The escalating rise of antibiotic-resistant bacterial pathogens poses a critical threat to global public health, causing millions of annual fatalities [1]. Consequently, the development of alternative therapeutic strategies is of paramount importance. Mushrooms have been utilized for thousands of years as natural medicine to treat infections due to their rich composition of bioactive phytochemicals and secondary metabolites that strengthen the immune system and fight pathogens [2][3].

Several mushroom species, notably reishi (Ganoderma lucidum) and turkey tail (Trametes versicolor), are recognized for their potent antimicrobial properties. These effects are largely driven by high concentrations of bioactive compounds, such as triterpenes and polysaccharides (e.g., beta-glucans), which not only directly inhibit pathogens but also modulate the immune system and act as prebiotics to support gut health [4]. While most research has independently examined the antimicrobial actions of reishi and turkey tail, limited studies have investigated their combined effects [5]-[8]. However, other studies suggest that combined mushroom extracts can exhibit potent synergistic antimicrobial activity against S.aureus and E.coli, while also enhancing the efficacy of antibiotics against drug-resistant pathogens [9].

Research suggests that synergistic combinations of medicinal mushrooms, such as Ganoderma lucidum and Lentinulaedodes, reduce MIC values for superior pathogen inhibition [10]. This study addresses a gap in the literature regarding the combined effect of reishi (aqueous extract) and turkey tail (ethanol extract). This approach harnesses diverse phytochemicals; ethanol typically extracts potent terpenoids and sterols, while water extracts polar polysaccharides, allowing for a broader, synergistic antimicrobial spectrum.

Bacterial infections, particularly from superbugs like Staphylococcusaureus, are a major global health threat, causing millions of deaths, complicated by rapidly growing antibiotic resistance, requiring urgent development of new treatments, with natural sources [13]. Mushrooms are being investigated as new sources for antimicrobial agents due to the diverse bioactive compounds (such as phenolics, polysaccharides, terpenoids, and alkaloids) they naturally produce [14][15]. These compounds combat drug-resistant pathogens by interfering with cell walls, membranes, and metabolic functions [16][17].

This study investigated the synergistic antimicrobial effects of turkey tail (Trametes versicolor) and reishi (Ganoderma lucidum) mushroom extracts against S.aureus. The research aimed to fill a significant gap, as prior studies concentrated on individual mushroom benefits or general antibacterial properties, not the combined potential of these two specific extracts against this resistant bacterium [8][12]. We found that both the alcohol extracts of turkey tail mushroom and the aqueous extracts of reishi mushroom demonstrated antimicrobial activity against the bacterium S.aureus. These mushroom extracts demonstrated potent and rapid antimicrobial action, beginning within 3 hours of exposure. The rapid antimicrobial effect of mushroom extracts, sometimes occurring within just a few hours, primarily stems from the direct interaction of their potent bioactive compounds, such as beta-glucans, ganoderic acid, and triterpenoids, with essential microbial structures and processes [5][18]. This interaction includes disrupting cell membranes and inhibiting vital enzymes, leading to swift cellular dysfunction and death [5][12][18].

The presence of these key antimicrobial compounds was confirmed through High Performance Liquid Chromatography (HPLC) analysis, consistent with findings in another research [19]. HPLC analysis is crucial for the study of bioactive compounds in mushrooms because it enables their accurate identification, separation, and quantification. Moreover, this versatile technique provides essential data for quality control, pharmacological research, and the development of mushroom-based food supplements and pharmaceuticals. Higher peaks for beta (1 - 3) glucan and ganoderic acid in a mushroom extract’s HPLC analysis indicate elevated concentrations of these bioactive compounds. This increased concentration is associated with greater potential antimicrobial activity, according to research published by Akamu et al. [12].

Alcohol extracts of turkey tail demonstrated greater antimicrobial efficacy than an aqueous extract derived from reishi mushroom. The stronger antimicrobial activity observed in alcohol extracts of turkey tail compared to aqueous reishi extracts stems from a combination of the mushrooms’ inherent chemistries and the solvent used for extraction. Turkey tail is rich in protein-bound polysaccharides, terpenes, and high concentrations of polyphenols, while reishi is primarily known for its triterpenoids and polysaccharides [20][21]. The key difference in antimicrobial effectiveness lies in the extraction solvent. Alcohol, being less polar than water, is more efficient at dissolving a broader spectrum of organic, non-polar, and semi-polar bioactive compounds (such as triterpenoids, sterols, and polyphenols), which typically exhibit more potent antimicrobial properties [22]. In contrast, water extracts primarily yield polysaccharides and water-soluble proteins, whose antimicrobial effects are often less potent or differ in mechanism, thus explaining the superior activity of the alcohol-based turkey tail extraction in this comparison [23]. In this study, it was observed that plain solvent ethanol exhibited a stronger antimicrobial activity against S.aureus than mushroom extracts because ethanol (typically 70% - 90%) acts as a potent, immediate bactericidal agent that denatures proteins and dissolves cell membranes, whereas mushroom extracts are complex mixtures containing lower concentrations of bioactive compounds. Ethanol kills quickly (seconds), while extracts require higher concentrations for inhibiting bacterial growth [24].

Reishi and turkey tail mushrooms exhibit greater antimicrobial activity when combined due to synergistic effects, where their diverse bioactive compounds like polysaccharides (beta-glucans), triterpenes, and phenolics enhance each other’s effects [17][25]. The study confirmed a synergistic effect between alcohol extract of turkey tail and aqueous extract of reishi using the Fractional Inhibitory Concentration (FIC) index. The data indicated synergy because the FIC index was found to be less than 1. This suggests that when used together, the two extracts have a greater inhibitory effect than the sum of their individual effects.

Polysaccharide-K (PSK) and Polysaccharide Peptide (PSP) target bacteria by damaging cell envelopes and disrupting metabolism, while also acting as a prebiotic that reduces harmful gut bacteria, for example, S.aureus and E.coli [25]. Reishi’s action complements this by disrupting microbial cell walls, inhibiting pathogen replication, and inducing oxidative stress [17][26]. This multi-pathway approach makes their combination a powerful functional food effective against a broader range of bacteria, fungi, and viruses than either mushroom alone [17][25]. It has been reported that combining polysaccharides with phenolic compounds creates a potent synergistic antifungal treatment that is more effective at inhibiting the growth and biofilm formation of C.albicans than either compound alone [27]. This powerful effect stems from a dual mechanism [17]: the polysaccharides work to disrupt the structural integrity of fungal cell walls, while the phenolic compounds simultaneously induce damaging oxidative stress within the cells. Furthermore, the phenolic compounds help regulate the accumulation of Reactive Oxygen Species (ROS) to optimal cytotoxic levels, and the polysaccharides enhance the host’s immune response, leading to a robust and effective antifungal action.

Genomic analysis of Staphylococcusaureus (shown in Table 3) identified two key mutations linked to antibiotic resistance: an ATP-binding protein (KQ76_RS04770) and glutathione peroxidase (KQ76_RS13475). ATP-binding proteins are crucial bacterial proteins that use ATP hydrolysis to drive antibiotic efflux, directly causing resistance, allowing bacteria to survive high concentrations of antibiotics, while glutathione peroxidase provides protection against oxidative stress [28][29]. Mushroom extracts target antibiotic resistance mechanisms, including ATP-Binding Cassette (ABC) transporters and oxidative stress defenses like glutathione peroxidase, primarily through the action of bioactive compounds that inhibit these systems or create a synergistic effect with conventional antibiotics. These extracts often work by inhibiting the efflux pumps that bacteria use to eject antimicrobials, thereby increasing the intracellular concentration of antibiotics. Furthermore, mushroom extracts effectively induce oxidative stress in bacteria, leading to membrane damage and reduced viability. Targeting the genes responsible for these proteins offers a potential strategy for creating new therapies to treat S.aureus infections. Additionally, the study highlights that extracts from turkey tail and reishi mushrooms show high antibacterial effectiveness, suggesting they could serve as alternative treatments to conventional antibiotics.

In this study, other mutations in S.aureus were associated with adaptation and cellular functions such as in the case of the following genes:DUF1433 domain-containing protein (KQ76_RS09235); DNA-binding heme response regulator (hssR); alpha/beta hydrolase (KQ76_RS13020); glutathione peroxidase (KQ76_RS13475); ribosome biogenesis GTPase (ylqF); ECF-type riboflavin transporter substrate-binding protein (KQ76_RS13825); tRNA uridine 5-carboxymethylaminomethyl(34) synthesis enzyme (mnmG); D lactate dehydrogenase (KQ76_RS12955); M23 family metallopeptidase/HAD IIB family hydrolase (KQ76_RS11280/KQ76_RS11285); BglG family transcription antiterminator (KQ76_RS10985); serine tRNA ligase/AzlC family ABC transporter permease (serS/KQ76_RS00050); hypothetical protein (KQ76_RS09255). DUF1433 domain-containing protein (KQ76_RS09235) has unknown function; DNA-binding heme response regulator (hssR) regulates DNA-binding activity and controls gene expression [30]; alpha/beta hydrolase (KQ76_RS13020) is responsible for the hydrolysis of ester and peptide bonds [31]; Glutathione peroxidase (KQ76_RS13475) protects the cell from oxidative damage by reducing lipid hydroperoxides to alcohols and hydrogen peroxide to water [29][32]; ribosome biogenesis GTPase (ylqF) is involved in ribosome assembly, translation, and signal transduction [33]; ECF-type riboflavin transporter substrate-binding protein (KQ76_RS13825) is a transmembrane protein that help uptake micronutrients, such as B-type vitamins and cations, into cells [34]; tRNA uridine 5-carboxymethylaminomethyl(34) synthesis enzyme (mnmG) is involved in tRNA modification [35]; D lactate dehydrogenase (KQ76_RS12955) is necessary for glycolysis [36]; M23 family metallopeptidase/HAD IIB family hydrolase (KQ76_RS11280/KQ76_RS11285) is used by bacteria to lyse cell walls of other bacteria and nematodes [37]; BglG family transcription antiterminator (KQ76_RS10985) controls the expression of carbohydrate transporters [38]; serine tRNA ligase/AzlC family ABC transporter permease (serS/KQ76_RS00050) catalyzes the attachment of serine to tRNA(Ser) [39]; hypothetical protein (KQ76_RS09255) is associated peptidoglycan metabolism, cell wall organization, ATP hydrolysis, and outer membrane fluidity [40]. Mutations in exonuclease SbcCD subunit D (KQ76_RS09465) and conserved phage C-terminal domain-containing protein (KQ76_RS07500) play a role in DNA repair, environmental sensing, and regulating genes [41][42]. It can be argued that these two selective sweeps displayed in specific genes are associated with DNA repair, environmental sensing, and bacterial growth enhancement.

Whole-genome sequencing of control cells revealed that most mutations occurred in genes controlling gene expression, ester bond hydrolysis, micronutrient uptake, tRNA modification, carbohydrate metabolism, DNA repair, and environmental sensing. Specifically, mutations affecting protein synthesis, gene expression, and carbohydrate metabolism can drive antibiotic resistance in bacteria. These modifications allow bacteria to adapt by altering their physiology, reducing drug uptake, or modifying target sites.

Several limitations to this study should be noted. First, the investigation was limited to Gram-positive bacteria, excluding tests against Gram-negative strains. Second, molecular analysis did not include whole-genome sequencing of the treated samples. Finally, the scope of the study was restricted to a limited selection of bacterial strains rather than a broad, diverse panel.

The escalating crisis of antimicrobial resistance has galvanized the search for alternative antimicrobial agents, including the exploration of the mycochemicals derived from medicinal mushrooms for novel bioactive compounds with potential therapeutic applications. This study examined the antimicrobial efficacy of an alcohol-based extract from the turkey tail mushroom (Trametes versicolor) and an aqueous extract from the reishi mushroom (Ganoderma lucidum). The study also examined S. aureus for genes that confer resistance to antimicrobial drugs. The key finding was that the combined extracts demonstrated a stronger inhibitory effect against Staphylococcus aureus compared to either extract used individually, suggesting a synergistic relationship. Chemical analysis via High Performance Liquid Chromatography (HPLC) revealed that the antimicrobial activity is attributed to shared bioactive compounds in both extracts, including ganoderic acids, triterpenoids, and beta (1 - 3) glucans. These compounds are believed to exert their effect by interfering with vital microbial cellular processes necessary for survival, such as oxygen uptake, oxidative phosphorylation, and DNA synthesis. Following the analysis, sequencing revealed multiple resistance-related genes in S. aureus. The observed synergistic effect offers promise for developing novel treatment options for infectious diseases. Future work will focus on testing the antimicrobial potential of reishi and turkey tail extracts against various Gram-positive and Gram-negative bacteria. Furthermore, we will sequence both the control and treated bacterial cells to pinpoint specific mutations that confer resistance to these extracts.

The authors sincerely thank the Department of Biological Sciences, Winston-Salem State University, for all the logistics.

The Genomic Research and Data Science Center for Computation and Cloud Computing (GRADS-4C) (211512) funded this project.