosa with 7.33mm+/ In (Table 5 and figure 1) all the mean zone of inhibition with standard deviation of fungal fruit bodies

5 16 Table 5. Mean and SD of Inhibition zone of Acetone, Methanol, Aqueous extracts and Gentamycin sulphateof fungal fruit bodies against bacterial pathogens in mm. Test Organism Zone of inhibition in mm (Mean+/-SD) Ethanol Aqueous Acetone extract extract extract (50 µg /ml) (50µg/ml) (50µg/ml) Gentamycin sulphate (50µg /ml) B. subtilis / / / /-0.55 S. aureus / / / /-0.89 E. coli / / / /-1.03 P. aeruginosa / / / /-0.89 Figure 1. Bar diagram showing zone of inhibition of different extracts and antibiotic against different Microorganism [1: B. subtilis, 2: S. aureus,3: E. coli,4: P. aeruginosa] against bacteria strains were shown. The observations of antimicrobial study are more likely to be due to the fact that gram negative bacteria possess an outer lipid membrane that acts as a barrier to many environmental substances including antibiotics (Jigna et al., 2005). Therefore it may be a reason that the mushroom extracts were more active against gram-positive microorganisms than gram-negative microorganisms. The minimum inhibitory concentration of different extract against different microorganism was shown in Table 6. The acetone extract had shown lowest MIC and aqueous extract had shown maximum MIC among the three extracts. Among the microorganisms Bacillus subtilis had shown lowest and Pseudomonas aeruginosa had shown highest MIC respectively. Bacillus subtilis had shown minimum MIC of 20 µg/ml for acetone extract and 30 and 40 µg /ml for ethanol and aqueous extract respectively. Staphylococcus aureus had shown minimum MIC of 30 µg /ml for acetone extract and 30 and 40 µg /ml for ethanol and aqueous extract respectively. Both acetone and ethanol extract were having same MIC. Escherichia coli had shown minimum MIC of 40 µg /ml for acetone extract and 40 and 50 µg/ml for ethanol and aqueous extract respectively. Both acetone and ethanol extract were having same MIC. Pseudomonas aeruginosa had shown minimum MIC of 40 µg/ml for acetone extract and 50 and 50 µg /ml for ethanol and aqueous extract respectively. Both aqueous and ethanol extract were having same MIC. In the present investigation, the results of detail activity of acetone, ethanol and aqueous extract of the isolated fruiting body of wood decaying mushroom Stereum ostrea along with the activity profile with standard commercial antibiotics (gentamycin) was presented in Table 5. the antibacterial activity of fruiting body of Stereum ostrea against two Gram positive bacteria (Baccilus subtilis and Staphylococus aureus) and two Gram negative bacteria( E.coli and P. aeruginosa) shown in Table 1,2,3,4. In general the result of the

6 17 Table 6. Minimum inhibitory concentration of different extracts of Stereum ostrea. Acetone extract (µg /ml) Ethanol extract (µg /ml) Aqueous extract (µg /ml) Organism B. subtilis (MTCC 1789) S. aureus (MTCC 187) E. coli (MTCC P. aeruginosa (MTCC 779) [+ve sign indicates growth, and ve sign indicates no growth] present study indicate that the antibacterial activities of different extract ( Acetone extract, ethanol extract and aqueous extract) were having maximum zone of inhibition of mm, mm and mm respectively against Bacillus subtilis and minimum zone of inhibition of mm, 8.50 mm and 7.33 mm respectively against P. aeruginosa (Table 1-4). It was observed that extract were more effective against Gram positive than Gram negative bacteria. In this investigation, the fungus mushroom Stereum ostrea showed significant invitro antimicrobial effect. The proportion of the isolates with antibacterial activities maximum in acetone extract, followed by ethanol and aqueous extract were mm, mm and mm respectively for Bacillus subtilis, mm, mm, 8.50mm for Staphylococus aureus and mm mm, 7.83 mm for Escherichia coli and mm, mm, 7.33 mm for Pseudomonas aeruginosa respectively for acetone, ethanol, aqueous extract which is comparable with those published earlier (Praveen et al., 2012). CONCLUSION Results obtained from the antimicrobial study by paper disc diffusion method in the present study revealed that different extracts of Stereum ostrea showed inhibitory effect against all the tested microbial strains but had more antimicrobial activity against gram positive as compared to gram negative strains. The zone of inhibition study also suggests that the extracts had shown antimicrobial activity in a concentration dependent manner against the test microorganisms and was comparable with the standard drugs. Further, from the antimicrobial study it was observed that the order of activity was in the sequence of Acetone extract > Ethanol extract > Aqueous extract. The results presented in this project are only based on different extract and did not specify any defined antibacterial substances. In present study the use of different solvent extract are capable to extract the various biochemical compound from stereum ostrea show various beneficial effects which was supported by a variety of literature. From previous studies in addition to the current it could be concluded that Stereum ostrea contain a potential metabolite and can be further utilized for its antimicrobial applications in agriculture, medicine and other area of interest. ACKNOWLEDGMENT The authors are grateful to the management and staff of the Institute of Pharmacy and Technology, Salipur and College of basic science, OUAT, Bhubaneswar for their support and generosity for carrying out the research. REFERENCES Jun SK., Eunye K., Kyeong NY., Jong-Ho K., Sung JP., Hu JL., So HK., Young KP., Yong HP., Cheol-Yong H., Yong- Kwon K., Yoon-Sik L., Dae HJ and Myung-Haing C (2007). Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine 3:

7 18 Anke T., Kupka J., Schramm G and Steglich W (1980). Antibiotics from basidiomycetes. X. Scorodonin, a new antibacterial and antifungal metabolite from Marasmiusscorononius. Journal of Antibiotics 33: Kim DH., Shim SB., Kim NJ and Jang IS (1999). β-glucuronidase-inhibitory activity and hepatoprotective effect of Ganodermalucidum. Biological Pharm Bulletin 22: Hatvani N (2001). Antibacterial effect of the culture fluid of Lentinusedodes mycelium grown in submerged liquid culture. International Journal of Antimicrobial Agents 17: Mustafa Y and Fatma B (2006). Antimicrobial Activities of Fruit Bodies and/or Mycelial Cultures of Some Mushroom Isolates. Pharmaceutical Biology 44: Praveen K., Usha KY., Naveen MB., Rajasekhar R(2012). Antibacterial Activity of a Mushroom - Stereum ostrea. Journal of Biology. Agriculture and Healthcare 2:1-5. Li GH., Li L., Duan M and Zhang KQ (2006). The chemical constituents of the fungus Stereum sp. hem & Biodiversity 3: Prusty AK., Ghosh T and Sahu SK (2008). Anthelmintic, antimicrobial and antipyretic activity of various extracts of Clerodendruminfortunatum Linn. Leaves. Oriental Pharmacy and Experimental Medicine 8: Ahmed I., Chandana J., Geon WL and Tae SL (2007). Antibacterial and Antifungal Activities of Stereum ostrea, an Inedible Wild Mushroom. Mycobiology 35: Norrel SA and Messley KE (1997). Microbiology Laboratory Manual Principles and Applications. Prentice Hall, Upper Saddle River, New Jersey. Jigna P., Rathish N and Sumitra C (2005). Preliminary screening of some folklore medicinal plants fromwestern India for potential antimicrobial activity. Indian J Pharmacol 37: