Antimicrobial Activity of Pleurotus squarrosulus on Clinical Pathogenic Bacteria and Fungi

Aim: To evaluate the antimicrobial activities of Pleurotus squarrosulus mushroom extracts on bacterial and fungal isolates. Study Design: Pleurotus squarrosulus was obtained from different sources in Umuahia North Local Government, Abia state, Nigeria and identified in the Department of botany, University of Nigeria, Nsukka. Place and Duration of Study: Antimicrobial activities of Pleurotus squarrosulus was carried out in the department of microbiology between January 2016 and August 2016 Methodology: Pleurotus squarrosulus was extracted using ethanol, methanol and aqueous. Antimicrobial susceptibility tests were carried out by agar disc diffusion technique using National Committee of Clinical Laboratory Standard. Qualilative phytochemical analysis was carried out using standard methods. Results: Methanol, ethanol and aqueous extracts of Pleurotus squarrosulus were tested against E. coli, B. cereus, S. aureus, P. aeruginosa, C. albicans and C. glabrata. The different test microorganisms showed varied susceptibility to the test extracts. All the test organisms were Original Research Article Kalu and Kenneth; JAMB, 4(3): 1-9, 2017; Article no.JAMB.34644 2 inhibited by methanol, ethanol and aqueous extract at varied concentrations ranging between 500 mg/ml and 125 mg/ml. Statistically, inhibition of the antibacterial and antifungal control for the test organisms were significantly higher (P < 0.05) than that of the extracts. The phytochemical analysis revealed the presence of saponin, carbohydrates, tannins, flavonoids and proteins in all the extracts while glycoside and alkaloids, were found in some. Conclusion: The finding of this result suggest that Pleurotus squarrosulus possess broad-spectrum antimicrobial activity. The potential of developing antimicrobials from plants appear rewarding.


INTRODUCTION
Pleurotus species is one of the choice edible mushrooms which can be cultivated in many countries in the subtropical and temperate zones. Generally Pleurotus is referred to as "oyster mushroom" over the world while in China it is known as Abalone mushroom" and "Dhingri" in India. Pleurotus species have been used by the people in all over the world for their nutritional value, medicinal properties and other beneficial effects [1].
Oyster mushrooms are easy to grow and process and do not need huge investment. Mushroom farming is being practiced in more than 100 countries and its production is increasing at the rate of 7 per cent per annum. Production of mushroom has already crossed 5 million metric tons annually in the world and is expected to reach around 7 million metric ton in next ten years. India had been known world over for its exotic mushrooms and total mushroom production in India was 48,000.00 tons in 2005. Oyster mushroom cultivation has increased during the last decade [2].
Mushrooms have been used as food supplement from times immemorial not only for their flavor, aroma and nutritive values but also for their medicinal properties [3][4][5]. Wild mushroom holds a variety of bioactive compounds that have made it possible to be used as an impending source for the improvement of medicine and nutraceuticals [6]. These bioactive compounds have been employed as immune-modulator, anti-fibrotic, anti-inflammatory, anti-diabetic, anti-viral, antioxidant and antimicrobial agents [7]. Besides, mushroom has been used extensively in traditional medicine for curing of various types of diseases [8][9][10]. For centuries, mushrooms have been prescribed for treatment of diseases such as gastro-intestinal disorder, bleeding, high blood pressure and various bacterial infections [11]. While some of the medicinal values associated with mushroom must have arisen from surperstitious beliefs and myths, they have provided information for curiosity research studies. Research has shown that some of these claims are not mere myth but are authentic [12,13]. Besides medicinal and nutritional use, mushroom can be used as natural dyes for fabrics [14].

Collection and Identification of Materials
Pleurotus squarrosulus was collected from different sources of Umuahia North Local Government area, Abia state and identified by a botanist in the Department of botany, University of Nigeria, Nsukka.

Sample Preparation and Extraction
Fresh Pleurotus squarrosulus mushrooms were thoroughly washed with distilled water, cut into pieces, air-dried at room temperature and pulverized using manual grinder. Fifty grams of each of the ground samples was soaked in 500 ml ethanol, cold water, and methanol for 24 hours with intermittent shaking. Each sample was filtered using Whatman №1 filter paper. The filtrate was dried with a rotary evaporator in order to obtain the extract which was scooped and poured into well-labeled sample bottles and stored at 4ºC [15].

Determination of Antimicrobial Activity of Mushroom Extracts
Antimicrobial activity of mushroom extracts was determined according to the National Committee of Clinical Laboratory Standards [16]. Agar disc diffusion method on SDA and Muller-Hinton agar were used for fungi and bacteria respectively. A micropipette was used to introduce 100 µL of the inoculum onto the agar plate, and spread with glass rod spreader under sterile conditions. The paper discs of 6 mm diameter soaked in 10 µL of different concentrations of the extracts (500, 250, 125, 62.5, 31.25, 15.63 and 7.81 mg/mL) was applied on the agar plate. Similarly, for control plates, paper discs of 6 mm with dilute dimethylsulfoxide were used as negative control and antibiotics discs of tetracycline (10 µg/mL) and ampicillin (10 µg/mL) were used for Gram negative bacteria isolates, oxacillin (5 µg/mL) was used for Gram positive bacteria isolates whereas antifungi disc of nystatin (20 µg/mL) oxoid disc was used as positive control.
This procedure was carried out in triplicate for the entire test organisms and allowed to stand for 30 min on the bench after which they were incubated for 24 h at 37 ± 2ºC for bacteria and 72 at 28 ± 2ºC for yeast. After incubation, the inhibition zone diameters produced by the different concentrations of the crude extracts were measured (in millimeter) using transparent meter rule.

Statistical Analysis
Experimental values were given as means ± standard deviation (SD). Statistical significance of data were analyzed at P ≤ 0.05 (Independent-Samples T Test) using statistical package for social sciences (SPSS, Armonk, NY, USA) version 20.

RESULTS AND DISCUSSION
Natural products not only provide valuable components but also an important source of bioactive compounds that provide lead information for developing useful synthetic compounds. Mushrooms contain a large number of biologically active components that impart health benefits and protection against degenerative diseases. They have been traditionally used in all over world for treatment of variety of chronic disease. Antimicrobial activity of the crude extract of Pluerotus squarrosulus as well as phytochemical characteristics were studied. Table 1 Table 3 shows the phytochemical analysis that revealed the presence of bioactive compounds which were present at varying levels. Saponins, protein and carbohydrate were detected in all the extracts while glycosides, alkaloids, tannins and flavonoids were found in some. Fig. 1 shows the antimicrobial activity of Pleurotus squarrosulus methanol extract on the test organisms. The mean inhibition zone diameter varied directly with increase in extract concentration. E.coli was inhibited at different concentration of 500, 250 and 125 mg/ml, P. aeruginosa and B. cereus were inhibited at different concentration ranging from 500 mg/ml to 62.5 mg/ml, also S. aureus was inhibited at different concentrations ranging from 500 mg/ml to 31.25 mg/ml and C. albicans were inhibited at different concentrations of 500 mg/ml to 31.25 mg/ml whereas C. glabrata was not inhibited by the extract even at the highest concentration of 500 mg/ml. However, inhibition of the antibacterial and antifungal control for the test organisms were significantly higher (p < 0.05) than that of the extract.  inhibited at concentrations of 500 mg/ml and 250 mg/ml. However, inhibition of the antibacterial and antifungal control for the test organisms were significantly higher (p < 0.05) than that of the extract. albicans and C. glabrata were well inhibited by the extract at concentrations ranging from 500 to 125 mg/ml. E. coli and P. aeruginosa were not inhibited even at the highest concentration of 500 mg/ml. However, inhibition of the antibacterial and antifungal control for the test organisms were significantly higher (p < 0.05) than that of the extract. The results indicated that extracts from mushroom has similar antimicrobial properties as reported by Nwachukwu and Uzoeto [15]. The sensitivity of isolates to the mushroom extracts implies that intrinsic substance in the extracts is unknown to the microorganisms which made it impossible for them to resist. The variations in the antimicrobial activities of Pleurotus squarrosulus extracts may be due to the differences in their bioactive compositions or concentrations, methods of extraction and mechanism of action of active ingredients [17]. The results of the present study strengthened the outcomes of earlier works done by others that showed mushrooms produced a great variety of antimicrobial agents. For instance, it is known that the extract from fruit bodies of several Lactarius sp. [18,19]; Fomitopsis sp. [20]; Boletus sp. [21]; Cortinarius sp. [22]; Ganoderma lucidum, Navesporus floccosa and Phellinus rimosus [23]; Pleurotus tuber-regium [ [25]; Lactarius deliciosus, Sarcodon imbricatus and Tricholoma portentosum [26]; Russula delica [27]; Pleurotus eryngii var. ferulae [28]; Infundibulicybe geotropa, Lactarius controversus, Lactarius delicious and Phellinus hartigii [29]; Lactarius indigo [30] and Stereum

CONCLUSION
This research has further illuminated the medicinal value of Pleurotus squarrosulus found in Umuahia North Local Government, Abia State Nigeria. From the present study, the sensitivity of isolates to the mushroom extracts implies that intrinsic substance in the extracts is unknown to the microrganisms, which made it impossible for them to resist.