Evaluation of the Antibacterial Activity of Pleurotus spp. Cultivated on Different Agricultural Wastes in Chiro, Ethiopia

In the present study, mushrooms, Pleurotus ostreatus and Pleurotus florida, were cultivated on different agricultural wastes namely coffee straw (CS), pea straw (PS), Sorghum Grain Residue (SGR), and Wheat Grain (WG) for the evaluation of antibacterial activity. Antimicrobial activity evaluation was carried out against human pathogenic microorganisms, namely, Escherichia coli, Bacillus subtilis, Streptococcus faecalis, Pseudomonas aeruginosa, and Salmonella typhi by using the disc diffusion method. Methanolic extracts of P. ostreatus cultivated on a Sorghum grain residue substrate were recorded for the highest antibacterial activity against E. coli (19.8 mm) and P. aeruginosa (16.4 mm), and methanolic extracts of P. florida cultivated on a wheat grain substrate were recorded for the highest antibacterial activity against E. coli (18.6 mm) and S. faecalis (14.8 mm). Therefore, results suggested that P. ostreatus and P. florida cultivated on the coffee straw and Sorghum grain substrate were found with the highest antimicrobial activity in comparison to other substrates. The results supported that the methanolic extracts of P. ostreatus and P. florida might indeed be potential sources of antibacterial agents.


Background and Justification.
Mushrooms are a nutritious food source, being rich in protein, vitamins, and minerals. ey are also known to contain substances that enhance the immune system, fight infectious diseases, and lower blood pressure and cholesterol levels. Mushroom is being used as a valuable food source and traditional medicine around the world since ancient times, especially in China and Japan. Mushrooms are rich sources of bioactive compounds such as ß-glucan, proteoglucan, lectin, phenolic compounds, flavonoids, polysaccharides, triterpenoids, diatery fibre, lentinan, schizophyllan, lovastatin, pleuran, steroids, glycopeptides, terpenes, saponins, xanthones, coumarins, alkaloid, purin, purimidin, kinon, fenil propanoid, kalvasin, volvotoksin, flammutoksin, porisin, AHCC, maitake D-fraction, ribonucleas, and eryngeolysin. Pharmacological and nutritional aspects make mushroom as an important tool for the ailment of severe diseases such as microbial and viral infections, cancer, tumor, inflammation, cardiovascular diseases, and immunomodulating diseases [1].
Pleurotus spp. is promising as medicinal mushroom, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic, and immunomodulation activities [2]. e oyster mushroom may also be considered as medicinal mushroom for its hypocholesterolic property because it contains statins such as lovastatin which reduces cholesterol [3]. e oyster mushroom, Pleurotus spp., is widely cultivated on a wide range of substrates which are composed of lignin and cellulose. Cultivation of Pleurotus spp. supports a broad range of temperatures (15-30°C) on different ranges of substrates such as agro waste residues, weeds, and wastes after the production of food, feed, vitamins, enzymes, and a number of pharmaceuticals in addition to their waste degradation and detoxification properties [4,5]. e bioactive compounds present in Pleurotus spp. make it a medicinally important mushroom [4].
In the recent years, high scale usage of synthetic antibiotic has led to the emergence of multidrug resistance pathogens and is now posing a threat to the world. erefore, a search for natural plant-based antimicrobial agents is in need. is development is the consequence of the limited effectiveness of synthetic products to fight against newer and drug-resistant bacteria. For this purpose, the antimicrobial properties of many natural compounds from a wide variety of plant species have been assessed [6]. erefore, this study was conducted to evaluate the effect of different substrates on the antimicrobial activity of P. ostreatus and P. florida.
In Ethiopia, no studies have been conducted for the evaluation of the antibacterial activity of Pleurotus spp. cultivated on different agricultural wastes, and little is known of the biology and potential antibacterial sources of Pleurotus species grown on different agricultural waste in spite of its nutritional importance. Hence, due to this importance, attention needs to be given to the antibacterial agents which could serve as an input for efficiently managing bacterial disease. erefore, this study was undertaken with the objective of evaluation of the antibacterial activity of Pleurotus species grown on different agricultural wastes in the West Hararge Zone, in Chiro, Ethiopia.

Agricultural Wastes Utilized.
Coffee straw, Sorghum grain residue, pea straw, and wheat grain.

Spawn Collection.
e mother spawn of Pleurotus florida and Pleurotus ostreatus were collected which were cultivated and sold as edible mushrooms from Addis Ababa University, Ethiopia.

Spawn Preparation.
Spawn was prepared by using method of Bano and Shrivastava [7] with slight modifications. One kg of wheat grain was cooked for 40 min and, after that, washed in tap-water. Grain was drained and supplemented with 2 g lime and 8 g gypsum and mixed manually. en, grain was filled in poly propylene (PP) bags of 1 kg capacity and sterilized in an autoclave at 121oC for 15 min. After cooling, a PP bag was inoculated with freshly prepared mycelium (previously prepared PDA plate) and incubated at 25oC for two weeks in an incubator.

Preparation of the Mushroom Extract.
e present study was carried out to know the antimicrobial activity of Pleurotus spp. (P. ostreatus and P.florida) mushrooms cultivated on different agricultural wastes. Freshly harvested fruiting bodies from P. ostreatus and P. florida were shadedried and finely powdered. Twenty grams of the powder was extracted with 200 ml of 95% solvent methanol, ethanol, and aqueous separately using a soxhlet apparatus at 25°C at 150 rpm for 24 hours and filtered through Whatman no. 4 paper. e remaining extract was filtered and evaporated by vacuum distillation; the filtrate, thus, obtained was used as mushroom extract [8]. For the entire analysis, compounds of the extract were dissolved in dimethylsulfoxide (DMSO), and filter-sterilization was carried out through a 0.22-μm membrane filter. Extracts were kept in the dark at 4°C for not more than 1 week prior to use. e extraction was repeated twice.

Pathogenic Microorganisms Used for the Antimicrobial
Assays. In this experiment, five bacterial strains were used for antimicrobial assay. e preserved strains were obtained from the Addis Ababa University Microbiology Department. e pathogens were maintained on nutrient agar, Cezpek Dox agar, Mueller Hinton agar, and Mueller Hinton broth.

Antibacterial Assay.
e antibacterial activity of the methanolic extract of mushrooms was determined by the agar well diffusion method [9,10] with slight modification to suit the conditions of this experiment. Briefly, the methanol extracts were dissolved in 3% dimethylsulfoxide (DMSO) to a final concentration of 10 mg/ml and filter sterilized through a 0.45-μm membrane filter. Small wells (6 mm in diameter) were made in the agar plates by using a sterile cork borer. 20-100 microliters of the extract of each isolate of mushrooms was loaded into the different wells. An overnight culture of each microbial isolate was emulsified with nutrient broth to a turbidity that was equivalent to 0.5 McFarland (10 8 cfu/ml). In order to determine the antimicrobial efficacy of the fractions, an aliquot of the test culture (100 μl) was evenly spread over the surface of the solidified agar. Bacteria were cultured on Mueller Hinton agar. Streptomycin (20-100 μl/well) was used as positive control for test microorganisms. All the preloaded plates with the respective extract and test organism were incubated at 37°C, for 24 hours. After the incubation period, the zones of inhibitions were measured in millimeters [11].

Minimum Inhibitory Concentration (MIC).
e standard agar dilution protocol with doubling dilution was used. e extract was incorporated into nutrient agar at concentrations ranging from 200 µl to 1000 µl. A control without the extract was also prepared. 10 μL of each test organisms, previously diluted to 10 CFU/ml, was used to inoculate the plates.
ese were incubated at 37°C for 24 h in the first instance and for another 24 h before the growth was observed and recorded. e minimum inhibitory concentrations (MICs) of the extract for each test microorganism were considered the agar plate with the lowest concentrations without growth [12].

Data Entry and Analysis.
All data were analyzed and expressed as mean ± standard deviations of three separate determinations (n � 3). e statistical analysis was carried out by using SAS for Windows, version 20.0. One-way analysis of variance (ANOVA) and LSD comparisons were carried out to detect the significant difference (p < 0.05) between the mean values that had more than two groups.

Antibacterial Effects of Various Extracts against Pathogenic Microorganisms.
e antimicrobial activity of Pleurotus spp. (P. ostreatus and P. florida) was carried out against pathogenic microorganisms, namely, Escherichia coli, Bacillus subtilis, Streptococcus faecalis, Pseudomonas aeruginosa, and Salmonella typhi.

Effects on E. coli.
e methanolic, ethanolic, and aqueous extracts of mushroom species have shown antibacterial effects on the growth of the cultures of E. coli. Furthermore, the antibiotic effect of methanolic and ethanolic extracts of P. ostreatus and P. florida is more than that of the aqueous extracts of the two species. In addition, it was also found that their effects with all extracts were found more on the methanolic extract than the others. A higher effect on the methanolic extract than on the others may be due to the higher diffusion rate of the extract into the methanolic than in the others as the solid disc of +ve control has variation with these effects. e antibiotic effect of all extracts is not because of individual secondary metabolites but is the combined effect of all metabolites, as mentioned in Tables 1-3. e maximum activity recorded against E. coli. was 19.8 mm showed by the methanolic extract of P. ostreatus when cultivated on the Sorghum grain residue substrate and the minimum was 7.2 mm by the aqueous extract, as shown in Tables 1 and 3. e obtained results showed similarity with the findings of Menaga et al. [13]. In the present study, methanolic extracts of Pleurotus spp. showed the activity against E. coli.  Tables 2, 8-11. Mushrooms obtained from the coffee straw and Sorghum grain residue substrate were found with an excellent antimicrobial activity, whereas the mushrooms obtained from the pea straw and wheat grain substrate were recorded with a poor antimicrobial activity because of low production of bioactive compounds. Methanolic extracts of P. ostreatus from the Sorghum grain residue substrate gave best results against E. coli (19.8 mm), and methanolic extracts of P. florida from Sorghum grain gave best results against B. subtilis (15.5 mm) and streptococcus faecalis (14.8 mm).
On the other hand, aqueous extracts showed the antimicrobial activity against E. coli (7.2 mm-17.4 mm), B. subtilis (6.3 mm-14.7 mm), Streptococcus faecalis (6.2 mm-14.5 mm), Pseudomonas aeruginosa (7.1 mm-15.2 mm), and Salmonella typhi (4.6 mm-14.5 mm), as given in Tables 3, 12-15. Mushrooms obtained from the coffee straw and Sorghum grain substrate were found with an excellent antimicrobial activity, whereas the mushrooms obtained from the pea straw substrate were recorded with a poor antimicrobial activity because of low production of bioactive compounds. e results of Akyuz and Kirbag [18] on the antimicrobial activity of the methanolic extract of Pleurotus spp. against B. megaterium, E. coli, K. pneumonia, S. aureus, C. albicans, C. glabrata Epidermophyton spp., and Trichophyton spp. explained that petroleum ether and acetone extracts of P. ostreatus were found effective against Staphylococcus spp. Jagadish et al. [19] reported that the ethanol extract of P. florida and P. aureovillosus did not exhibit an antimicrobial effect against K. pneumoniae, P. vulgaris, P. aeruginosa, and C. albicans, but showed activity against S. aureus  [16] found that the inhibition zone ranging from 3.5 mm-20 mm was formed by the extract of P .ostreatus during an antimicrobial study. Its methanolic extracts gave the best results against E. coli (15.2 mm), and S. aureus (16.6 mm) was very close to the present study. Surekha et al. [17] reported the antimicrobial activity of P. ostreatus against pathogenic bacteria E. coli (15 mm), S. aureus (24 mm), and P. vulgaris (18 mm).
illaimaharani et al. [18] reported that the antibacterial activity of different extracts of P. Florida was tested against 8 human bacterial pathogens, namely, E. coli, S. typhi, K. pneumoniae, V. parahaemolyticus, K. oxytoca, P. murabilus, V. cholarae, and Streptococcus spp., and the antibacterial activity of the ethanol extract of P. florida was found maximum (23 mm) against Streptococcus spp. and minimum (4 mm) against V. parahaemolyticus. e antimicrobial activity against E. coli was found to be 11 mm. Akyuz and Kirbag [18] reported the same results for the ethanol extract of P. eryngii that it showed the maximum antifungal activity against C. albicans (7.7 mm), C. glabrata (7.7-9.3 mm), Epidermopyton sp. (7.7-8 mm), and Trichophyton spp. (7.7-8.7 mm).
A previous study of Menaga et al. [13] on the antimicrobial activity of the ethanolic extract of P. florida exhibited the highest activity against Pseudomonas spp. and Campylobacter spp., whereas the methanol extract showed higher activity against the E. coli, Salmonella typhi, Staphylococcus aureus, Camphylobacter sp., and Vibrio sp. aqueous extract, also revealing a high zone formation against Vibrio sp. of 24 mm. Menaga et al. [13] concluded that the methanol extract showed an activity against E. coli (21 mm), Salmonella typhi (20 mm), Staphylococcus aureus (20 mm), International Journal of Microbiology          International Journal of Microbiology      (20 mm). In a previous study, Jonathan [5] reported that the sporophore methanolic extract of Pleurotus florida showed an activity in E. coli (13 mm) and Klebsiella spp. (20 mm) and no activity against Bacillus spp., Pseudomonas, and Proteus spp. (30 mm). Menaga et al. [13] reported the zone formation in Pseudomonas spp., (20 mm), Salmonella spp., (20 mm), and Klebsiella pneumonae (13 mm), whereas the ethanolic extract of mycelium showed zone formation in Staphylococcus aureus (16 mm), Streptococcus mutans (14 mm), Escherichia coli (12 mm), Micrococcus luteus (16 mm), and Bacillus subtilis (9 mm) and no zone formation against Pseudomonas aeruginosa, Salmonella abony, Klebsiella pneumoniae, Proteus vulgaris, and Candida albicans.

Effects on Bacillus subtilis.
e methanolic, ethanolic, and aqueous extracts of mushroom species have shown antibacterial effects on the growth of the cultures of B. subtilis.
e maximum activity was recorded against B. subtilis with 15.5 mm showed by the methanolic extract of P. florida when cultivated on the SGR substrate, and the minimum was 6.3 mm by the aqueous extract, as shown in Tables 4 and 12. Furthermore, the antibacterial effect of methanolic and ethanolic extracts of P. ostreatus and P. florida is more than that of aqueous extracts of the two species. In addition, it was also found that their effects with all extracts were found more on the methanolic extract than the others. A higher effect on the methanolic extract than on the others may be due to a higher diffusion rate of the extract into the methanolic than in the others, as shown in Tables 4,  8, and 12. e obtained results showed similarity with the findings of Menaga et al. [13]. In the present study, methanolic extracts of Pleurotus spp. showed the activity against B. subtilis (6.3 mm-15.5 mm). Coffee straw and Sorghum grain residue substrates provide the highest zone of inhibition, and aqueous extracts have showed the lowest zone of inhibition.

Effects on Streptococcus faecalis.
e highest zone of inhibition was recorded in methanolic extracts of both Pleurotus species at the coffee straw and Sorghum grain residue (14.8 mm) in both species. e least zone of inhibition was recorded in aqueous extracts of pea straw (6.9 mm) of P. Florida species (Tables 5 and 13). is result was in agreement with the research conducted in [19].

Effects on Pseudomonas aeruginosa. All mushroom extracts have shown an antibacterial effect with the cultures of
Pseudomonas aeruginosa on all substrates. Furthermore, the antibiotic effect of the coffee straw and Sorghum grain residue extract is more than that of the extracts of pea straw. In addition, it was also found that the zone of growth inhibition was found more on the coffee straw and Sorghum grain residue (Tables 6, 10, and 14). us, selected mushroom species are a potentially good source of traditional medicines and therapeutics. e presence of bioactive compounds such as alkaloids, steroids, flavonoids, tannins, saponins, phenols, and phlobotannins in mushroom species can be used for the treatment of different ailments such as malaria, diarrhoea, skin burn, and antimicrobial agents. e antimicrobial activity showed the highest zone of growth inhibition by the coffee straw extract of P. ostreatus (16.4 mm) on Pseudomonas aeruginosa. It should be pointed out that the majority of the extracts present an antimicrobial activity against the tested bacterial species, which is in agreement with the results previously reported by our research group concerning E. coli, K. pneumonia, and P. aeruginosa [20], even when higher extract concentrations were used [21]. Nevertheless, a recent study describes methanolic extracts obtained from mushroom as having high antibacterial activity against E. coli, assessed by the disc diffusion method [22]. e extraction solvent or even the mushrooms' origin, as well as the bacterial strain, could explain the differences in the antimicrobial activity reported by different authors for the same species.
3.6. Effects on Salmonella typhi. Pea straw methanolic, ethanolic, and aqueous extracts of both mushroom species have showed the lowest antimicrobial effects on Salmonella typhi . e highest antimicrobial effect was recorded in methanolic extracts of P. ostreatus cultivated on the coffee straw substrate (16.9 mm, as shown on Table 7), while the lowest antimicrobial effect was noted in ethanolic extracts of pea straw (3.2 mm, as indicated in Table 11). e present study coincides with the work of Menaga et al. [13] which concluded that the methanol extract showed activity against E. coli In the aqueous extract of Pleurotus spp., the highest effect on Salmonella typhi was recorded by using coffee straw in both species (14.5 mm), and the lowest antibacterial effect was observed in P. ostreatus by using pea straw (4.6 mm) (Table 15).

Antibacterial Assay and MIC.
e present study has revealed the antibacterial activity of the mushroom extract. e results of the antibacterial activity are presented in Table 16. All the mushrooms used in this study were found to exhibit various degrees of antimicrobial effects against the tested microorganisms. e zone of inhibition exhibited more than 10 millimeters was considered as highly active for extracts. P. ostreatus has a broad-spectrum antibacterial and antifungal activity. Similar antimicrobial potentials have been observed in the culture extracts of Irpex lacteus [23] and Agrocybe sp. [24,25]. e best in vitro antibacterial activity was shown by P. florida (17 ± 0.2 mm) followed by Pleurotus ostreatus, and against Esherichia coli, Pleurotus florida also showed best antimicrobial activity than Pleurotus ostreatus.
is antimicrobial efficacy of Pleurotus florida and L. edodes were similar to that in the study of Kuznetsov et al. [26]. Ishikawa et al. [27] reported data similar to the results obtained in this work, showing the antibacterial action of Pleurotus florida against B. cereus, S. aureus, and E. coli. Komemushi et al. [28] also reported that L. edodes and P. florida inhibited the growth of Gram-positive and Gramnegative bacteria. e revealed information regarding the strong antimicrobial activity of P. ostreatus, P. florida, and Hypsizigus tessulatus against pathogenic isolates has similarity to that in [29]. Pseudomonas aeruginosa showed more resistant to mushroom extracts comparatively than other isolates.
Among the bacterial isolates, the MIC of all mushroom extracts was high for Pseudomonas aeruginosa. e MICs of P. florida against all isolates were comparatively lower than those of P .ostreatus.
e Lowest MIC was observed for Salmonella typhi, Streptococcus faecalis, Esherichia coli, and P. florida. is quite similar result was also observed by [21]. All the mushrooms also revealed an antimicrobial activity showing different MICs for each microorganism (Table 17).

Conclusions.
e results of the study showed that the mushroom extracts indicate the presence of a potent antibacterial activity, which confirms its use against microbial pathogens including antibiotic-resistant bacteria. e results of these extracts for their antibacterial activities against 5 bacterial species showed high degrees of variation between the extraction solvents used and the species of test microorganisms. e extracts from the aqueous solvent had the lowest activity, indicating the possibility that the active antimicrobial components may not be of secretive nature. e results clearly indicated that the methanolic extracts from fruiting bodies had the widest spectrum and highest growth inhibitory effect against the test organism. e most sensitive bacterial species to this methanolic extract were E. coli (19. is result is in accordance with the fact that the methanolic extracts of fruiting bodies from both Pleurotus ostreatus and Pleurotus florida contained antibacterial compounds against certain fungal and bacterial pathogens [29]. e result obtained from the study is expected to show the antibacterial activity of Pleurotus species and which Pleurotus species has a high content of antibacterial activity so that they can be used to treat several bacterial diseases. So, the Pleurotus species are employed as an alternative source of medicine to mitigate the diseases associated with the microorganism.
In general, the resent finding encourages their use in human diets, which, in turn, might serve as protective agents to microbial diseases. Further researches are needed to be conducted in order to evaluate the antibacterial activity of other pathogenic bacterial species.

Recommendation.
Out of selected mushroom species used for this study, all of them showed high antibacterial activity against the test pathogen, but there is limited scientific evidence on those mushroom species. So, further study is necessary to prove their potency. Further investigation is also needed to evaluate the effect of these mushrooms on a wide range of pathogenic bacteria.

Data Availability
e dataset used to support the findings of this study is available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.