Abstract
Context: The production of antimicrobial compounds by macrofungi is not unexpected because they have to compete with other organisms for survival in their natural hostile environment. Previous studies have indicated that macrofungi contain secondary metabolites with a range of pharmacological activities including antimicrobial agents.
Objective: To investigate macrofungi for antimicrobial activity due to the increasing need for new antimicrobials as a result of resistance in the bacterial community to existing treatments.
Materials and methods: Forty-seven different specimens of macrofungi were collected across Queensland, Australia. Freeze-dried fruiting bodies were sequentially extracted with three solvents: water, ethanol, and hexane. These extracts were tested against representative Gram+ve, Staphylococcus aureus and Gram−ve, Escherichia coli bacteria.
Results and discussion: Overall water and ethanol extracts were more effective against S. aureus than E. coli, whereas a small number of hexane extracts showed better results for their antimicrobial potential against E. coli at higher concentrations only. Encouraging results were found for a number of macrofungi in the genera Agaricus (Agaricaceae), Amanita (Amanitaceae), Boletus (Boletaceae), Cantharellus (Cantharellaceae), Fomitopsis (Fomitopsidaceae), Hohenbuehelia (Pleurotaceae), Lentinus (Polyporaceae), Ramaria (Gomphaceae), and Strobilomyces (Boletaceae) showing good growth inhibition of the pathogens tested.
Conclusion: The present study establishes the antimicrobial potential of a sample of Australian macrofungi that can serve as potential candidates for the development of new antibiotics.
Introduction
The accidental discovery of penicillin from fungi (CitationFleming, 1929) widely attracted scientific attention for the potential role of fungi as antimicrobial agents and lead to the discovery and development of other antibiotics. However, the emergence and subsequent spread of antibiotic resistant bacterial strains is of increasing concern as reviewed by CitationLevy and Marshall (2004). Currently, this problem presents a significant challenge to medicine because of the therapeutic failure of life-saving drugs (CitationAlfonso, 2005) and hence, more and better antibiotics are needed as indicated by the “10 × ‘20 Initiative” to develop 10 new antibiotics by 2020 by CitationInfectious Diseases Society of America (2010).
Most fungi-derived pharmaceuticals have been sourced from Ascomyceteous fungi where most (but not all) species produce microscopic fruiting bodies; for example, those used in pharmaceuticals include Penicillium, Aspergillus, and so on, whereas perhaps fewer pharmaceuticals, certainly in an industrial context, have been derived from the higher phyla of fungi, the Basidiomycota. The Basidiomycota contains an abundance of species that produce large fruiting bodies including typical mushrooms, coral fungi, puffballs, bracket fungi, and so on. Some species are frequently used as a food source such as the common field mushroom, Agaricus bisporus (J. E. Lange) Imbach (Agaricaceae); others have been used chiefly in medicine (CitationBoa, 2004; CitationMolitoris, 1994), whereas some are known for their notoriously toxic properties such as Amanitas.
According to CitationChang and Buswell (1996), the Romans perceived mushrooms as “Food of the Gods,” the Chinese treasured them as a health food, whereas Lentinula edodes (Berk.) Pegler (Marasmiaceae), the shiitake mushroom, was highly prized by Japanese emperors as an aphrodisiac and was cultivated at secret and heavily guarded locations. In accordance with their traditional uses, macrofungi have been extensively investigated for their therapeutic significance resulting in the discovery of an antibiotic pleuromutilin from Pleurotus mutilis (Fr.) Sacc. and Pleurotus passeckerianus Pilat (Pleurotaceae) (CitationKavanagh et al., 1951). A number of pleuromutilin derivatives have since been developed for veterinary use in the treatment of Mycoplasma infections (CitationDrews et al., 1975; CitationWerner et al., 1978; CitationHannan et al., 1997; CitationHunt, 2000; CitationJones et al., 2002; CitationXu et al., 2009). Furthermore, retapamulin (Altabax) has emerged as an antibiotic for human use for the topical treatment of Gram+ve bacterial skin infections including methicillin-resistant Staphylococcus aureus (CitationJones et al., 2006; CitationNovak & Shlaes, 2010).
The therapeutic potential of mushrooms has been extensively reviewed by CitationWasser and Weis (1999) and CitationLindequist et al. (2005). Extracts of various fungal fruiting bodies such as Pleurotus ostreatus (Jacq.) P. Kumm. (CitationIwalokun et al., 2007), Pholiota adiposa (Fr.) P. Kumm. (Strophariaceae) (CitationDulger, 2004), Coprinus digitalis (Batsch) Fr. (Agaricaceae) (CitationEfremenkova et al., 2003), Podaxis pistillaris (L.) Fr. (Agaricaceae) (CitationAl-Fatimi et al., 2006), Lycoperdon pusillum Batsch [now Bovista pusilla (Batsch) Pers.], and Lycoperdon giganteum Batsch [now Calvatia gigantea (Batsch) Lloyd] (Lycoperdaceae) (CitationJonathan & Fasidi, 2003) have shown activity against a range of different Gram+ve and Gram−ve bacteria and also fungi. CitationStamets (2006) mentioned that macrofungi produce numerous novel pharmaceuticals. However, only a small proportion (10%) of the total estimated number of macrofungi species on Earth (140,000) has been described (CitationHawksworth, 2001). It means that there is an enormous inherent scope for the nutritional and medicinal value among macrofungi that still needs to be discovered. The same is true for the Australian macrofungi. Among 10,000 estimated Australian macrofungi, <4000 have been described, thus leaving behind a large proportion of macrofungi yet to be named indicating the scarce taxonomical information available (CitationMay, 2003).
Macrofungi have been used as food and medicine by indigenous Australians (CitationKalotas, 1996) but only limited research has been carried out for the evaluation of their antimicrobial potential (CitationOvenden et al., 2005; CitationBeattie et al., 2010). Therefore, considering the previous reports on the antimicrobial potential of macrofungi and in view of the continuous need for the development of new antimicrobials, the present study aimed to evaluate a sample of Australian macrofungi for their antibacterial activity against sensitive strains of Staphylococcus aureus (Gram+ve) and Escherichia coli (Gram−ve). The agar well diffusion and disc diffusion methods are commonly used for antimicrobial activity testing but these methods have some limitations. For example, some compounds may be more diffusible and can produce a greater zone of inhibition despite their lower activity in comparison with less diffusible compounds that might be more active but may produce smaller zone of inhibition (CitationJanes et al., 2007; CitationLund et al., 2009). Therefore, in the present study, a high-throughput 96-well microplate bioassay procedure was used.
Materials and methods
Sample collection
Macrofungi fruiting bodies (47 different species) were collected from a range of natural environments across Queensland, Australia during May 2008 to October 2009 (). The fruiting bodies were identified based on sporocarp morphology and macroscopic characters. The collections were freeze-dried and stored at −80°C until extraction.
Table 1. List of macrofungi collected between May 2008 and October 2009 across Queensland, Australia for evaluation of their antibacterial activity.
Preparation of macrofungi extracts
Freeze-dried macrofungi (500 mg) were macerated in 25 mL distilled water and then extracted for 1 h in an ultrasonic water bath. Following centrifugation (15,000 rpm for 15 min), the supernatant was removed. The residue was re-extracted with 25 mL water in the sonicating water bath for 30 min, centrifuged, and the supernatant was pooled with that from the first extraction, collectively forming 50 mL of water extract, which was filtered through 0.45-µm membrane filter and freeze-dried. The remaining insoluble material was extracted sequentially with 100% ethanol and n-hexane, respectively, following the same procedure as described for the water extracts. The ethanol extracts were evaporated to dryness under reduced pressure in a rotary evaporator at 40°C, whereas the hexane extracts were evaporated overnight in a fume hood.
Preparation of mushroom extracts concentrations
The water extracts were each dissolved in 2 mL of distilled water. The ethanol and hexane extracts were first dissolved in 400 µL absolute alcohol, sonicated for 10 min, and made up to 2 mL with distilled water. This stock solution was diluted with tryptone soya yeast extract broth (TSYEB) to make a concentration of 50% that was serially diluted three times with TSYEB to obtain the respective concentrations of 25, 12.5 and 6.25% for all the extracts tested in the antibacterial assay.
Test organisms and culture conditions
The sensitive strains of clinically important S. aureus strain 6571 [National Collection of Type Cultures (NCTC), Health Protection Agency Centre for Infection, London, UK] and E. coli strain 9001 (NCTC) were used for the screening tests. The organisms were grown in TSYEB (CM0129 with the addition of 6 g/L Yeast LP0021, Oxoid, Basingstoke, UK) for 24 h. The overnight growth of the culture was quantified to an absorbance reading of 0.5 at 540 nm using a spectrophotometer (Unicam, HeliosAlpha, UK) by diluting with TSYEB, to obtain a standard inoculum with 105 CFU/mL for use in the assay.
Antibacterial activity assay
A high-throughput 96-well microplate bioassay procedure was used according to the method of CitationSultanbawa et al. (2009), with some modifications. Within each sterile 96-well plate, the first two rows contained 200 µL media only (serving as a sterility check). Test samples were loaded in the next rows with respective concentrations of 50, 25, 12.5 and 6.25%, all comprising 50 µL culture + 150 µL extract. The last row contained 50 µL bacterial culture and 150 µL media serving as negative control. The experiment was replicated three times in separate plates and the same procedure was followed for all three extracts. The standard antibiotics, penicillin G, and oxytetracycline hydrochloride (Sigma-Aldrich, St. Louis, MO) were used as the positive controls against S. aureus and E. coli.
After loading the samples, the plates were read at 640 nm (Tecan, Sunrise, Austria) to determine the absorbance (t0). Then the plates were incubated at 37°C for 22 h, after which the solutions in the plates were mixed using a pipette before measuring the absorbance again (t22). The percent inhibition was calculated using the formula (CitationSultanbawa et al., 2009):
where C0 is the absorbance value of the corresponding negative control well at t0 and C22 is the absorbance value of the corresponding negative control well at t22.
Results
Overall 141 extracts were prepared, comprising of three replicate samples from each of 47 mushroom species extracted using three solvents (water, ethanol, and hexane). The fruiting bodies used represented fungi from 21 families within nine orders from the three subclasses (Agaricomycetidae, Phallomycetidae, and Agaricomycetes incertae sedis) of the class Agaricomycetes. Due to meager taxonomical information available for Australian macrofungi, many of those collected could be identified to genus level only. Extracts were evaluated at four different dilutions (50%, 25%, 12.5%, and 6.25%) against two contrasting microorganisms, Gram+ve S. aureus and Gram−ve E. coli.
The water and ethanol extracts showed differential activity against the bacteria (). Ten macrofungi, namely Agaricus sp. 1, Amanita sp., Amanita ochrophylla (Cooke & Massee) Cleland; Amanitaceae, Boletus sp. subsect. luridi (Boletaceae), Cantharellus sp. (Cantharellaceae), Fomitopsis lilacinogilva (Berk.) J. E. Wright & J. R. Deschamps; Fomitopsidaceae, Hohenbuehelia sp., Lentinus sp. 3, Ramaria sp. 1, and Strobilomyces sp. showed excellent inhibition against both bacteria tested in the present study with water and/or ethanol extracts. For example, the ethanol extract of F. lilacinogilva completely inhibited S. aureus and showed good activity against E. coli at all the test concentrations (). On the other hand, 11 water extracts, 17 ethanol extracts, and 21 hexane extracts possessed either no or weak antibacterial activity against both pathogens. Only a small number of hexane extracts exhibited any activity at all; these were Tylopilus sp. 2, Cantharellus sp., Psathyrella sp., Cyathus striatus (Huds.) Pers. (Nidulariaceae), and Chlorophyllum molybdites (G. Mey.) Massee (Agaricaceae), which were effective against E. coli only at higher concentrations (data not shown).
Table 2. Growth inhibition (%) of S. aureus and E. coli with water and ethanol extracts of various macrofungi belonging to the class Agaricomycetes.
Discussion
Ethanol has previously been noted as the solvent responsible for extracting components with maximum antimicrobial activity (CitationJonathan & Fasidi, 2003; CitationDulger et al., 2004). In the present study, ethanol was similar to water in terms of producing extracts with antimicrobial activity. This might be due to the sequential extraction procedure adopted here, which would have allowed components that are soluble in both ethanol and water to have been extracted in water, whereas for other studies the same active components could be contained within two different solvents.
Gram−ve bacteria are generally more resistant to antibiotics than Gram+ve, due to the structural complexity of their cell wall being less permeable, so consequently antimicrobials are often less effective against Gram−ve bacteria (CitationCosterton & Cheng, 1975; CitationWalsh, 2003). Likewise, Gram−ve bacteria have been reported to be less sensitive to extracts from macrofungi (CitationYamac & Bilgili, 2006; CitationBarros et al., 2007; CitationKaraman et al., 2009). Similarly, in the present study, the extracts were generally less effective toward the Gram−ve bacteria (E. coli) as compared with Gram+ve bacteria (S. aureus). However, this relationship does not hold for every macrofungi. For example, CitationGbolagade et al. (2007) and CitationTambekar et al. (2006) concluded a better inhibitory activity against Gram−ve than Gram+ve bacteria from extracts of A. bisporus, Pleurotus sajor-caju [now Lentinus sajor-caju (Fr.) Fr.], Pleurotus florida (Mont.) Singer, and Polyporus giganteus [now Meripilus giganteus] (Pers.) P. Karst. In the same way, the ethanol extracts of three macrofungi in the present study, Amanita sp. 1, Amanita flavella E. J. Gilbert & Cleland (Amanitaceae), and Boletus sp. subsect. luridi, showed better activity against Gram−ve E. coli rather than Gram+ve S. aureus. The better activity of these macrofungi against Gram−ve bacteria suggests promising antibacterial potential.
Antibacterial activity was observed amongst representatives across all the subclasses and nine orders assessed in this study (). Previous screening of macrofungi in the order Agaricales (subclass Agaricomycetidae) indicated a higher percentage of active isolates (CitationSuay et al., 2000). In the present study, certain macrofungi from the Agaricales namely, Hohenbuehelia, Amanita, and Agaricus revealed strong antibacterial activity. Both the water and ethanol extracts of Hohenbuehelia sp. exhibited strong inhibition of S. aureus at all four test concentrations, whereas E. coli inhibition decreased in a dose-dependent manner with the ethanol extract from this fungus. Indeed, activity against S. aureus has previously been noted for methanol extracts of the fermentation broths of a different species (not yet reported in Australia), Hohenbuehelia mastrucata (Fr.) Singer; Pleurotaceae collected in Spain (CitationSuay et al., 2000). The water extracts of the three Amanita spp. in the present study were variable in activity: A. ochrophylla was more inhibitory against S. aureus than E. coli, whereas Amanita sp. 1 was more effective against E. coli, but A. flavella was ineffective against both bacterial species. The ethanol extracts from all three Amanita spp. caused greater inhibition of E. coli than S. aureus. In contrast, the methanol extract of another Amanita species, Amanita virosa (Fr.) Bertill. was ineffective against these same bacterial species (CitationJanes et al., 2007). Previous studies have indicated that Agaricus may be a lucrative genus to investigate with regards to antibiotic activity; water extract of A. bisporus show activity against Gram−ve bacteria and the ethanol extract of Agaricus brasiliensis Wasser, M. Didukh, Amazonas & Stamets has been shown to be effective against Gram+ve bacteria (CitationTambekar et al., 2006; CitationLund, 2009). The water extract of Agaricus sp. 1 in this study exhibited good activity against both Gram+ve and Gram−ve bacteria but the water and ethanol extracts of the other four Agaricus spp. showed either weak or zero activity against the test pathogens. These examples from the Agaricales highlight the large variation in activity between species within each genus but point to the potential for using taxonomic relationships as a lead in the hunt for antimicrobial activity.
The genus Ramaria (subclass Phallomycetidae) has previously indicated potential antimicrobial activity, with the methanol extract of Ramaria largentii Marr & D. E. Stuntz from Slovenia and ethanol extract of Ramaria flava (Schaeff.) Quél. (Gomphaceae) from Turkey showing weak activity against S. aureus while being ineffective against E. coli (CitationGezer et al., 2006; CitationJanes et al., 2007). In the present study, the ethanol extracts of all three Australian Ramaria sp. tested inhibited the growth of E. coli and the water extract of Ramaria sp. 1 revealed strong activity against S. aureus. Some interesting activity was also recorded for macrofungi within subclass Agaricomycetes incertae sedis. Complete inhibition of S. aureus was achieved with the ethanol extract of Cantharellus sp., which also showed strong inhibition of E. coli at higher concentrations. While CitationDulger et al. (2004) concluded that the ethanol extract of Cantharellus cibarius was more effective against E. coli than S. aureus, our results are in accordance with CitationSantoyo et al. (2009) and CitationBarros et al. (2008). Based on previous tests, Russula may not be expected to provide antibacterial compounds, as CitationKeller et al. (2002) found that the methanol extracts from five different species of European Russula did not possess any activity against either Gram+ve nor Gram−ve bacteria. Indeed, in our tests extracts of Russula erumpens Cleland & Cheel showed only weak activity. Using taxonomic relationships as a guide toward bioactivity is only useful where records exist. The ethanol extract of F. lilacinogilva exhibited complete inhibition of S. aureus and moderate inhibition of E. coli even at the lowest concentration. Likewise, CitationPopova et al. (2009) and CitationLiu et al. (2010) reported that the chloroform extract of Fomitopsis rosea (Alb. et Schw. Fr.) Karst and triterpene isolated from dichloromethane extract of Fomitopsis pinicola (Swartz ex Fr.) Karst inhibited S. aureus and Bacillus cereus. The antimicrobial potential of different species in this genus suggests that it may be worthwhile further investigating closely related species.
In conclusion, this is the first report on the screening evaluation of different Australian macrofungi species. From the results of this study, a small number of Australian macrofungi have been identified with promising antibacterial activity against S. aureus and E. coli that can serve as potential candidates for much needed new antibiotics. Further work is needed toward the evaluation of their antimicrobial potential against a wider range of microorganisms and finally the identification and isolation of the active compounds responsible for this activity could provide new starting material for the development of novel antibiotics.
Declaration of interest
The authors report no conflicts of interest.
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