Antimicrobial Activities and Time-Kill Kinetics of Extracts of Selected Ghanaian Mushrooms

The rapid rise of antimicrobial resistance is a worldwide problem. This has necessitated the need to search for new antimicrobial agents. Mushrooms are rich sources of potential antimicrobial agents. This study investigated the antimicrobial properties of methanol extracts of Trametes gibbosa, Trametes elegans, Schizophyllum commune, and Volvariella volvacea. Agar well diffusion, broth microdilution, and time-kill kinetic assays were used to determine the antimicrobial activity of the extracts against selected test organisms. Preliminary mycochemical screening revealed the presence of tannins, flavonoids, triterpenoids, anthraquinones, and alkaloids in the extracts. Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea showed mean zone of growth inhibition of 10.00 ± 0.0 to 21.50 ± 0.84, 10.00 ± 0.0 to 22.00 ± 1.10, 9.00 ± 0.63 to 21.83 ± 1.17, and 12.00 ± 0.0 to 21.17 ± 1.00 mm, respectively. The minimum inhibitory concentration of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea ranged from 4.0 to 20, 6.0 to 30.0, 8.0 to 10.0, and 6.0 to 20.0 mg/mL, respectively. Time-kill kinetics studies showed that the extracts possess bacteriostatic action. Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea exhibited antimicrobial activity and may contain bioactive compounds which may serve as potential antibacterial and antifungal agents.


Introduction
Infectious diseases pose serious threats to the existence, health, and survival of mankind [1]. The World Health Organization (WHO) survey in 2008 indicated that infectious diseases caused 32% of deaths worldwide with 68% of the deaths occurring in Africa [2]. Infectious diseases still account for a great proportion of death globally and in some regions remain the most important cause of ill health [2]. Apart from affecting the health of individuals directly, infectious diseases have heavy impact on whole societies and economies [3]. The discovery of penicillin and subsequent development and synthesis of other antibiotics had been a milestone in the history of medicine. However, this medical breakthrough is being lost to the development and rapid spread of bacterial resistance to antimicrobial agents [4].
Globally, the emergence of antimicrobial resistant bacterial strains [5] is increasingly limiting the potency of current drugs and significantly causing failure of treating infections [6]. This situation shows that the potencies of prevalent antibiotics are decreasing steadily [7]. Therefore, there is a great need to develop novel drugs to combat pathogenic microorganisms that have developed widespread microbial resistance to antibiotics [8]. Since multidrug resistance of microorganisms is a major medical concern, screening of natural products in search for new antimicrobial agents is the need of the hour [9]. The use of natural products has been extremely successful in the discovery of new medicine, and mushrooms could be a source of natural antimicrobials [10].
Reports indicate that mushrooms contain many biologically active components that offer health benefits and protection against diseases [11] and are responsible for their antitumor [12], anti-inflammatory [13], antioxidant [14], and antimicrobial activities [15]. Mushrooms have been reported in several studies to be one of the most promising sources for obtaining natural compounds that can act as anti-infective agents. Some mushrooms, including Pleurotus ostreatus [16,17], Ganoderma lucidum [18], Russula delica [19], Phellinus hartigii [20], and Stereum ostrea [21], have been reported to exhibit wide range of antimicrobial activity against different Gram-positive, Gram-negative bacteria, and also fungi. However, the antimicrobial properties of some mushrooms have 2 Evidence-Based Complementary and Alternative Medicine not been studied, while others have limited data as far as their antimicrobial activity is concerned. This has necessitated the need for the continuous screening of mushrooms for their antimicrobial activities.

Preparation of Methanol Extracts.
The fruit bodies of the mushrooms were dried in an oven (Gallenkamp, London, UK) at 30 ∘ C for two hours to a constant weight. The various mushroom samples were ground to fine powder using a lab mill machine (Christy and Norris, Chelmsford, UK). Two hundred grams of each sample was weighed and successively extracted with 1 L each of 70% v/v methanol. The mixture was allowed to stand at room temperature (28 to 30 ∘ C) for three days with frequent agitation and homogenized and the supernatant was filtered using Whatman filter paper (number 10) (Sigma-Aldrich, Michigan, USA). The filtrates were concentrated in a rotary evaporator (Rotavapor BÜCHI R-200 with heating bath B-490, Büchi, Konstanz, Germany) at 40 ∘ C under reduced pressure and lyophilized. The yield of the extract, related to the dried powdered mushroom materials of T. gibbosa, T. elegans, S. commune, and V. volvacea, was 28, 25, 20, and 13% w/w, respectively. The extracts were kept in air tight containers, labelled, and stored in a desiccator until required for use.

Mycochemical
Screening. The methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea were screened to detect the presence or otherwise of secondary metabolites such as tannins, flavonoids, triterpenoids, and alkaloids, following standard procedures [43,44].

Thin-Layer Chromatography (TLC) of Methanol Extracts.
The methanol extracts of V. volvacea, T. gibbosa, T. elegans, and S. commune were investigated using TLC described by Marica et al. [45]. Three hundred milligrams (300 mg) of each extract was dissolved in 2 mL chloroform in a beaker and applied as spots with the aid of capillary tubes on a silicagel coated plate (Qingdao Marine Chemical Plant, Qingdao, China) about 1 cm from the base. The spotted plates were run in a developed solvent system of 100% chloroform. The developed plate was observed under ultra-violet light using both short and long wavelengths (254 and 365 nm) and then sprayed with anisaldehyde (Sigma-Aldrich, London, UK) in order to reveal compounds present in the extracts. Distances between the spots were measured and the retention factor ( ) values were calculated, using the following:

Determination of Antimicrobial Activity Using the Agar
Well Diffusion Method. The antimicrobial activity was evaluated using the agar well diffusion method described by Agyare et al. [46]. Twenty millilitres of sterile nutrient agar and potato dextrose agar stabilized at 45 ∘ C for 15 minutes were seeded with 100 L of 1 × 10 6 colony forming units (CFU)/mL of test bacteria and fungus, respectively, and aseptically poured into a sterile Petri dish and allowed to set. Four wells (8 mm) equidistant from each other were created with a sterile cork borer (number 4). The wells were filled with 200 L of 7.5, 10, 20, and 30 mg/mL of methanol extracts of the mushrooms dissolved in sterile distilled water. The plate was made to stand on the bench for 30 minutes to 1 h to allow diffusion of the extract. The zones of growth inhibition (including diameter of well) were measured after 24 h of incubation at 37 ∘ C for bacteria and 72 h after incubation at 28 ∘ C for the fungus. Ciprofloxacin and ketoconazole (Sigma-Aldrich, Michigan, USA) were used as reference antimicrobial agents against test bacteria and fungus, respectively. The procedure was performed in independent triplicates and the mean zones of growth inhibition were determined.

Determination of Minimum Inhibitory Concentration (MIC).
The minimum inhibitory concentrations (MICs) of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea against the test organisms were determined using the broth microdilution method described by Agyare et al. [46]. Each well of a 96-well microtiter plate was filled with 100 L double strength nutrient broth. Stock solutions of extracts within the range of 0.25 to 50 mg/mL were prepared and 20 L of inoculum (1 × 10 6 CFU/mL) was added to each well. The plate was incubated at 37 ∘ C for 24 h. After incubation, 20 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1.25 mg/mL) was added to each well. The plate was incubated again at 37 ∘ C and growth was observed as purple coloration, while clear/yellow coloration indicated no growth after 30 min of incubation. The MIC was determined as the lowest concentration which showed no visible growth upon the addition of MTT. Ketoconazole at concentration ranging from 1.0 to 10.0 g/mL and ciprofloxacin at concentration range of 0.01 to 1.0 g/mL were used as reference standards. The procedure was performed in independent triplicates to validate the results.

Determination of Minimum Bactericidal and Fungicidal
Concentrations. The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea against the test organisms were determined using the method described by Cos et al. [47]. Each well of a 96well microtiter plate was filled with 100 L double strength nutrient broth. A specified volume of the stock solution was added to each well to obtain a twofold serial dilution of the extract within the range of 1 to 50 mg/mL. A volume of 20 L of 1.0×10 6 CFU/mL of the test organism was added to appropriately labelled wells and incubated at 37 ∘ C. After 24 h after incubation, aliquots (10 L) were taken from each well and inoculated into freshly prepared 200 L nutrient broth and incubated at 37 ∘ C for 24 h. Twenty microliters (20 L) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added after incubation and growth was observed as purple coloration, while clear/yellow coloration indicated no growth. The MBC or MFC was determined as the least concentration of the extract that exhibited no bacterial/fungal growth upon the addition of 20 L of MTT (1.25 mg/mL).
The procedure was carried out in independent triplicates to validate the results.

Time-Kill Kinetics Assay.
Time-kill kinetics of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea was carried out following the procedure described by Tsuji et al. [48]. Concentrations equal to MIC, twice the MIC, and four times the MIC of the extracts were prepared. An inoculum size of 1.0 × 10 6 CFU/mL was added and incubated at 37 ∘ C. Aliquots of 1.0 mL of the medium were taken at time intervals of 0, 1, 2, 3, 4, 5, 6, 12, and 24 h, for bacteria, and 0, 6, 12, 30, 36, 48, 54, and 72 h for fungi and inoculated aseptically into 20 mL nutrient agar and incubated at 37 ∘ C for 24 h. A control test was performed for the organisms without the extracts or reference antibiotic. The colony forming unit (CFU) of the organisms was determined. The procedure was performed in triplicate (three independent experiments) and a graph of the log CFU/mL was plotted against time.
2.11. Statistical Analysis. Graph Pad Prism Version 7.0 for Windows (Graph Pad Software Inc., San Diego, CA, USA) was used to analyze data obtained from study by using oneway ANOVA followed by Dunnett's post hoc test.

Mycochemical Analysis.
The methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea were found to contain secondary metabolites such as tannins, flavonoids, triterpenoids, glycosides, and alkaloids. However, coumarins were absent in all four mushroom extracts, and anthraquinones were present in T. gibbosa, T. elegans, and S. commune but absent in V. volvacea, while saponins were absent in T. gibbosa, T. elegans, and S. commune but present in V. volvacea (Table 1).

TLC of Methanol Extracts.
The values of the identified bands that eluted from T. gibbosa, T. elegans, S. commune, and V. volvacea spots on the TLC plates were calculated ( Figure 1). T. gibbosa, T. elegans, and S. commune extract each resulted in ten (10) bands that eluted from the spot, while seven (7) bands eluted from V. volvacea extract. However, all four extracts had some bands that were similar as they had the same characteristic fluorescence at both 254 nm and 365 nm ( Figure 1).      Evidence-Based Complementary and Alternative Medicine     followed by a gradual rise up to the 24th h for E. coli and S. aureus and 72nd h for C. albicans when compared to the control ( Figure 4). The time-kill kinetics profile of extract of V. volvacea against the test organisms; E. coli, S. aureus, and C. albicans at test concentrations showed reduction in number of viable cells over the first 6, 8, and 42 hours, respectively, followed by a gradual rise up to the 24th h for E. coli and S. aureus and 72nd h for C. albicans when compared to the control ( Figure 5). The course of antimicrobial action was however observed to be bacteriostatic and concentration dependent for extracts of all four mushrooms (T. elegans, T. gibbosa, S. commune, and V. volvacea) studied. The area under the curve (AUC) for T. gibbosa, T. elegans, S. commune, and V. volvacea against E. coli, S. aureus, and C. albicans at concentrations studied revealed that the number of cells was significantly ( < 0.0001) reduced when compared to the control ( Figures  2-5).

Discussion
Research into the pharmacological activities of mushrooms especially their antimicrobial activity has attracted attention recently. The methanol extracts of T. gibbosa, T. elegans, and S. commune and V. volvacea were found to contain secondary metabolites such as tannins, flavonoids, triterpenoids, glycosides, and alkaloids. However, the absence of anthraquinones in T. gibbosa, T. elegans, and S. commune extracts and the presence of saponins in V. volvacea extract (Table 1) may be due to the differences in substrates from which the various samples were collected [49,50]. The presence of saponins, tannins, terpenoids, flavonoids, and alkaloids in T. gibbosa is similar to the report by Mbayo et al. [27] that indicated the presence of tannins, terpenoids, anthocyanins, flavonoids, and alkaloids in T. gibbosa. The presence of phenolic compounds in T. elegans has been reported by Awala and Oyetayo [14]. The presence of flavonoid in S. commune is reported to be responsible for its antimicrobial activity [51].
TLC profile of the extracts, visualized under visible light, indicated the number of bands that eluted from the various extracts. Since silica gel retains the more polar compounds, the nonpolar compounds eluted first and moved further up the TLC plate. Hence, the more polar the compound, the lower the (Retention factor) value and the less polar the compound the larger the value. Thus, it could be said that the mushroom extracts studied have both polar and nonpolar compounds. However, all four extracts (T. gibbosa, T. elegans, S. commune, and V. volvacea) showed bands that were similar as they had the same characteristic fluorescence and value (0.89) (Figure 1). This may be due to the fact they belong to the same class, Basidiomycetes [52], and therefore may possess similar compounds or secondary metabolites.
T. gibbosa, T. elegans, and S. commune and V. volvacea have antimicrobial agents or principles present in their extracts though the extent of growth inhibition varied. The antimicrobial activity of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea may be due to the presence of the secondary metabolites that act individually or in synergism to inhibit the growth of the test organisms [53]. The differences in the diameter zone of inhibition for T. gibbosa and T. elegans at the highest concentration of 30 g/mL and the next highest concentration of 20 mg/mL (Table 2) may be as a result of the effect of diffusion of the bioactive agents within the medium [54]. Compared with the reference antibiotics, T. gibbosa had broad spectrum of activity against the test organisms. This is however in contrast to the findings of Ga and Kaviyarasana [24] who reported that methanol extract of T. gibbosa exhibited narrow antibacterial activity against S. aureus, B. subtilis, and Micrococcus flavus, but rather in agreement with the report by Mbayo et al. [27] which revealed that methanol extract of T. gibbosa exhibited broad spectrum activity against Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Shigella sonnei.
Methanol extract of T. elegans inhibited the growth of all test bacteria (Table 2). Though reports on the antimicrobial activity of T. elegans are limited, its inhibitory activity against the test organisms could be attributed to the presence of bioactive compounds and secondary metabolites such as flavonoids, tannins, and triterpenoids [55]. The inhibitory action of T. elegans is not surprising as members of the same genus have been reported to possess antimicrobial activity [56].
Microbial growth inhibition was exhibited by extract of S. commune against the test bacteria except B. subtilis. The high zone of growth inhibition exhibited by S. commune extract against C. albicans (Table 3) is in agreement with the results previously reported by Ooi and Liu [57]. The inhibition of growth by extract of S. commune against E. coli, S. aureus, K. pneumonia, P. aeruginosa, and S. pyogenes is in agreement with the findings of Matsuyama et al. [58].
V. volvacea extract inhibited the growth of S. aureus, K. pneumoniae, P. aeruginosa, and S. pyogenes (Table 3) but showed no antimicrobial activity against E. coli and reported that polypores and gilled mushrooms are found to exhibit more pronounced antibacterial than antifungal activity.
To overcome the drawbacks of the agar diffusion test including the inability of some extracts to diffuse into agar and to distinguish bactericidal and bacteriostatic effects, the broth dilution method was employed to determine the potency of the extracts. From the MIC results, all four mushroom extracts inhibited the growth of Gramnegative and Gram-positive bacteria (Tables 4 and 5). The above observations support the findings of the broad spectrum antimicrobial activity of some mushrooms. For example, Pleurotus ostreatus and Meripilus giganteus have been found to exhibit broad spectrum antimicrobial activity [61,62].
Antimicrobials are usually regarded as bactericidal if the MBC/MIC or MFC/MIC ratio is ≤4 and bacteriostatic if >4 [63]. The ratios obtained for all the test organisms were above 4 which indicated that all four mushroom extracts were bacteriostatic and fungistatic in action against test organisms ( Table 6). The bacteriostatic action of these mushroom extracts was also confirmed by the time-kill kinetic studies. Bacteriostatic or fungistatic antimicrobial agents only inhibit the growth or multiplication of pathogenic microorganisms and thus require the host immune system to aid in the elimination of the pathogen [64,65].
Time-kill kinetic studies indicate that methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea exhibited bacteriostatic actions. There are few reports on the time-kill kinetic studies of mushrooms, and several reports of the natural product extracts have been reported [64,66]. However, the findings in this study are in contrast with the study of Tinrat [67] who determined the time-kill kinetic activity of the mushrooms Pleurotus sajor-caju, Hypsizygus tessellatus, Lentinus edodes, Flammulina velutipes, and Pleurotus eryngii and found them to exhibit bactericidal activity. There is the need to isolate and characterize the bioactive compounds in the various extracts responsible for the antimicrobial activity.

Conclusion
Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea exhibited antimicrobial activity but were not active against A. niger, A. flavus, and A. tamarii. Methanol extracts of T. elegans and T. gibbosa exhibited static activity against E. coli, S. aureus, and C. albicans and hence there is a need to isolate and purify the agents or molecules from the extracts responsible for the antimicrobial properties which may serve as potential antibiotics.

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