Antimicrobial Potential of Trichaptum Biforme and Bjerkandera Adusta from Pennsylvania, USA

The antimicrobial potential of Trichaptum biforme and Bjerkandera adusta extracts were evaluated on some selected bacteria and Candida albicans. Our results showed that aqueous, methanol and ethanol extracts of T. biforme and B. adusta exhibited no antimicrobial activity against Candida albicans. However, some extracts of B. adusta were very effective against three Gram-negative bacteria ( Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa ) and three Gram-positive bacteria ( Staphylococcus aureus, Staphylococcus epidermidis and Bacillus cereus ). Likewise, extracts of Trichaptum biforme were also effective against all three Gram-positive bacteria and only one Escherichia coli . The effectiveness of these two fungi as antibacterial agents should be well explored with development of optimized methods for mass production of these macrofungi in controlled environments. Keywords: Macrofungi; extracts; bacteria; explored; effectiveness; optimized method DOI: 10.7176/JNSR/11-16-01 Publication date: August 31 st 2020

The emergence of drug resistant microorganisms has attracted the attention of scientists into the exploration of antimicrobial drugs from fungi and plants. Antimicrobial drug resistance is one the biggest threats to human existence. Pathogenic microorganisms of humans and animals are rapidly evolving mechanisms of resistance against existing antimicrobial agents due to the abuse, overuse and misuse of antimicrobial agents. This study was therefore carried out to explore the potential use of extracts of T. biforme and B. adusta as antimicrobial agents against Gram-positive and Gram-negative bacteria, and the fungus, Candida albicans.

Methods and materials
Two test fungi used in this study were Trichaptum biforme and Bjerkandera adusta. The fruit bodies were collected from the forest adjacent to Ivory Nelson Center for the Sciences at Lincoln University, Pennsylvania (latitude 41°16'14.28"S and longitude 173°17'2.27"E). They were identified by the color of their sporocarps, spore print, types of pore, and other standard descriptions of Alexopolous et al. [17] Webster and Weber [21]. The fruit bodies were air-dried on the laboratory bench and oven dried to constant weight at 45 o C. The dried samples were pulverized in a Waring blender. Powdered samples were placed in sterile bottles and refrigerated at 4 o C until used.
Ground sample of Trichaptum biforme and Bjerkandera adusta were extracted using sterile distilled water, 95 % ethanol and 95 % methanol. Extraction was carried out by mixing 1.0 gram of each of the fungal samples with 10 ml of each of the three solvents in sterile plastic tubes. The tubes were placed in a gyrating incubator which was set at 150 rpm and 25 o C for 48 hrs. Fungal extracts were then obtained by centrifugation at 25 o C and 3,000 rpm for 20 minutes. The supernatant of each extract obtained after centrifugation was sterilized by passing through a 0.22 μm membrane filter. The fungal extracts were then dried at 45 o C before re-suspension with 5ml of the same solvent used for extraction to obtain a dilution of 1/5 (200 mg/ml). Fig. 2: Bjerkandera adusta The assay for antimicrobial activities was achieved using three Gram-positive bacteria; Staphylococcus aureus (85W1941), Staphylococcus epidermidis (85W1940) and Bacillus cereus (85W1815); and three Gramnegative bacteria, Escherichia coli (85W1860), Salmonella typhimurium (85W1956) and Pseudomonas aeruginosa (85W1903). The only fungus used for the study was Candida albicans (85W4150). These microorganisms were obtained from WARD'S Natural Science, Rochester, New York. USA (Latitude 43.1547222; Longitude -77.6158333).

Fig. 1: Trichaptum biforme
Overnight cultures of Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and Candida albicans were each suspended in tubes of sterile normal saline solution to a turbidity equivalent to 0.5 McFarland. Sterile cotton swabs were dipped in each suspension, and used to smear the surfaces of Mueller-Hinton agar until dry. The antimicrobial effects of aqueous, ethanol and methanol extracts of the fungal extracts were evaluated on each of the smeared microorganism using the agar well diffusion technique. For each organism smeared on Muller-Hinton agar, three wells were punched using sterile 6.0 mm cork-borer. The agar within the wells was carefully and aseptically removed, and 50 μL of the appropriate solvent (distilled water; 95 % ethanol; 95 % methanol) was pipetted into the center well to serve as control. Fifty microliters (50 μL) each of the extract of same solvent was also pipetted into the remaining wells. The plates were left on laboratory bench for one hour before incubation at 37 o C for 24 hours. Zones of inhibition were measured with a caliper and metric ruler.
Minimum bactericidal concentration of each extract was determined on the susceptible bacteria by doubling dilution (1/5 to 1/1028) of the extracts using same solvent of extraction and the agar well diffusion technique. Cultures of the test microorganisms: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa were each suspended in tubes of sterile saline solution to a turbidity of equivalent to 0.5 McFarland. Sterile cotton swabs were dipped in each suspension, and used to thoroughly smear the surfaces of Mueller-Hinton agar until dry. For each bacterial smear on Muller-Hinton agar, nine holes were punched using 6mm cork-borer. The agar within the holes were carefully and aseptically removed, and 50μl of the 1/5 dilution (10mg) was placed into the center well. The other extract dilutions of same solvent were pipetted into the remaining wells. The plates were allowed to sit at room temperature for one hour before incubation at 37 o C for 24 hours. Zones of inhibition were measured with a caliper and metric rule.

Results
Results of this study show that aqueous, methanol and ethanol extracts of Trichaptum biforme and Bjerkandera adusta exhibited no antimicrobial activities against Candida albicans. Some extracts of B. adusta were however effective against all three Gram-negative bacteria and three Gram-positive bacteria. Some extracts of Trichaptum biforme were also effective against all three Gram-positive bacteria and only one Gram-negative bacterium, Escherichia coli. Table 1 shows that the methanol, ethanol and aqueous extracts of B. adusta were effective against all the three Gram positive bacteria; Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, and one Gram negative bacterium, Escherichia coli. Pseudomonas aeruginosa was susceptible against methanol and ethanol extracts, but not the aqueous extract. On the other hand, Salmonella typhimurium was susceptible to the aqueous and methanol extracts of B. adusta, but was resistant to the ethanol extract.
The methanol extract of T. biforme was effective against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus and Escherichia coli. The ethanol extract was only effective against Bacillus cereus. Salmonella typhimurium and Pseudomonas aeruginosa were not susceptible to the aqueous, methanol and ethanol extract of T. biforme. Table 2 shows the minimum inhibitory concentration of aqueous, methanol and ethanol extracts of Trichaptum biforme and Bjerkandera adusta on Staphylococcus aureus. The results show that only the methanol extract of Trichaptum biforme was effective against Staphylococcus aureus with MBC of 0.0391 mg; the aqueous and ethanol extracts had no antibacterial effect against Staphylococcus aureus. On the other hand, the aqueous, methanol and ethanol extracts of Bjerkandera adusta showed antibacterial activity against Staphylococcus aureus. The MBC of the aqueous extract of was 0.313mg; that of the methanol and ethanol extracts was 0.0391. Table 3 Table 5 shows the results of the MBC of aqueous, methanol and ethanol extracts of T. biforme and B. adusta on E. coli. All three extracts of B. adusta were effective against Escherichia coli; however, the aqueous and ethanol extracts were more effective with MBC of 0.1563 mg; MBC of methanol extract was higher at 0.313mg.
On the other hand, aqueous and ethanol extracts of T. biforme had no antibacterial activity against E. coli. The methanol extract of T. biforme was effective with MBC of 0.0781mg. Table 6 shows the results of the MBC of aqueous, methanol and ethanol extracts of T. biforme and B. adusta on P. aeruginosa. None of the extracts of T. biforme had any antibacterial activity against P. aeruginosa. The aqueous extract of B. adusta also did not have any antibacterial activity against P. aeruginosa. Methanol and ethanol extracts of B. adusta were however effective against P. aeruginosa; ethanol extract was more effective with MBC of 0.313mg while the methanol extract was only effective at 10 mg. Table 7 shows the results of the MBC of aqueous, methanol and ethanol extracts of T. biforme and B. adusta on Salmonella typhimurium. The results show that none of the extracts of T. biforme was effective against Salmonella typhimurium. On the other hand, the aqueous and methanol extracts of B. adusta exhibited antibacterial activity against Salmonella typhimurium. The MBC for the aqueous extract was 2.5 mg while that of the methanol extract was 1.25 mg.

Discussion
Species composition and richness of some polyroid macrofungi have been studies in the US [22][23][24][25], and this group of fungi has generally been used as ecosystem indicators. However, therapeutic effects of many macrofungi have been well documented. The natural defensive mechanism of fungi against different pathogenic organisms can be associated with their capabilities in producing different antibiotics and accumulation of different phytochemicals in their tissues. These chemicals have been extracted by different scientists, many of which have proven to inhibit several viruses, bacteria or fungi pathogens with low or no cytotoxicity to human or other animal hosts.
Our results indicate that Candida albicans was not susceptible to the aqueous, ethanol and methanol extracts of Trichaptum biforme and Bjerkandera adusta. This implies that T. biforme and B. adusta extracts lack antifungal activity, but possess antibacterial activity. Similar resistance of Candida albicans to methanol extracts of Boletus lupinus, Flammulina velutypes, Phellinus igniarius, Sarcodon imbricatus, Tricholoma aurantium and Xerocomus ichnusanus have been reported by Nedelkoska et al. [27]. Another report from Nigeria also showed that Candida albicans was not susceptible to the methanol extracts of Fomes lignosus, Marasmius jodocodo, Pleurotus florida, Plurotus tuber-regium, Psathyrella atroumbonata, Termitomyces microcarpus and Termitomyces robustus [26].
In an in-vitro study, extracts of several polypore mushrooms were tested and 75 percent of them were confirmed to show antimicrobial activities against wide range of microorganisms [28]. Similar observation was made by Ranadive et al. [29], but reported that little has been reported among verse species of fungi that possess antimicrobial activities. Our results however, showed clear antibacterial effects of Trichaptum biforme and Bjerkandera adusta extracts. This is in agreement with the reports of other authors that pathogenic bacteria are generally more susceptible to mushrooms and other macrofungal extracts compared with fungi [6,10].

Conclusion
The nutritive effect of many mushrooms have been well identified, their therapeutic effect has also been identified for many years however, antimicrobial potentials of many polypore mushrooms have not been well documented and exploited. In this study, extracts of Trichaptum biforme and Bjerkandera adusta showed strong antibacterial effects against selected Gram positive and Gram negative bacteria. Further studies are planned on the appropriate utilization of these extracts as antimicrobial agents coupled with techniques for mass cultivation of these fungi in controlled environments.

Declaration of competing interest
Authors declare no conflict of interest on this manuscript