Methanol of analytical grade purity was from Lab-Scan (Lisbon, Portugal). The culture media Brain Heart Infusion Agar (BHIA), Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), and all the antibiotics were obtained from Oxoid (Hampshire, UK). The saline solution was prepared with NaCl from Merck (Darmstadt, Germany). The dye resazurin and crystal violet were purchased from Sigma-Aldrich (St Louis, Mosby, USA). All the organic solvents were HPLC grade. Ultrapure water from purified system (Isopad Isomantle, Gemini BV, Pr Beatrixlaan 301, 7312 DG Apeldoorn, Netherlands) was used. External standards caffeic acid, catechin, chlorogenic acid, ferulic acid, gallic acid, (-)-epicatechin, naringin, p-hydroxybenzoic acid, quercitrin, and rutin were purchased from Extrasynthese (Lyon Nord, Genay Cedex, France). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), non-essential amino acids, penicillin, streptomycin, and trypsin–EDTA were obtained from Invitrogen Corporation (Life Technologies, SA, Madrid, Spain).

Boletus edulis and Neoboletus luridiformis mushrooms were collected in a mixed stand at Sabugal, Guarda, located at the Center of Portugal. The morphological identification of the wild macrofungi was made till species according to macro- and microscopic characteristics (8). Representative voucher specimens were deposited at the mycological herbarium of Universidade de Trás-os-Montes e Alto Douro. After taxonomic identification, the mushrooms were immediately stored at −20°C, freeze dried (Dura Dry TM μP, −41°C and 500 mTorr), and grounded to a fine powder. The samples were kept in the dark in hermetically sealed plastic bags up to analysis.

The microorganisms used were clinical isolates from patients hospitalized in various departments of Hospital Center of Trás-os-Montes and Alto Douro (CHTMAD)—these are located in the cities of Lamego, Peso da Régua, Chaves, and Vila Real, a Portuguese north province of Trás-os-Montes and Alto Douro. Ethical approval for this study was granted by the Ethics Committee of Hospital Vila Real (CHTMAD), according to a research collaboration protocol established in 2004. These strains belong to MJH and MJMC collections and are stored at −70°C in aliquots of BHI medium with 15% (v/v) glycerol in the Medical Microbiology Laboratory at Department of Veterinary Sciences — Antimicrobials, Biocides and Biofilms Unit at University of Trás-os-Montes and Alto Douro (UTAD). Several Gram-positive and Gram-negative bacteria isolated from wound exudates were used to screen the antimicrobial activity of the mushroom extracts. All the strains were identified by morphological and biochemical tests (morphological identification of colonies, Gram staining, conventional biochemical identification methods, and MicroScan WalkAway identification panels), followed by Kirby–Bauer antibiotic sensitivity assays, using different antibiotics. Escherichia coli (E. coli) (CETC 434) and Staphylococcus aureus (S. aureus) (CETC 976) strains were obtained from Spanish Type Culture Collection (CETC). Ethics approval for this study was granted by the Ethics Committee of Hospital Vila Real.

Briefly, bacterial suspension with the turbidity adjusted to 0.5 McFarland standard units, were spread with a sterile cotton swab onto Petri dishes (90 mm of diameter) containing 4 mm of Mueller-Hinton agar. Six-millimeter diameter sterile paper discs were dispensed on the seeded agar plates and imprinted with 12 μL of 1000 μg.mL⁻¹ extracts solution (in Dimethyl Sulfoxide (DMSO) 10%). Incubation for 24 h at 37°C, followed by diameter measurement (mm) of the clear inhibitory zones around the discs imprinted with the extracts. In all experiments, a negative control (12 μL DMSO) and a positive control [standard commercial antibiotic gentamicin (10 μg)] were included.

The antimicrobial activity was classified according to the following scheme: noneffective (−): inhibition halo = 0; moderate efficacy (+): 0 < inhibition halo < antibiotic inhibition halo; good efficacy (++): antibiotic inhibition halo < inhibition halo < 2x inhibition halo; strong efficacy (+ + +): inhibition halo > 2x antibiotic inhibition halo (6). Minimum inhibitory concentration (MIC, lowest concentration of mushroom extract able to inhibit bacterial growth) was evaluated by a resazurin microdilution assay (6). Bacteria tested were picked from overnight cultures in Brain Heart Infusion. A small portion of bacteria was transferred into a bottle with 50 mL of Mueller Hinton Broth (MHB), capped and placed in an incubator overnight at 37°C. After 16 h of incubation, bacterial suspension was adjusted to an optical density of 0.5 measured at OD₅₀₀ nm. The resazurin solution (3.4 mg.mL⁻¹) was prepared in sterile distilled water. A 96-wells sterilized microplate was used and a volume of 100 μL of MHB was used in each well, together with 200 μL of extract solution, or positive control. From the first well (belonging to the first horizontal line) 100 μL was taken and added to the next well and then this step was repeated to each of the following wells in the vertical line, allowing a serial fold dilution of decreasing concentration (range of 1000 μg.mL⁻¹ to 7.81 μg.mL⁻¹). In addition, 20 μL of bacterial suspension and 20 μL of resazurin solution was added to each well. Microplates were incubated at 37°C for 18–24 h. All tests were performed in triplicate and MIC was then assessed visually by the color change of resazurin in each well (blue to pink in the presence of bacteria growth). For the determination of minimum bactericidal concentration (MBC, the lowest concentration of mushroom extract at which bacterial growth by at least 99.0%), the content from each well without changes in color was plated on Mueller-Hinton Agar and incubated at 37°C for 24 h. The lowest concentration that yielded no growth after this subculturing was taken as the MBC.

The microtiter biofilm assay was used to assess the ability of the mushrooms extracts to control adhered cells of S. aureus CECT 976 and E. coli CECT 434 biofilms. Briefly, 96-well polystyrene microtiter plates were filled with 200 μl of bacterial suspension at OD₆₂₀ nm of 0.04 ± 0.02 and incubated for 24 h (biofilm formation) at 37°C and 150 rpm. After the incubation period, the content of each well was aspirated and washed once with 200 μl of saline solution 0.85% (w/v). Then, 180 μl of fresh medium (MH broth) and 20 μl of extracts solution (to a final concentration at MIC, 5 × MIC, and 10 × MIC) were applied on biofilms. Concentrations ≥ MIC were used considering that biofilm-associated cells are usually 10–1,000-fold more resistant than in the planktonic state. Controls wells containing 10% (v/v) dimethyl sulfoxide (DMSO) were included. After 24 h of exposure at 37°C and 150 rpm, the effects of extracts were analyzed in terms of biomass and metabolic activity.

Phytochemical characterization of mushroom extracts was achieved by analysis of total phenols, orthodiphenols content, and antioxidant activity as well as by high-performance liquid chromatography-diode array detector (HPLC-DAD).

Human foreskin fibroblasts-1 (HFF-1) was purchased from ATCC (ATCC Number: SCRC-1041; ATCC, Manassas, Virginia, USA). Cell line were grown in DMEM medium (Carlsbad, California, USA) fortified with L-glutamine, 10% inactivated fetal calf serum (FBS), antibiotic–antimitotic mixture (final concentration of 100 U/ml penicillin and 100 U/ml streptomycin) maintained in 5% CO₂ incubator (Cell Culture^® CO₂ Incubator, ESCO GB Ltd., UK). At 90–95% confluence, cells were trypsinized and plated in microtiter dishes. The viable cells were counted using trypan blue dye (Gibco) in hemocytometer. Cell viability was assessed using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)-2H-tetrazolium (MTT purchased from Promega, Madison, Wisconsin, USA) conversion assay. Cells were cultured in 96-well microtiter plate at a density of 25 × 10³ cells per ml culture medium for 24 h. Then, cells were incubated with 1 μg.mL⁻¹ to 10 mg.ml⁻¹ of both the extracts and its corresponding formulation for 24 h at 37°C. After the removal of samples from the wells, cells were washed in phosphate-buffered saline, followed by addition of fresh medium. The microtiter plates were then incubated in a humidified atmosphere of 5% CO₂ at 37°C for 24 h. To evaluate the number of viable cells, 100 μl of MTT solution was added into each well and incubated for 4 h at 37°C in the dark. Afterward, the medium was removed, the intracellular formazan crystals were solubilized, and extracted with 100 μl DMSO. After 15 min in continuous stirring at room temperature, the absorbance was measured at 490 nm with background subtraction at 630 nm in the Synergy HT Microplate Reader (BioTek Instruments Incorporation, Winooski, Vermont, USA; (11)).

Data are expressed as mean ± SD and were statistically analyzed by the one-way ANOVA, followed by the Holm-Sidak's multiple comparison test. Statistical analyses were performed using the GraphPad Prism software (San Diego, CA) for Windows (version 7) and differences were considered significant when p < 0.05 (95% significance).

Data on screening of antimicrobial activity of the B. edulis and N. luridiformis against Gram-positive and Gram-negative bacteria are shown in Tables 1, 2, respectively. Aqueous extracts from both the species of mushrooms represented high activity against most of the sensitive and resistant clinical isolates, while the methanolic extract showed a lesser activity against all the isolates. Concerning to aqueous extracts, the bacterial inhibition halos from Gram-positive varied between 8 and 25 mm and between 8 and 18 mm for B. edulis and N. luridiformis, respectively (Table 1). Noteworthy, aqueous extract also showed high bacterial inhibition halos in P. aeruginosa, with values varying between 13 and 21 mm and 7 and 17 mm for B. edulis and N. luridiformis, respectively (Table 2). Methanolic extracts from both the species showed lower bacterial inhibition halos values for all the clinical isolates. On the other hand, aqueous and methanolic extracts of both the species of mushrooms were not effective against K. pneumoniae.

The MIC and MBC of extracts against Gram-positive and Gram-negative clinical isolates are given in Tables 3, 4, respectively. The MIC values for Gram-positive bacteria ranged between 15.63 and 62.5 μg.ml⁻¹ and 62.5 and 250 μg.ml⁻¹ for aqueous B. edulis and N. luridiformis, respectively. The MIC values for P. aeruginosa varied from 7.81 to 62.5 μg.ml⁻¹ and 31.25 to 125 μg.ml⁻¹ for aqueous B. edulis and N. luridiformis, respectively. Methanolic extracts from both the species showed lower MICs values for all the clinical isolates. Similar to diffusion test, aqueous and methanolic extracts of both the species were not effective against K. pneumoniae.

Concerning to MBC, the lowest concentration with bactericidal effect of aqueous and methanolic extracts of B. edulis against Methicillin-resistant Staphylococcus aureus (MRSA) were 250 and 500 μg.ml⁻¹, respectively. For P. aeruginosa, the values were 125 μg.ml⁻¹ for aqueous B. edulis extract and 500 μg.ml⁻¹ for methanolic extract. With respect to N. luridiformis, the MBC of aqueous and methanolic extracts against MRSA was 500 μg.ml⁻¹. Similar results were achieved for P. aeruginosa with N. luridiformis aqueous and methanolic extracts.

The efficacy of the different extracts at MIC, 5 × MIC, and 10 × MIC was tested against S. aureus CETC 976 and E. coli CETC 434 for 24 h and the biofilms were characterized in terms of biomass and metabolic activity (Figures 1, 2, respectively). The higher biomass removal was obtained for the aqueous extract of B. edulis in S. aureus (78.0 ± 5.02%) and in E. coli (94.0 ± 8.0%) at 10 × MIC (Figures 1A,B). Concerning the remaining analyzed extracts, the biofilm removal in both the bacterial species presented the following order: N. luridiformis aqueous > B. edulis methanolic > N. luridiformis methanolic for each concentration tested (Figures 1, 2). Concerning to the biofilm inactivation, the treatments with the different extracts caused the highest inactivation and presented the following order for both the biofilms (S. aureus and E. coli): B. edulis aqueous > N. luridiformis aqueous > B. edulis methanolic > N. luridiformis methanolic for each concentration tested (Figures 1, 2). The percentage of biofilm inactivation with the different extracts at 5 × MIC and 10 × MIC was higher than with MIC.

Data of Figure 6 indicate that the cell viability of HFF-1 cell lines, after exposure to all extracts in the different concentrations, showed a cell survival rate of ~90%.