Discovery of Antifungal and Biofilm Preventative Compounds from Mycelial Cultures of a Unique North American Hericium sp. Fungus

Edible mushrooms are an important source of nutraceuticals and for the discovery of bioactive metabolites as pharmaceuticals. In this work, the OSMAC (One Strain, Many Active Compounds) approach was used to isolate two new compounds (1 and 2) along with seven known compounds (3–9) from a mycelial culture of a unique North American edible mushroom Hericium sp. The fruiting body was collected in Marine on St. Croix, Minnesota (USA), and mycelial cultures were grown on four different solid and liquid media. Extracts from the mycelial cultures were screened for antimicrobial activity and only the extract from the Cheerios substrate culture exhibited antifungal activity. Bioassay guided fractionation and HPLC analysis were used to isolate nine pure compounds and the structures of the known compounds were established by analysis of the NMR and mass spectrometry data and comparison to published reports. Compound 1 is a new erinacerin alkaloid and 2 is an aldehyde derivative of 4-hydroxy chroman. Four chlorinated orcinol derivatives (3–6), a pyran (7), erinaceolactone (8), and erinacine (9) were identified. Compound 4 showed antifungal activity against C. albicans and C. neoformans (MIC = 31.3–62.5 μg/mL, respectively). Compound 4 also inhibited biofilm formation of C. albicans and C. neoformans at 7.8 μg/mL. These results suggest that mycelial cultures of edible fungi may provide useful, bioactive compounds.


Introduction
The fruiting bodies (mushrooms) of Basidiomycota have been used as tea, health enhancing foods, and medicines for thousands of years as an integral component of traditional folk medicine cultures globally, including Traditional Chinese Medicine (TCM) [1][2][3][4]. Although production of important clinical antibiotics such as penicillin, cephalosporin, and griseofulvin by filamentous Ascomycota fungi is well known [5], a number of potent, biologically active secondary metabolites have been isolated from Basidiomycota and these compounds may contribute to their medicinal usage [6]. For example, psilocybin and psilocin from a wide range of mushroom species are being clinically tested for treatment-resistant depression and anxiety disorders [7,8]. Due to their chemical diversity and historical success as both medicines and sources of bioactive compounds, mushrooms remain an attractive resource for natural products drug discovery.
The fruiting bodies and substrate mycelia of mushroom forming fungi are spatially and ecologically separate, with different environmental pressures and defensive strategies [9]. It is likely that these morphologically distinct forms differentially upregulate secondary metabolite pathways [10].
( Figure 1) and tested for biological activities. Except for shared compound peaks with retention times (tR) between 10-12 min, extracts from mycelial cultures contained several compounds that were not found from the fruiting body of Hericium sp. WBSP8. Extracts from the fruiting body primarily yielded peaks (tR = 44-50 min) corresponding to relatively nonpolar compounds. Liquid cultures resulted in similar chromatographic profiles for both mycelial and supernatant extracts.
Cultures grown on rice or Cheerios also produced a range of compounds with medium polarity (tR = 20-30 min), including a prominent peak at tR = 28 min present only in the Cheerios extract. Cultures grown on rice or Cheerios also produced a range of compounds with medium polarity (tR = 20-30 min), including a prominent peak at tR = 28 min present only in the Cheerios extract.

Analysis of Antimicrobial Activities of Hericium sp. WBSP8 Extracts
The seven extracts in Figure 1A were screened for activity against a panel of six bacterial and two fungal human pathogenic strains using the microbroth dilution method (Figure 2). Extracts were tested at a wide range of concentrations up to 1 mg/mL because we are aware that many people consume dried mycelial and mycelial extract formulations from edible mushrooms that might contain comparable quantities of active metabolites [25]. Only the crude extract from the Cheerios culture showed activity against both yeast pathogens Candida albicans and Cryptococcus neoformans with an MIC of less than 250 µg/mL. Extracts from soy and malt culture supernatants showed activity against C. neoformans with MICs comparable to the Cheerios extract, while extracts from rice cultures were only weakly active (MIC > 500 µg/mL). Several extracts (rice, malt mycelia and supernatant, and soy supernatant) exhibited weak antibacterial activity (MIC > 500 µg/mL) against S. aureus and the soy culture supernatant extract was also active against E. faecalis (MIC > 500 µg/mL). The fruiting body extract was inactive against all of the strains tested.

Analysis of Antimicrobial Activities of Hericium sp. WBSP8 Extracts
The seven extracts in Figure 1A were screened for activity against a panel of six bacterial and two fungal human pathogenic strains using the microbroth dilution method (Figure 2). Extracts were tested at a wide range of concentrations up to 1 mg/mL because we are aware that many people consume dried mycelial and mycelial extract formulations from edible mushrooms that might contain comparable quantities of active metabolites [25]. Only the crude extract from the Cheerios culture showed activity against both yeast pathogens Candida albicans and Cryptococcus neoformans with an MIC of less than 250 μg/mL. Extracts from soy and malt culture supernatants showed activity against C. neoformans with MICs comparable to the Cheerios extract, while extracts from rice cultures were only weakly active (MIC > 500 μg/mL). Several extracts (rice, malt mycelia and supernatant, and soy supernatant) exhibited weak antibacterial activity (MIC > 500 μg/mL) against S. aureus and the soy culture supernatant extract was also active against E. faecalis (MIC > 500 μg/mL). The fruiting body extract was inactive against all of the strains tested.

Bioassay-Guided Isolation of Secondary Metabolites from Hericium sp. WBSP8 Grown on Cheerios
We used bioassay-guided fractionation on the most active extract from the Cheerios culture to isolate, identify, and characterize the inhibitory compounds. In addition, the HPLC profile of this extract displayed a prominent, unique peak at tR = 28 min, which was likely related to its antifungal activity. The Cheerios culture was extracted successively with ethyl acetate (EtOAc), acetone, and methanol (MeOH) (3 × 400 mL each solvent). The extracts were combined, dried, and subject to a modified Kupchan partition scheme. The active EtOAc fraction was separated using reverse-phase and Sephadex LH-20 column chromatography, followed by semi-preparative RP C18-HPLC to provide metabolites 1−9 (Figures 3 and S2).

Bioassay-Guided Isolation of Secondary Metabolites from Hericium sp. WBSP8 Grown on Cheerios
We used bioassay-guided fractionation on the most active extract from the Cheerios culture to isolate, identify, and characterize the inhibitory compounds. In addition, the HPLC profile of this extract displayed a prominent, unique peak at tR = 28 min, which was likely related to its antifungal activity. The Cheerios culture was extracted successively with ethyl acetate (EtOAc), acetone, and methanol (MeOH) (3 × 400 mL each solvent). The extracts were combined, dried, and subject to a modified Kupchan partition scheme. The active EtOAc fraction was separated using reverse-phase and Sephadex LH-20 column chromatography, followed by semi-preparative RP C 18 -HPLC to provide metabolites 1−9 ( Figure 3 and Figure S2).
Compound 2 was obtained as a white amorphous solid with a molecular formula of C12H14O3. The 1 H-NMR spectrum of 2 indicated the presence of two methyl groups, one aldehyde proton, and a 1,3,4-trisubstituted benzene ring appearing as an ABX system ( Table 2). Analysis of the 13 C, DEPT, and HMQC NMR data indicated two methyls, one methylene, five methines, three SP2 carbons, and one quaternary carbon. The two methyl groups were correlated to each other (H-3′/C-2′, H-2′/C-3′) and to C-2 in the HMBC spectra, indicating their geminal position at C-2 ( Figure 4). The HMBC correlations between H-1′/C-6 and H-5/C-1′ established the position of the aldehyde group at C-6. Collectively, these data indicated a dimethyl chromane structure very similar to the plant derived compound 1-[(4S)-3,4-dihydro-4-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl]-ethanone [27], but with an aldehyde at C-6 instead of an ethanone group. In addition, 2 was optically active with a positive specific rotation value of [α]D + 10.0 (c 0.12, MeOH). We compared this data to the published absolute stereochemistry analysis of closely related R and S C-4 hydroxy chromane enantiomer pairs with a methoxy or ethanone at the C-6 position [28]. In this prior work, these 4R/S enantiomers were isolated as mixtures from the plant, and could only be separated by chiral HPLC [28]. The optical rotation values of only the 4S enantiomers were positive, suggesting the 4S configuration for 2 (Table  S1). However, as we were not able to analyze compound 2 by chiral HPLC, it is not known if it exists as an enantiomeric mixture, and the absolute stereochemistry of C-4 remains tentative. Thus, 2 was determined to be a novel aldehyde derivative of 4-hydroxy chroman, 4-hydroxy-2,2-dimethyl chromane-6-carbaldehyde.
Compound 2 was obtained as a white amorphous solid with a molecular formula of C 12 H 14 O 3 . The 1 H-NMR spectrum of 2 indicated the presence of two methyl groups, one aldehyde proton, and a 1,3,4-trisubstituted benzene ring appearing as an ABX system ( Table 2). Analysis of the 13 C, DEPT, and HMQC NMR data indicated two methyls, one methylene, five methines, three SP 2 carbons, and one quaternary carbon. The two methyl groups were correlated to each other (H-3 /C-2 , H-2 /C-3 ) and to C-2 in the HMBC spectra, indicating their geminal position at C-2 ( Figure 4). The HMBC correlations between H-1 /C-6 and H-5/C-1 established the position of the aldehyde group at C-6. Collectively, these data indicated a dimethyl chromane structure very similar to the plant derived [27], but with an aldehyde at C-6 instead of an ethanone group. In addition, 2 was optically active with a positive specific rotation value of [α] D + 10.0 (c 0.12, MeOH). We compared this data to the published absolute stereochemistry analysis of closely related R and S C-4 hydroxy chromane enantiomer pairs with a methoxy or ethanone at the C-6 position [28]. In this prior work, these 4R/S enantiomers were isolated as mixtures from the plant, and could only be separated by chiral HPLC [28]. The optical rotation values of only the 4S enantiomers were positive, suggesting the 4S configuration for 2 (Table S1). However, as we were not able to analyze compound 2 by chiral HPLC, it is not known if it exists as an enantiomeric mixture, and the absolute stereochemistry of C-4 remains tentative. Thus, 2 was determined to be a novel aldehyde derivative of 4-hydroxy chroman, 4-hydroxy-2,2-dimethyl chromane-6-carbaldehyde.

Antibacterial and Antifungal Activity of Isolated Compounds
Compounds 1-9 were evaluated for their antimicrobial activities against a panel of eight human pathogens. Compound 4 showed inhibition against C. albicans and C. neoformans, with MIC values of 62.5 and 31.25 µg/mL, respectively ( Figure 2). Compounds 4, 7, 8, and 9 exhibited weak antibacterial activity against several pathogens ( Figure 2). Compounds 1-3 and 5-6 were inactive in all antimicrobial assays.
2.6. Antibiofilm Activity of Compounds 1-9 In addition to identifying inhibitors of planktonic microbial pathogens, we are interested in the discovery of compounds that inhibit or disrupt fungal biofilms. Compounds 1-9 and the crude extract from the Cheerios culture were tested (0.125-1000 µg/mL) in a biofilm prevention assay with C. neoformans and C. albicans. Compound 4 reduced biofilm formation by 59.4% and 48.6% for C. neoformans and C. albicans, respectively, at 7.8 µg/mL ( Figure 5A). The crude Cheerios culture extract showed weak activity (23.6% biofilm prevention) at the same concentration (7.8 µg/mL) against C. neoformans. In comparison, compound 4 and the crude extract showed no inhibition of planktonic cultures (<10%) at the same concentration ( Figure 5C). The same samples were also tested for their ability to disrupt established (24 h) biofilms of C. neoformans and C. albicans at 1 mg/mL. Compound 4 and the crude extract reduced preformed C. neoformans biofilms by 45.0% and 36.1%, while the control compound nystatin reduced biofilm mass by 60.6% at 1 mg/mL ( Figure 5B). None of the test samples significantly reduced established C. albicans biofilms.

Antibacterial and Antifungal Activity of Isolated Compounds
Compounds 1-9 were evaluated for their antimicrobial activities against a panel of eight human pathogens. Compound 4 showed inhibition against C. albicans and C. neoformans, with MIC values of 62.5 and 31.25 μg/mL, respectively ( Figure 2). Compounds 4, 7, 8, and 9 exhibited weak antibacterial activity against several pathogens (Figure 2). Compounds 1-3 and 5-6 were inactive in all antimicrobial assays.

Antibiofilm Activity of Compounds 1-9
In addition to identifying inhibitors of planktonic microbial pathogens, we are interested in the discovery of compounds that inhibit or disrupt fungal biofilms. Compounds 1-9 and the crude extract from the Cheerios culture were tested (0.125-1000 μg/mL) in a biofilm prevention assay with C. neoformans and C. albicans. Compound 4 reduced biofilm formation by 59.4% and 48.6% for C. neoformans and C. albicans, respectively, at 7.8 μg/mL ( Figure 5A). The crude Cheerios culture extract showed weak activity (23.6% biofilm prevention) at the same concentration (7.8 μg/mL) against C. neoformans. In comparison, compound 4 and the crude extract showed no inhibition of planktonic cultures (<10%) at the same concentration ( Figure 5C). The same samples were also tested for their ability to disrupt established (24 h) biofilms of C. neoformans and C. albicans at 1 mg/mL. Compound 4 and the crude extract reduced preformed C. neoformans biofilms by 45.0% and 36.1%, while the control compound nystatin reduced biofilm mass by 60.6% at 1 mg/mL ( Figure 5B). None of the test samples significantly reduced established C. albicans biofilms.

Discussion
In this work, we compared the chemical profiles and antimicrobial properties of extracts from the fruiting body and in vitro mycelial cultures of Hericium sp. WBSP8 cultivated under different conditions. Analysis of extracts by HPLC showed that different cultivation media significantly influenced the production of secondary metabolites. The mycelial culture on Cheerios provided the only antifungal extract and led to the isolation of compound 4 as the major antifungal component (Figure 1), in addition to two novel compounds (1 and 2). These results confirm that the secondary metabolome of Hericium sp. WBSP8 is distinct when grown under different conditions, consistent

Discussion
In this work, we compared the chemical profiles and antimicrobial properties of extracts from the fruiting body and in vitro mycelial cultures of Hericium sp. WBSP8 cultivated under different conditions. Analysis of extracts by HPLC showed that different cultivation media significantly influenced the production of secondary metabolites. The mycelial culture on Cheerios provided the only antifungal extract and led to the isolation of compound 4 as the major antifungal component (Figure 1), in addition to two novel compounds (1 and 2). These results confirm that the secondary metabolome of Hericium sp. WBSP8 is distinct when grown under different conditions, consistent with other metabolomic studies of fungi [33,34]. These findings also support the use of Cheerios as a useful cultivation substrate to promote secondary metabolite expression in Basidiomycota fungi, which has been previously shown to enhance the production of novel and diverse compounds in filamentous fungi [35]. Compounds 3-9 have been previously isolated from mycelial cultures of Hericium erinaceus [36]. Interestingly, we isolated 4 as the primary antifungal component, but it was first isolated from H. erinaceus and reported as active against Bacillus subtilis and inactive against all fungi tested including Saccharomyces cerevisiae IFO0304, Aspergillus niger JCM 5697, Fusarium oxysporum J1, and Verticillium dahliae at 200 µg/disk in diffusion assays [29]. Compound 4 was previously isolated from liquid cultures of H. erinaceus with yields of 6.5 mg/L [29], but our cultivation system on Cheerios cereal supplemented with glucose increased this yield five-fold to 38 mg/Kg.
Although compound 4 displayed modest activity against planktonic C. albicans and C. neoformans, the most significant activity identified was prevention of C. neoformans biofilms at sub-inhibitory concentrations. Many pathogenic microbes exist in a biofilm state, which can lead to an increase in virulence, upregulation of toxin production, and increase in drug resistance [37]. C. neoformans and C. albicans are important pathogens associated with human biofilm infections, and demonstrate significant resistance to clinical antifungals [38,39]. Due to the relatively high toxicity and nonspecificity of most antifungal drugs, fungal biofilm infections are especially challenging to eradicate, and there is a critical need for new biofilm-specific treatments. Compound 4 prevented C. neoformans biofilm formation when added to cultures after 6 h of growth, but only disrupted established (24 h) biofilms at very high concentrations (1 mg/mL), suggesting a mechanism related to attachment, cell adhesion, or early signaling [40]. Compound 4 also exhibited no cytotoxicity towards mammalian cells (HaCAT, IC 50 > 100 µM), in contrast to most clinical antifungal agents. Biofilm preventative compounds similar to 4 could be developed as a safer, less toxic component of coatings for in vivo materials or devices to prevent fungal biofilm growth.
Overall, this study demonstrated that Basidiomycota are a viable source for antifungal compounds and that secondary metabolite production can be elicited in mycelial cultures using similar growth strategies established for other fungi. This work also showed that close taxonomic relatives of well characterized edible mushrooms offer opportunities for new compound discovery.

General Experimental Procedures
Reagents were purchased from Fisher Scientific (Waltham, MA, USA) unless otherwise noted. Optical rotations were measured on a Rudolph Research Analytical Autopol III polarimeter (Hackettstown, NJ, USA). IR spectra were obtained using a JASCO 4100 FT-IR spectrophotometer (Oklahoma City, OK, USA). High resolution mass analyses were performed using an Agilent 6230 TOF LC/MS mass spectrometer (Santa Clara, CA, USA) with a dual ESI and APCI source. Standard 1D and 2D NMR spectra were recorded on a Bruker 400 MHz spectrometer (Billerica, MA, USA). Proton and carbon chemical shifts are reported in ppm and referenced with the 1 H and 13 C signals of solvents. TLC separations were performed using Whatman silica gel 60 F 254 aluminum backed TLC plates. HPLC separations were performed with an Agilent 1200 instrument (Santa Clara, CA, USA) with a PDA detector system. Pure fungal cultures were obtained by sub-cultivating outgrowing mycelia. The remaining fruiting body was cut into small pieces (2 × 2 cm) and dried at 40 • C for 72 h. The dried fungal material was stored in a dry and dark environment at room temperature until extraction.

Mushroom Collection and Identification
The morphology of the specimen most closely resembled Hericium americanum (branching fruiting body with long spines), but molecular analysis suggested that it was distinct from this species. For formal strain identification by internal transcribed spacer (ITS) sequencing, a piece of a well grown mycelia was excised, ground in liquid nitrogen, washed with EtOH, and processed for genomic DNA extraction with buffer based on the protocol established by Saitoh et al. [41]. PCR was performed on genomic DNA with primers ITS1F (5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and ITS4 (5 -TCCTCCGCTTATT GATATGC-3 ) for amplification of the ITS region [42]. The PCR product was gel purified (Promega Wizard ®® SV Gel and PCR Clean-Up System; Promega, Madison, WI, USA) and submitted for Sanger sequencing (University of Minnesota Genomics Center). The resulting ITS sequence was compared by BLASTn analysis to reference sequences in the NCBI Genbank. Analysis of the ITS sequence data show similarity to H. alpestre DQ206987 (a European species), H. abietis JQ716938 (a species found in the US Pacific Northwest on conifers), and H. americanum DQ206987, but is more distant to most other H. americanum entries. Due to the complexity of the genus and misidentification of many specimens in the literature and databases, we can only identify our specimen as a unique member of the Hericium complex. The fungal culture was incorporated in our Basidiomycota library (University of Minnesota) under the reference 161009-WBSP-0008 and the ITS sequence was deposited in GenBank (accession number: MN243091). All cultivations were carried out at 25 • C in the dark for 28 days as static cultures (Rice and Cheerios) and with shaking (150 rpm) for liquid cultures (Malt and Soy).

Microbroth Dilution Assay
Compounds and extracts were tested for planktonic microbial growth inhibition using a broth dilution assay [43]. The extracts or compounds were dissolved in DMSO at a stock concentration of 10 mg/mL and kept at 4 • C for the bioassays. Exponentially growing cultures (OD 600 = 0.03-0.06) of each strain were prepared from overnight cultures. Cultures were diluted 1:10 in broth (C. neoformans was used directly without dilutions) and added to a 96-well plate (195 µL/well). Extracts and pure compounds were tested at a final concentration of 250 to 3.9 µg/mL with two-fold dilutions. The samples with inhibition below 50% at 250 µg/mL were tested at a final concentration of 1 mg/mL. Tetracycline (Sigma, St. Louis, MO, USA; 10 µg mL −1 in DMSO) was used as positive control against S. aureus, B. subtilis, E. coli, P. aeruginosa, A. baumannii, and K. pneumonia. Penicillin G (Sigma, St. Louis, MO, USA; 10 µg mL −1 ) served as positive control against VRE. Nystatin (Sigma, St. Louis, MO, USA; 10 µg mL −1 ) was used as positive control for C. albicans and C. neoformans. Plates were read at 600 nm after incubation for 24 h for bacteria and 48 h for fungi. Inhibition was calculated by subtracting the absorbance of the blank wells, dividing by the average value for the DMSO only wells and multiplying by 100.

Tissue Culture Cytotoxicity Assays
Pure compounds were tested against human fibroblast cells (HaCaT) using a standard MTT cell viability assay [44]. HaCaT human fibroblast cells (AddexBio T0020001, San Diego, CA, USA) were maintained in growth media: DMEM (Invitrogen 11965-092, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen 16000-044), 1% penicillin/streptomycin (Invitrogen 15140-122), 1% sodium pyruvate (Invitrogen 11360), and 1% Glutamax-1 (Invitrogen 35050-061). 96-well plates were coated with 0.01% collagen overnight and rinsed with PBS prior to seeding with 25 × 104 cells/mL HaCaT cells and allowed to adhere overnight. After 24 h, compounds were added in triplicate at nine, 3x dilutions from 100 µM final concentration in growth media. Plates were incubated for 72 h at 37 • C in a 5% CO 2 /95% air humidified atmosphere after which time the media was removed and MTT was added in a RPMI phenol red free media (Invitrogen 11835-030). The MTT was removed after 3 h, formazan crystals were solubilized with 200 µL of isopropanol and plates were read on an i3 spectrophotometer (Molecular Devices, San Jose, CA, USA) at 570 nm for formazan and 690 nm for background subtraction. EC 50 values were calculated by fitting the data in the GraphPad Prism software (version 5.04).

Extractions and Compound Isolation
The dried fruiting body was ground to a fine powder, extracted three times with methanol at room temperature overnight, and filtered through Whatman filter paper. The combined filtrates were concentrated, dried with a rotary vacuum evaporator at 40 • C, and stored at −20 • C until use.
The mycelia and substrate from four Cheerios or Rice cultures were combined and successively extracted with ethyl acetate (EtOAc), acetone, and MeOH (3 × 400 mL) with maceration at RT. The organic extracts were combined and evaporated under reduced pressure to afford the dried extracts (6.0 g Cheerios cultures, 18.0 g rice cultures).
Extracts from liquid malt or soy cultures were prepared by combining two 125 mL cultures for each medium and removing the mycelium by filtration through qualitative P8 grade filter paper (Fisher Scientific). The supernatant was extracted three times with an equal volume of EtOAc and then evaporated to dryness. The filtered mycelium was successively extracted with EtOAc, acetone, and MeOH (150 mL) and the combined organic solvents evaporated until dryness.

Biofilm Inhibition and Disruption Assays
The fungal biofilm prevention and disruption assays were performed based on published protocols [45] with minor revisions. Biofilms were cultured in flat-bottom, nontissue culture-treated 96-well plates. Overnight cultures of C. albicans were grown in YM broth in 15 mL culture tubes at 30 • C on a shaker at 250 rpm for 15 h, cells were diluted to an OD 600 of 0.5, and then diluted 1:1000 in a RPMI medium (with L-glutamine, without sodium bicarbonate; Gibco, supplemented with 34.5 g/L morpholinepropanesulfonic acid, Sigma and adjusted to pH 7.0 using NaOH). Using the same method, SDB was used as the medium for biofilm formation with C. neoformans for all growth steps. Extracts and compounds 1-9 were tested at 0.125, 0.5, 2, 7.8, 31.25, 250, and 1 mg/mL in triplicate in both assays. DMSO was used as a negative control for all assays (5 µL).
Prevention of biofilm formation: Diluted cultures of each strain (195 µL) were added to 96-well plates and transferred to a static incubator at 30 • C. Compounds and extracts (5 µL) were added after 6 h, the plates were sealed with parafilm and incubated under static conditions for an additional 42 h at 30 • C.
Biofilm disruption assays: Diluted cultures of C. albicans and C. neoformans were added to 96-well plates (195 µL) and incubated at 30 • C under static conditions. After 24 h, the planktonic cells and medium were aspirated from the wells and 195 µL of fresh medium was added to the well, followed by 5 µL of test extracts or compounds in DMSO. Plates were sealed and incubated at 30 • C for an additional 24 h.
For both of the above assays, at the end of the final incubation period the planktonic cells and medium were aspirated and wells were washed twice with sterile saline (250 µL). The remaining attached biofilms were fixed with 95% ethanol per well (200 µL), and after 20 min plates were emptied and left to dry. Plates were stained for 15 min with 0.1% crystal violet (100 µL). Excess stain was then removed by rinsing the plate with water. The plates were allowed to dry, and the crystal violet-stained biofilm was solubilized with 200 µL of 30% glacial acetic acid solution per well. The OD of each well was measured at 550 nm with a microplate reader. The percentage of biofilm inhibition was calculated using the following formula:

Statistical Analysis
Data are presented as the mean ± standard error (SE). The statistical significance of the data was tested by one-way analysis of variance (ANOVA) using Graphpad Prism 5.0 (Graphpad software Inc., version 5.04, La Jolla, CA, USA). The least significant differences (P < 0.05) were calculated to compare differences between the means following determination of significant effects by ANOVA.
Author Contributions: X.S. contributed to the chemistry and bioassay work, and drafted the manuscript; F.G. coordinated the collection, cultivation, and extraction of the mushroom, and revised the manuscript; C.S.-D. and C.E.S. contributed to the conception and design of the study and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.