Restoration of Antibiotic Effectiveness with P. hartigii Extract Against Multidrug-Resistant E. coli  ^†

Abstract

Antibiotic resistance poses a critical threat to global health, largely driven by bacterial efflux pumps that expel antibiotics and reduce their efficacy. This study investigated the potential of Phellinus hartigii ethyl acetate extract (Ph-Ace) to inhibit efflux pumps and restore antibiotic activity against multidrug-resistant Escherichia coli strains. In vitro assays demonstrated that Ph-Ace effectively inhibited efflux pumps, enhancing the efficacy of resistant antibiotics. GC/MS analysis identified key components such as nonadecane and octacosane. These findings suggest Ph-Ace as a promising natural efflux pump inhibitor. Further molecular and clinical studies are required to confirm its therapeutic potential.

1. Introduction

For nearly a century, antibiotics have been fundamental to modern medicine, saving millions of lives by effectively treating bacterial infections. Unfortunately, the emergence and spread of antibiotic resistance pose a serious threat, risking a return to the pre-antibiotic era and leading to higher mortality rates. In 2019, antibiotic resistance was linked to over one million deaths worldwide [1,2].

Understanding the underlying mechanisms of antibiotic resistance is crucial for the development of effective therapies [3]. Microorganisms utilize efflux pumps to evade antimicrobial agents and survive in hostile environments [4]. These efflux pumps reduce the intracellular concentration of drugs without altering the antibiotic molecules themselves [5,6]. Efflux pumps are present in both prokaryotic and eukaryotic cells [7]. Efflux pumps are membrane proteins that expel toxic compounds, including antibiotics, from bacterial cells. Found in both Gram-positive and Gram-negative bacteria, they are classified into five important major families: RND (Resistance–Nodulation–Division), MFS (Major Facilitator Superfamily), ABC (ATP-Binding Cassette), SMR (Small Multidrug Resistance), and MATE (Multidrug And Toxic compound Extrusion). While most are single-component systems, the RND family, found only in Gram-negative bacteria, functions as a tripartite complex: an inner membrane transporter, a membrane fusion protein, and an outer membrane channel. This structure enables direct drug efflux and plays a crucial role in multidrug resistance [8].

The use of combination therapies that pair efflux pump inhibitors (EPIs) with antibiotics has attracted significant interest as a promising strategy to restore antibiotic efficacy [9,10]. Despite the many EPIs discovered, clinical use is limited due to toxicity, formulation, and pharmacokinetic issues [11]

Medicinal compounds derived from mushrooms, particularly those belonging to the Phellinus genus, are widely recognized and utilized globally for their diverse bioactivities, including antioxidant, antidiabetic, anti-inflammatory, antimicrobial, antiallergic, and hepatoprotective properties [12].

The main aim of this study was to investigate the potential of Phellinus hartigii (P hartigii) extracts, particularly P. hartigii acetone (Ph-Ace), to inhibit efflux pumps and thereby restore the efficacy of antibiotics against multidrug-resistant Escherichia coli (E. coli) strains. By evaluating the biological activities of the extract and its ability to reverse resistance mechanisms, this study seeks to contribute to the development of alternative therapeutic strategies to combat antibiotic resistance.

2. Materials and Methods

2.1. Access to Phellinus hartigii

The Phellinus hartigii (Allesch. & Schnabl) Pat. specimen used in this study was collected from Ilgaz Mountain National Park, located in Kastamonu Province, Türkiye, a region known for its rich biodiversity and diverse fungal flora.

2.2. Extraction

Dried P. hartigii sample (1 g) was extracted with 100 mL acetone by shaking at 125 rpm for 2 days at room temperature. After filtration and vacuum evaporation at 50 °C, extract stocks (4 mL) of P. hartigii (Ph-Ace) were prepared in DMSO.

2.3. GC-MS Analysis

GC/MS analysis was carried out using a Shimadzu-QP 2010 Ultra system (Shimadzu, Kyoto, Japan). Compounds that accounted for more than 5% of the total signal intensity in the chromatogram were considered major components. These compounds were further evaluated in terms of their chemical structure and potential biological activity.

2.4. Preparation of the Inoculum

E.coli strains were selected based on prior resistance profiling at Kastamonu University, Department of Biology, Microbiology Research Laboratory [13]. The BD Phoenix system (Becton Dickinson, Sparks, MD, USA) was employed for bacterial identification. Resistance profiles indicate that E. coli 3 is resistant to Aztreonam (ATM) and Cefixime (CFM); E. coli 7 resists Amoxicillin-Clavulanate (AMC) and Piperacillin-Tazobactam (TZP); E. coli 8 shows resistance to AMC, TZP, and Ceftriaxone (CRO); and E. coli 10 is resistant to ATM. Strains were activated in LB and TSB broths (Merck, Darmstadt, Germany), and pure colonies were obtained on corresponding agars. Exponentially growing cultures were prepared, and bacterial suspensions were adjusted to 0.5 McFarland standard in 0.9% sterile saline using a spectrophotometer.

2.5. Minimum Inhibition Concentration (MIC) Test

The antimicrobial activity of Ph-Ace extract against selected multidrug-resistant strains was assessed by MIC determination using 96-well microplates. To minimize toxicity, the original extract stock (50% DMSO) was diluted to a 1% DMSO working solution. Each well contained sterile Mueller Hinton Broth (Merck, Germany) inoculated with 100 mg/L bacterial suspension adjusted to 0.5 McFarland. Plates were incubated at 37 °C for 24 h, and the MIC was recorded as the lowest concentration inhibiting visible growth. Additionally, EPI application of verapamil, phenyl arginine beta napthylamide (PaβN) and thioridazine hydrochloride (Sigma Aldrich, St. Louis, MO, USA) was performed on these strains in previous studies [13]. All assays were conducted in triplicate to ensure reproducibility and reliability.

2.6. Efflux Pump Inhibition Test

The efflux pump inhibitory activity of Ph-Ace extract was evaluated using ethidium bromide (EtBr) dye. The extract (2.0 mg/L, MIC/2) was added to TSB agar plates with EtBr concentrations ranging from 0 to 2.5 mg/L (0, 0.5, 1, 1.5, 2, and 2.5 mg/L). After incubation at 37 °C for 24 h, the plates were examined under 366 nm UV light to assess intracellular EtBr accumulation [14].

2.7. Combined Study with Antibiotics and Extract

To assess the potential efflux pump inhibitory activity of the Ph-Ace extract against multidrug-resistant Escherichia coli strains, a combination assay was performed using 24-well plates. Each well was filled with 1 mL of sterile Mueller–Hinton Broth (MHB), followed by the addition of 100 mg/L of the P. hartigii acetone extract (corresponding to half of its previously determined MIC value). An antibiotic disc—selected based on the specific resistance profile of the tested E. coli strain, was placed into each well. The antibiotics used included CFM, 5 μg, CRO, 30 μg, AMC, 30 μg, aztreonam ATM, 30 μg, and piperacillin-TZP, 36 μg. A standardized bacterial suspension (10⁶ CFU/mL) of each resistant E. coli isolate was then inoculated into the wells. The plates were incubated at 37 °C for 24 h under aerobic conditions. This setup aimed to observe any potential enhancement of antibiotic activity in the presence of the extract, indicating efflux pump inhibition [14].

2.8. Using OpenAI

During the manuscript preparation, the authors used ChatGPT (OpenAI, version July 2025) to improve language fluency and structure. All outputs were carefully reviewed and revised by the authors to ensure scientific accuracy and originality.

2.9. Statistical Analysis

A t-test (p = 0.05) was conducted to determine statistical significance, using R Studio 2024.04.2 for analysis.

3. Results

3.1. Resistance Profiles of E. coli Strains

The resistance profiles of E. coli strains are presented in (

Table 1. Resistance profile of E. coli.

Strains	Antibiotics	MIC (µg/mL)	Susceptibility Interpretation
E. coli 3	Aztreonam (ATM)	4	I
E. coli 3	Cefixime (CFM)	>2	R
E. coli 7	Amoxicillin-Clavulanate (AMC)	32/2	R
E. coli 7	Piperacillin-Tazobactam (TZP)	16/4	R
E. coli 8	Amoxicillin-Clavulanate (AMC)	32/2	R
E. coli 8	Piperacillin-Tazobactam (TZP)	16/4	I
E. coli 8	Ceftriaxone (CRO)	>4	R
E. coli 10	Aztreonam (ATM)	8	R

I (intermediate) and R (resistant).

) for comparison.

Minimum inhibitory concentrations (MICs) for selected antibiotics were determined against four multidrug-resistant E. coli strains. The following was found, according to the interpretative criteria.

E. coli 3 exhibited intermediate susceptibility to aztreonam (ATM, MIC = 4 µg/mL) and was resistant to cefixime (CFM, MIC > 2 µg/mL). E. coli 7 was resistant to both amoxicillin-clavulanate (AMC, MIC = 32/2 µg/mL) and piperacillin-tazobactam (TZP, MIC = 16/4 µg/mL). E. coli 8 showed resistance to AMC (32/2 µg/mL) and ceftriaxone (CRO, MIC > 4 µg/mL), while demonstrating intermediate susceptibility to TZP (16/4 µg/mL). E. coli 10 was resistant to ATM (MIC = 8 µg/mL).

These findings confirmed the multidrug-resistant profiles of the strains and were used to guide the selection of antibiotics for the efflux pump inhibition assays.

3.2. Minimum Inhibition Test

The MIC value for Ph-Ace was determined to be greater than 0.300 mg/mL. This indicates that concentrations above 0.300 mg/mL are required to effectively inhibit the growth of the tested bacterial strains. Such a high MIC suggests that while Ph-Ace exhibits antimicrobial activity, its potency may be moderate, necessitating further optimization or combination with other agents to enhance efficacy.

3.3. Efflux Pump Inhibition

As a result of evaluating the efflux pump inhibitory activity of the Ph-Ace extract using ethidium bromide (EtBr) dye, serially diluted plates were prepared and incubated at 37 °C for 24 h. After incubation, the plates were observed under UV light at a wavelength of 366 nm, and intracellular EtBr accumulation was assessed. Observations revealed that in the presence of the extract, the efflux of EtBr from the cells was reduced. Notably, at extract concentrations of 1.00 mg/L and above, the dye was retained within the cells. This indicates that the Ph-Ace extract inhibits the function of efflux pumps, preventing the extrusion of the dye from the cells, and thus demonstrates its potential as an efflux pump inhibitor.

Efflux pump inhibition assays demonstrated that both Phellinus hartigii extracts exhibited significant inhibitory effects on the efflux pump activity of multidrug-resistant E. coli strains. Building on these promising findings and considering the specific antibiotic resistance profiles of the tested E. coli strains, subsequent experiments were designed in which the extracts were co-administered alongside antibiotics to which these strains had previously exhibited resistance. Particular focus was placed on antibiotics whose resistance mechanisms are known to involve efflux pump activity, aiming to evaluate the potential of the extracts to restore antibiotic susceptibility by blocking efflux-mediated drug extrusion.

The antibiotics evaluated in combination with the extracts are summarized in the antibiotics list (

Table 2. Reversal of antibiotic resistance by extract co-treatment.

Strains	Antibiotics
E. coli 3	Aztreonam (ATM)
E. coli 7	Piperacillin-Tazobactam (TZP)
E. coli 8	Piperacillin-Tazobactam (TZP)
E. coli 10	Aztreonam (ATM)

), highlighting those whose efficacy was restored. These include agents for which resistance was notably overcome or reduced upon co-treatment, indicating that the extracts may effectively potentiate the antibacterial effects of these drugs by inhibiting the bacterial efflux pumps. This interaction underscores the therapeutic potential of the extracts as adjuvants to existing antibiotic regimens, especially in combating multidrug resistance.

3.4. GC/MS

Gas chromatography–mass spectrometry (GC/MS) analysis of the Ph-Ace extract was conducted to characterize its chemical composition. The analysis revealed several compounds, with the major constituents defined as those present at concentrations exceeding 10% of the total extract. Nonadecane (28.22%), octacosane (13.03%), and diacetone alcohol (12.79%) were identified as the predominant components (

Table 3. GC/MS results.

Major Compounds	Respective Quantities (%)
28.22	Nonadecane
15.95	Diacetone
13.03	Octacosane
8.18	1-Hexadecanol
5.31	Tetracosyl pentafluoropropionate

). These major compounds provide insight into the chemical profile and potential bioactivity of the extract.

4. Discussion

In this study, the Ph-Ace extract was tested in vitro on a group of E.coli strains exhibiting varying degrees of antibiotic resistance. The main aim was to evaluate whether the Ph-Ace extract could help overcome resistance by restoring the efficacy of antibiotics that had lost their potency, particularly through efflux pump inhibition.

The activity of certain antibiotics against E. coli strains 3, 7, 8, and 10 was successfully restored following treatment with the Ph-Ace extract (Table 2). This effect is believed to result from the extract’s ability to inhibit efflux pump membrane proteins that actively expel antibiotics from bacterial cells, thereby enabling resistance.

Efflux pumps are among the most common and effective bacterial resistance mechanisms. By reducing the intracellular concentration of antibiotics, they render treatments less effective or even completely ineffective. Therefore, inhibiting efflux pumps represents a critical strategy to overcome resistance and improve antibiotic efficacy.

The results of this study demonstrate that Ph-Ace extract can serve as a natural efflux pump inhibitor, restoring antibiotic susceptibility in resistant strains. This finding is significant because it suggests a way to enhance the effectiveness of existing antibiotics without the immediate need for the costly and time-consuming development of new drugs.

Using efflux pump inhibitors like Ph-Ace in combination therapies could potentially lower required antibiotic doses, reduce side effects, and slow the emergence and spread of multidrug-resistant bacteria. Consequently, this approach offers a promising strategy to address the growing global challenge of antimicrobial resistance by extending the useful lifespan of current antibiotics.

Further studies are warranted to elucidate the precise molecular mechanisms underlying the efflux pump inhibition by Ph-Ace extract. Detailed investigations at the genetic and proteomic levels could provide deeper insights into how the extract interacts with bacterial efflux systems. Additionally, in vivo studies and clinical trials will be necessary to confirm the efficacy and safety of Ph-Ace as an adjuvant therapy. Exploring the potential synergistic effects of Ph-Ace with a broader range of antibiotics could also expand its applicability in combating multidrug-resistant infections.

5. Conclusions

This study demonstrated that the Ph-Ace has promising potential as an efflux pump inhibitor against multidrug-resistant Escherichia coli strains. Although the extract showed moderate antimicrobial activity alone, with an MIC value exceeding 0.300 mg/mL, its combination with antibiotics significantly restored and enhanced antibiotic efficacy in resistant strains. This effect indicates that Ph-Ace could serve as a natural adjuvant to improve the effectiveness of existing antibiotics, potentially delaying the need for new drug development. Chemical analyses identified major bioactive compounds such as nonadecane, octacosane, and diacetone alcohol, which may contribute to the inhibitory effects of the extract. However, further molecular-level studies, including genetic and proteomic analyses, as well as in vitro experiments and gene expression assays, are required to fully elucidate the mechanism of action, evaluate safety, and confirm therapeutic potential. In particular, gene expression tests revealing how efflux pump genes respond to extract treatment are critical for understanding the extract’s mechanism. Overall, Ph-Ace represents a promising candidate to extend the useful lifespan of current antibiotics and address the issue of multidrug resistance.