Organic solvents (hexane, methanol, dimethyl sulfoxide (DMSO), acetone, and ethyl acetate) were purchased from Lennox laboratory supplies (Dublin, Ireland). The β-lactam antibiotics (penicillin, oxacillin, ampicillin, and amoxicillin), nitrocefin discs, Mueller-Hinton broth and agar, propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI) and iodonitrotetrazolium chloride (INT) were purchased from Sigma-Aldrich, Merck (Dublin, Ireland).

Commercially available mushroom species were sourced and purchased from Garryhinch Wood Exotic Mushrooms, Co. Offaly, Ireland in November 2021. A fresh mass of 1 kg of mushrooms Agaricus bisporus (portobello), Grifola frondosa (maitake), Hericium erinaceus (lions mane), Hypsizygus tessulatus (white beech), Lentinula edodes (shiitake), Pholiota adiposa (chestnut), Pholiota microspora (forest nameko), Pleurotus citrinopileatus (golden oyster), Pleurotus eryngii (king oyster) and Pleurotus ostreatus (oyster) was freeze dried (model FD 80; Cuddon Freeze Dry, Blenheim, New Zealand) at Teagasc Ashtown Food Research Centre, Dublin, Ireland and then blended into a fine powder (variable speed laboratory blender; Waring, Torrington, CT, USA). Samples were vacuum packed and stored at −20 °C until further use.

Each freeze-dried mushroom powder (20 g) was exhaustively extracted (1:10) using a sequence of solvents of increasing polarity (hexane<methanol<water). Samples were extracted with each solvent, under agitation (150 rpm) and at room temperature, for two consecutive two-hour periods, followed by one sixteen-hour extraction. Extracts were pooled according to their respective solvent and subsequently dried using a rotary evaporator (Rotavapor R-3000; Buchi, Flawil, Switzerland) at 40 °C, while water extracts were freeze dried (Alpha 1-2 LD plus; Martin Christ GmbH, Osterode am Harz, Germany) and transferred to amber vials. Dried methanol extracts were reconstituted in water and then subjected to three successive rounds of liquid-liquid partitioning using 250 mL of ethyl acetate in each case. The organic solvent and water were removed as previously described. All extracts were stored at −20 °C until further use.

Staphylococcus aureus (ATCC 25923) was purchased from the American Type Culture Collection (ATCC). A hospital-acquired (HA) MRSA isolate was donated by Sligo University Hospital (SUH), Sligo, Ireland. All strains were stored on ceramic beads in glycerol at −80 °C. Prior to use, beads were streaked onto fresh nutrient agar plates and grown for 24 h at 37 °C. Three colonies of similar morphology were inoculated into 15 mL of Mueller-Hinton broth and incubated for 16 h at 37 °C. The inoculum was adjusted to a final testing value of 5·10⁵ CFU/mL, prior to testing. The MRSA isolate was positively typed for the mecA gene by SUH. MRSA tested positive, while S. aureus tested negative for β-lactamase production, following nitrocefin application according to the manufacturer’s guidelines (Merck, Dublin, Ireland).

The broth microdilution method was used to determine the antimicrobial activity of extracts, as described previously by Smyth et al. (15). Stock solutions (8 mg/mL) of each sample extract were prepared by initially solubilising in sterile deionised water with φ(DMSO)=8 %. Extracts were sonicated (Elmasonic S180 sonicator (37 kHz); Elma Ultrasonic, Singen, Germany) for 10 min and centrifuged at 2600×g for 5 min using a Sorvall ST4R plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA).

Using a 96-well microtiter plate, the following controls were added in triplicate to lane one: 100 µL of Mueller-Hinton Broth (blank control), 100 µL of ampicillin (0.5 mg/mL, positive control), 100 µL of bacterial culture and a 4 % (V/V) aqueous DMSO solution (final concentration for lane 1, negative control). In the remaining wells, a 2-fold serial dilution of each extract was performed in triplicate across corresponding rows using Mueller-Hinton broth, resulting in a final extract concentration of 4 mg/mL. A volume of 100 µL of bacterial culture (5·10⁵ CFU/mL) was added to all corresponding wells, excluding the blank control. Plates were tested in triplicate and were incubated at 37 °C for 18 h. Following incubation, 40 µL of iodonitrotetrazolium dye (γ(INT)=0.2 mg/mL) was added to each well to assess cell viability, where viable bacterial cells are responsible for the witnessed colour change from clear to red. The wells where no colour change was observed after incubation were determined as the MIC using biological replicates (N=3).

Extracts were evaluated for their ability to modulate four β-lactam antibiotics (ampicillin, amoxicillin, oxacillin and penicillin) against an MRSA isolate using the fractional inhibitory concentration index (FICI), as described previously by Embaby et al. (16). Extracts were diluted to one quarter their respective MIC and added to subtherapeutic concentrations of antibiotic in triplicate, where they were then incubated with an inoculum of 5·10⁵ CFU/mL at 37 °C for 18 h. Equivalent extract volume fractions of DMSO (2 % final) were added to the negative controls containing subtherapeutic concentrations of antibiotic. Cell viability was assessed as previously described using biological replicates (N=3). Antibiotic modulation was classified as synergistic (<0.5), additive (0.5–1), indifferent (>1–4), and antagonistic (>4) according to the FICI (13):

The cell viability assay was used to determine cell membrane leakage, as previously described by Khamrai et al. (17) with minor modifications. A single MRSA colony was inoculated in Mueller-Hinton broth and incubated at 37 °C until mid-logarithmic stage absorbance A_{600 nm}=0.5 was reached. Next, 800 μL of culture were added to all Eppendorf tubes. The remaining 200 μL was comprised of the antibiotic/extract concentration, while volume fraction of 1 % DMSO was added to the negative control to serve as the vehicle control. Samples were incubated for 1 h at 37 °C under agitation at 150 rpm. Following incubation, samples were centrifuged at 8000×g for 8 min (Prism microcentrifuge; Labnet International, Edison, NJ, USA), where the supernatant was discarded and the pellet resuspended in 800 μL of Ringers solution. Then, 100 μL of DAPI solution (0.1 mg/mL) and 100 μL of PI (0.1 mg/mL) were added and incubated in the dark for 30 min at 37 °C under agitation (150 rpm). Following incubation, 10 μL of sample were analysed using a Canon EOS 1300D (Tokyo, Japan) camera attached to Nikon Eclipse 80i (Tokyo, Japan) for fluorescent microscopy. EOS utility software (v. 3.7.0.0) was used for capturing images (400× magnification) (ISO 100) with ND4 filter applied to remove background fluorescence. Daptomycin was used as a positive control. All samples and controls were tested as biological triplicates (N=3).

The total phenolic content of all crude extracts was evaluated (N=3) as previously described by Kenny et al. (18). Briefly, 100 μL of extract solubilised in methanol were added to a microcentrifuge tube, followed by 100 μL of methanol, 100 μL of Folin-Ciocalteu (2 N) reagent and 700 μL of sodium carbonate (20 % m/V). A volume of 100 μL of methanol was used in place of the extract for the blank control. Each microcentrifuge tube was vortexed and incubated in the dark, at room temperature, for 20 min. Samples were then centrifuged at 16 000×g (Prism microcentrifuge; Labnet International) for 3 min and the absorbance was measured at 735 nm using a spectrophotometer (model 6300; Jenway, Essex, UK). A gallic acid calibration curve (0.01–0.2 mg/mL) was used to express the results as gallic acid equivalents (GAE) on dry mass extract basis (mg/mg) following linear regression.

In this study, the ethyl acetate extract exhibited the strongest overall antimicrobial activity against S. aureus (MIC=0.8 mg/mL). The antimicrobial activity of an H. erinaceus ethanol extract against S. aureus has been previously reported (MIC=2.5 mg/mL) (19), with the authors attributing the activity to the total phenolic content present. In the present study, the H. erinaceus ethyl acetate extract demonstrated an antimicrobial effect against MRSA (MIC=4 mg/mL) and a total phenolic value, expressed as GAE on dry mass extract basis, of 0.03112 mg/mg, potentially suggesting that other compounds may be responsible for the outlined activity.

Despite the suggested use of H. erinaceus as an antibiotic modulator (20), its modulating potential remains unknown. In this study, H. erinaceus demonstrated synergism (FICI≤0.5) with amoxicillin, ampicillin and penicillin against an MRSA isolate, with a maximum 12.5-fold MIC reduction (FICI=0.33) in combination with ampicillin. These results indicate that H. erinaceus may serve as a novel source of antibiotic-modulating compounds.

In the present study, the ethyl acetate extract of Hypsizygus tessulatus demonstrated antimicrobial activity against both S. aureus and MRSA (MIC=4 mg/mL). Previous studies have reported weak antimicrobial activity (44 % inhibition) of water extracts derived from H. tessulatus stalks (1.2 mg/mL) (21).

In addition to the outlined antimicrobial activity, H. tessulatus demonstrated synergism (FICI=<0.5) with ampicillin (FICI=0.37), amoxicillin (FICI=0.41) and penicillin (FICI=0.49), resulting in 8.3-, 6.3- and 4.2-fold MIC reductions, respectively. To our knowledge, this is the first study to report on the antibiotic-modulating effects of H. tessulatus, highlighting it as a potential novel source of antibiotic-modulating compounds.

The ethyl acetate extracts of Pleurotus citrinopileatus were active against MRSA and S. aureus (MIC=1 mg/mL). Previous reports on P. citrinopileatus crude ethyl acetate extract indicate an antimicrobial effect against S. aureus (MIC=2.5 mg/mL) (22). The lipid metabolite glucosylceramide was suggested to be responsible for the outlined activity.

In the current study, despite showing no antimicrobial activity, P. eryngii ethyl acetate extract (1 mg/mL) reduced ampicillin MIC 3.6-fold (FICI=0.53) against MRSA, indicating additive properties (FICI=0.5–1). A previous study (23) reported synergism (FICI=<0.5) between a P. eryngii water extract (400 mg/mL) and ampicillin against a S. aureus isolate (FICI=0.19).

In this study, P. ostreatus extracts exhibited no antimicrobial or antibiotic-modulating properties. A previous study reported the antimicrobial activity of P. ostreatus mycelial extract (V(methanol):V(glycerol):V(water)=1:1:1) against S. aureus by zone-of-inhibition (disc diffusion) of (29.0±0.3) mm at an unreported concentration (24). However, it did not demonstrate antibiotic modulation with norfloxacin against B. subtilis. Aqueous ethanol (φ=60 %) extracts derived from P. ostreatus were evaluated for meropenem-modulating properties (25 mg/mL) against extended-spectrum β-lactamase-producing E. coli and K. pneumoniae isolates (14). A 4-fold reduction in meropenem MIC was reported; however, the extracts were not tested at subinhibitory concentrations, and synergism was defined using a FICI threshold of ≤1. In addition, the isolates exhibited no baseline resistance to meropenem prior to modulation. Similarly, a methanol (φ=80 %) extract derived from Fistulina hepatica (20 mg/mL) demonstrated synergistic activity (FICI=0.3) in combination with penicillin and ampicillin against MRSA (25). As in the previous study (14), subinhibitory concentrations of the mushroom extracts were not evaluated, reflecting differences in experimental design and criteria used to define synergism.

The antimicrobial and antibiotic modulating activity of species derived from the Pholiota family remain scarce. Thus, to the best of our knowledge, this is the first report on the antimicrobial (MIC=1 mg/mL) activity of Pholiota microspora.

In the present study, P. adiposa demonstrated inhibitory properties against both MRSA and S. aureus (MIC=4 mg/mL), while also exhibiting a 25-fold reduction in ampicillin MIC against MRSA (FICI=0.29), indicating synergism (FICI≤0.5). Previous studies evaluating the antimicrobial activity of ethanolic extracts derived from P. adiposa reported greater activity against MRSA (MIC=0.1 mg/mL) and S. aureus (MIC=0.2 mg/mL) (26). In that study, MIC values were determined using an agar dilution method, although the inoculum size was not specified. The relatively low abundance of phenolics in P. adiposa ethyl acetate extract, expressed as GAE on dry mass extract basis, ((0.0256±0.0002) mg/mg) in the present study suggests that non-phenolic compounds may be responsible for the reported activity.

In this study, A. bisporus ethyl acetate extract demonstrated inhibition against both S. aureus and MRSA (MIC=4 mg/mL). A. bisporus methanolic (MIC=0.6 mg/mL) and ethanolic (MIC=0.03 mg/mL) extracts have previously demonstrated antimicrobial activity against S. aureus (27). Notably, the bacterial inoculum tested was 10⁴ CFU/mL, which is lower than that in this study (5·10⁵ CFU/mL) and might explain the variance. The A. bisporus ethyl acetate extract contained phenolics, expressed as GAE on dry mass extract basis, (0.0332±0.0004) mg/mg, which may indicate that phenolics are not responsible for the observed activity.

A. bisporus ethyl acetate extract was the most effective antibiotic-modulating extract, lowering the MIC of penicillin, ampicillin and amoxicillin to 0.01 mg/mL, a 25-fold reduction (FICI=0.29), indicating antibiotic synergism. A previous study demonstrated synergistic effects between A. bisporus ethanol and acetone extracts (25 mg/mL) and the anti-staphylococcal drug, AFN–1252 (0.0005 mg/mL), with the authors noting the potential application of A. bisporus as an antibiotic modulator (28). It was suggested that unsaturated fatty acids, in particular linoleic acid, were responsible for the outlined activity, most likely by disrupting the cell membrane and cell wall of the MRSA isolate. Synergism was inferred without the calculation of the FICI value, as the MIC of the A. bisporus extract was not determined.

In this study, the ethyl acetate extract of A. bisporus has demonstrated increased cell membrane permeability through the cell viability assay (Fig. 1). When subtherapeutic concentrations of A. bisporus and ampicillin are tested individually, there is minimal effect on cell membrane damage. However, when both A. bisporus and ampicillin are used as a combination therapy, there is a notable increase in cell membrane leakage. Similarly, an ethanol extract derived from A. bisporus has previously shown cell membrane leakage against S. aureus (MIC=9.54 mg/mL) when used as an antimicrobial agent (29). This finding may illustrate the mechanism of action responsible for A. bisporus ability to modulate β-lactam antibiotics. It is suggestive that decreasing cell membrane integrity facilitates the localised concentration of antibiotic within the penicillin-binding protein domains, while simultaneously accelerating osmotic imbalance and subsequent cell lysis. However, no modulation was observed with oxacillin. Oxacillin resistance in MRSA is mediated by low β-lactam affinity for PBP2a while being resistant to the effects of β-lactamase, resulting in maximal activity to native PBPs. Consequently, an increase in membrane permeability or a reduction in β-lactamase activity would not be sufficient to enhance oxacillin activity, because the predominant resistance mechanism, PBP2a, remains unaffected.

A. bisporus is one of the most cultivated edible mushrooms globally, while it is estimated that 5–20 % of A. bisporus production generated is wasted due to physical abnormalities (30). While the repurposing of A. bisporus waste may serve as a sustainable source of effective antibiotic modulators, the approach has certain limitations for use in the food and biotechnology sectors. The active extract is obtained in low yields (0.48 % from dry mass; Table 3), which may cause difficulty for further purification and bioactivity-guided fractionation testing. In addition, the above results are obtained from a single MRSA isolate, which may not reflect the genetic and phenotypic diversity of MRSA strains, thereby limiting the broader applicability of the results. These factors indicate that further optimisation and testing against additional clinical isolates are needed before A. bisporus waste-derived antibiotic modulators can be translated into industrial or clinical applications.