The antibacterial effect of Tanacetum argyrophyllum essential oil on kanamycin-resistant Escherichia coli by disruption of energy metabolism and proton fluxes

Abstract

The essential oil (EO) of Tanacetum argyrophyllum harvested from Armenian flora (2080 m above sea level), characterized by a eucalyptol–camphor chemotype, and was investigated for its antibacterial activity, particularly against antibiotic-resistant bacterial strains. Chemical profiling revealed eucalyptol (35.0%), camphor (24.0%), and camphene (17.0%) as major constituents, alongside several minor terpenoids. The EO exhibited notable inhibitory effects against both wild-type Escherichia coli K-12 and kanamycin-resistant E. coli pARG-25 strains, with minimal inhibitory concentrations (MICs) reaching 100 µL/mL. . The combined influence of the EO with kanamycin could be described as synergistic, as Fractional Inhibitory Concentration Index (FICI) is 0.54. In this case the 62.5 µL/mL concentration of EO reduces the antibiotic MIC value fourfold. The investigation of colony-forming ability of bacteria under the influence of T. argyrophyllum EO revealed a reduction in bacterial viability by 30%. The changes in growth kinetics were also observed for both strains, which was indicated by a prolonged lag phase, suggesting impairment of early adaptation mechanisms. Further studies revealed that EO treatment significantly suppressed proton fluxes and ATPase activity in both strains. Particularly, total and DCCD-sensitive ATPase activities decreased by 1.5-fold, indicating a deviation in proton motive force maintenance and energy metabolism. The antibiotic-resistant E. coli pARG-25 strain exhibited higher ATPase activity compared to the wild-type, suggesting an elevated energy demand linked to resistance plasmid carriage, which was also targeted by the EO. These findings highlight that T. argyrophyllum EO disrupts bacterial energy homeostasis, representing a promising strategy for combating antibiotic-resistant pathogens. Overall, the results support the potential use of T. argyrophyllum EO as a natural antimicrobial agent.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-44036-z.

Introduction

The use of plant-derived compounds for combating microbial infections is deeply rooted in human history, with ethnobotanical practices forming the basis of numerous traditional medical systems. In Armenia, the medicinal application of plants, including essential oils and extracts, has been documented extensively in historical manuscripts and early printed texts. These sources detail the preventive and therapeutic use of botanical substances for managing microbial diseases¹.

In recent years, the convergence of historical pharmacognosy and modern science has led to novel applications of plant-based agents in fields beyond traditional therapeutics. One such example is the use of essential oils in cultural heritage conservation. At the Matenadaran essential oils of medicinal plants have been employed as natural, non-invasive antimicrobial agents for the disinfection of ancient manuscripts, representing an ecologically sustainable alternative to synthetic chemical treatments.

Simultaneously, increasing awareness of the limitations and side effects of synthetic pharmaceuticals has fueled renewed interest in natural remedies. This trend has reasserted the value of traditional botanical knowledge in contemporary biomedical research, particularly in the search for effective and biocompatible antimicrobial agents. Among the promising candidates is Tanacetum argyrophyllum, a species traditionally used in Armenian medicine for its antimicrobial properties against a broad spectrum of bacterial and fungal pathogens, highlighting the ongoing relevance of aromatic and medicinal plants in both historical and modern contexts^1–3.

The Tanacetum genus includes several species recognized for their medicinal properties and antibacterial effects. For instance, T. vulgare is considered as a poisonous plant and is traditionally used as an antiparasitic and antimicrobial remedy^4,5, T. parthenium is noted for its anti-inflammatory and antibacterial effects against oral pathogens^5,6. Another well-known species, T. cinerariifolium is primarily used for its insecticidal properties⁷. Among these, T. argyrophyllum stands out for its significant antibacterial activity.

Traditional approaches to finding new antimicrobial agents and drugs are becoming less effective due to the high-speed formation of bacterial resistance to such kind of preparations. The plant-origin metabolites are considered one of the most promising sources due to their high biological activity and low levels of side effects as well as no reported data of bacterial resistance to this kind of substances^8–10. It has been fascinating to study its antibacterial activity against antibiotic-resistant bacterial strains, as the significant emergence rate of antibiotic-resistance is a worldwide challenge for medicine and veterinary (WHO, 2023).

Antibiotics generally exert their bactericidal or bacteriostatic effects by targeting key cellular structures and processes, including the bacterial cell membrane, ion transport mechanisms, peptidoglycan biosynthesis enzymes, and the molecular machinery involved in DNA, RNA, and protein synthesis¹¹. A critical determinant of antibiotic resistance, particularly in Gram-negative bacteria such as Escherichia coli, is the presence of highly efficient and structurally diverse efflux pump systems. While both Gram-positive and Gram-negative bacteria possess efflux mechanisms, the latter often exhibit a broader repertoire and greater efficacy in actively exporting a wide range of antibiotics and toxic compounds, thereby contributing to multidrug resistance phenotypes¹².

Plant-derived antimicrobial agents are emerging as promising alternatives or adjuncts in the fight against antibiotic resistance, owing to their structural diversity and multifaceted mechanisms of action. These compounds frequently exhibit synergistic interactions with conventional antibiotics, enhancing their efficacy while disrupting bacterial viability through distinct biochemical pathways, including inhibition of ATPase activity^13,14. Furthermore, essential oils derived from medicinal plants have been shown to modulate ion fluxes across bacterial membranes and interfere with key resistance mechanisms, thereby attenuating the adaptive capacity of pathogenic microorganisms¹⁵.

Terpenes, a major class of plant-derived secondary metabolites, are among the most potent antibacterial constituents found in essential oils. These lipophilic compounds contribute significantly to the antimicrobial properties of plant essential oils through multiple mechanisms of action^9,16.

Terpenes exert their antibacterial effects primarily by disrupting microbial cell membranes, increasing membrane permeability, and altering ion homeostasis, ultimately leading to cell death. Their hydrophobic nature allows them to integrate into lipid bilayers, compromising membrane integrity and function. In addition to membrane disruption, some terpenes interfere with quorum sensing, enzyme activity, and efflux pump function, thereby enhancing the susceptibility of bacteria to antibiotics. The structural diversity of terpenes—including monoterpenes (e.g., thymol, carvacrol, and limonene) and sesquiterpenes—underlies their broad-spectrum antimicrobial efficacy and makes them promising candidates for the development of novel antimicrobial agents or adjuvants in antibiotic therapy^17,18.

While several mechanisms underlying the antimicrobial properties of plant-derived compounds have been characterized, many remain incompletely understood. In this study, we investigate the chemical composition of T. argyrophyllum essential oil (EO) and its antibacterial potential against a range of microorganisms, including yeasts, Gram-positive, and Gram-negative bacteria, with a particular emphasis on antibiotic-resistant strains. Special attention is given to the peculiarities of the chemical composition of EO extracted from T. argyrophyllum harvested from Armenian highlands and its antibacterial activity against kanamycin-resistant E. coli while providing mechanistic insight into EO effects on bacterial membrane-associated properties.

Materials and methods

Plant material and essential oil extraction

The plant material of T. argyrophyllum (C. KOCH) TZVEL was harvested from the Vayots Dzor Province of Armenia (Jermuk) at 2080 m above sea level, in May-June, during the early flowering period. The identification of plant species was carried out by Dr. Armen Sahakyan (Mesrop Mashtots Institute of Ancient Manuscripts, Matenadaran, Armenia). The plant collection and use were in accordance with all the relevant guidelines.

Essential oil has been extracted from the freshly collected aerial parts of the plant via the traditional hydro-distillation method, recipes were deciphered from Armenian medieval manuscripts by Dr. Armen Sahakyan. The oil was extracted using Clevenger-type apparatus as described before¹⁹. The extracted EO was stored in a dark and cool place (8–10 °C). The number of independent extractions reached 4–5.

Determination of EO chemical composition

The chemical composition of the T. argyrophyllum EO was determined by the Gas Chromatography Mass Selective analysis (GC-MS). The GC-MS analysis was performed using a Hewlett-Packard 5890 Series II gas chromatograph (“Hewlett-Packard Comp.”, “Agilent Technologies”, USA), equipped with a fused silica HP-5MS capillary column (30 m × 0.25 mm in diameter, 0.25 μm film thickness). The oven temperature varied from 40 to 250 °C with the scanning rate of 3 °C min^[–1. As carrier gas the helium (purity 5.6) was used with a flow rate of 1mL/min. The GC was equipped with Hewlett–Packard 5972 Series MS detector. Ionization voltage of 70 eV and ion source temperature of 250 °C were the MS operating conditions. One microliter of diluted EO (1/100, v/v in HPLC methanol) was manually injected. In order to prevent the GC column from being overloaded, EO was diluted 1:100 (v/v) in methanol. Peaks were tentatively identified using the National Institute of Standards and Technology (NIST)−2013 library database. For the HP-5MS column, the Relative Retention Index (RRI) was calculated. Under the same chromatographic conditions as for the measurement of EO, a mixture of homologues of n-alkanes (C9–C18) was utilized for the RRI computation².

Disk-diffusion and broth dilution assays, Investigated strains and growth conditions

The antimicrobial activity of T. argyrophyllum essential oil (EO) was initially assessed by agar disk-diffusion assay². For this, sterile cellulose disks (6 mm in diameter) were impregnated with EO different concentrations ranging from 3.125 to 200 µL/mL, diluted in 96% ethanol. Ethanol served as the negative, while kanamycin and tetracycline as positive controls. The assay was performed using solid peptone nutrient agar, and inhibition zone diameters were measured after the 24 h of incubation at 37 °C.

The minimum inhibitory concentrations (MIC) were determined using broth microdilution method in 96-well plates following standard protocols (using the abovementioned concentrations of EO)²⁰. The test panel included both Gram-negative and Gram-positive bacterial strains (Escherichia coli K-12, kanamycin-resistant E. coli pARG-25, Bacillus subtilis WT-A17, Staphylococcus aureus WDC 5233) as well as yeast species (Debaryomyces hansenii WDC M104 and Candida guilliermondii NP-4). The microbial strains were purchased from the Depository center of Scientific and Production Center “Armbiotechnology” of the National Academy of Sciences of Armenia.

For the investigation of EO mode of action E. coli K-12 (serving as a reference strain) and E. coli pARG-25 strains were applied. The latter carry a high-copy-cloning plasmid which contains a ColE1-type (pMB1-derived; pBR322/pUC) origin of replication, has a KanR cassette, providing resistance to kanamycin (plasmid size: 6.341 kb). Cells from one colony were transferred to the liquid medium and cultivated overnight at 37 °C. Then the fresh MP liquid nutrient medium (peptone − 20 g/L, glucose − 2 g/L, NaCl − 5 g/L, K₂HPO₄ − 2 g/L, pH = 7.5) was inoculated with bacteria and incubated for 18–22 h for using in further experiments. The same nutrient medium was applied both for the broth dilution and agar disk-diffusion assays (for the latter the medium was supplemented with agar (17 g/L)).

Determination of antibiotic modulatory activity of T. argyrophyllum EO

Antibiotic modulatory activity of T. argyrophyllum EO was explored by determining the minimal inhibitory concentrations of antibiotics in the presence and absence of EO at non-inhibitory concentrations²⁴. Broth microdilution assay was used for the determination of MIC values, as described previously²⁴. The decrease in MICs of antibiotic (ampicillin and kanamycin) in the presence of plant EO indicated antibiotic modulatory activity of the extract. To calculate the modulation factor (MF), which indicates modulating interactions, the following formula was used: MF = MICantibiotic/MICantibiotic+extract.

MF ≥ 2 indicated the occurrence of modulating interactions, if MICantibiotic/MICantibiotic+extract ratio = 1 meant no impact.

The antibiotic-modulatory activity of T. argyrophyllum EO was evaluated also using the checkerboard microdilution method to determine the Fractional Inhibitory Concentration Index (FICI). Minimum inhibitory concentrations (MICs) of the antibiotic and the test compound were first determined individually by the broth microdilution method according to CLSI guidelines.

For combination testing, two-fold serial dilutions of the antibiotic were prepared along the horizontal axis of a sterile 96-well microtiter plate, while serial dilutions of the test compound were prepared along the vertical axis, resulting in a matrix of concentration combinations. Each well was inoculated with a standardized bacterial suspension (approximately 5 × 10⁵ CFU/mL) prepared from overnight cultures and diluted in MP liquid nutrient medium. Plates were incubated at 35–37 °C for 18–24 h under aerobic conditions.

The MIC for each agent alone and in combination was defined as the lowest concentration showing no visible bacterial growth. The fractional inhibitory concentration (FIC) for each compound was calculated as:

FIC_A (EO) = MIC_A (in combination)/MIC_A (alone).

FIC_B (kanamycin) = MIC_B (in combination)/MIC_B (alone).

The FICI was calculated as:

FICI = FIC_A + FIC_B.

Interactions were interpreted as follows:

• FICI ≤ 0.5: Synergistic effect.

• 0.5 < FICI ≤ 1.0: Additive effect.

• 1.0 < FICI ≤ 4.0: Indifferent (no interaction).

• FICI > 4.0: Antagonistic effect.

All experiments were performed in triplicate, and mean FICI values were calculated²⁵.

Determination of Bacterial Colony-Forming Units (CFU)

The antibacterial activity of T. argyrophyllum essential oil was assessed by quantifying colony-forming units (CFU) using the standard plate count method^21,22. Serial dilutions (10⁶–10⁸-cell per mL) of bacterial suspensions were made and plated on peptone agar (composition detailed above). Following the addition of the bacterial suspensions, plates were incubated at 37 °C for 24 h. The essential oil was tested at its MIC. After the incubation, bacterial colonies were quantified, and CFU were calculated using the formula T = 10 × n × 10^m, where n represents the number of colonies, and m is the dilution factor²³.

Determination of bacterial growth parameters

The generation succeeding factor (G), the specific growth rate (µ) of bacteria, and the cell doubling time were determined by using the DEN-1 McFarland Densitometer (Biosan, Latvia). The µ was calculated by the following formula:

where,

lnN₂-lnN₁ – logarithmic difference of doubled optical reading,

t₂-t₁ – difference between doubling time.

The cell doubling time was determined by the ln2/µ Eq. 2¹.

Determination of proton flux

Cells in the stationary phase were harvested by centrifugation at 3500 × g for 10 min (Sorvall LYNX 6000 Superspeed Centrifuge, Thermo Scientific, USA), followed by two washes with distilled water. The cell pellet was resuspended in 150 mM Tris–HCl buffer (pH 7.5) containing 0.4 mM MgSO₄, 1 mM NaCl, and 1 mM KCl and the experiments were performed using the same buffer, at 37 ֯ C in a thermostatic vessel. Proton flux (J_H⁺) was measured as previously described²⁶, following the addition of glucose (2 g L^− 1) and T. argyrophyllum essential oil (100 µL/mL). pH changes were recorded using a pH-meter (Milwaukee MW151 MAX pH/ORP/Temp logging bench meter, USA) equipped with a selective H⁺ electrode (MA917). The method is based on the principle that changes in proton concentration in the external medium reflect the net proton flux across the bacterial membrane: an increase in external proton concentration indicates proton efflux, whereas a decrease indicates proton influx. Electrodes were calibrated by titration of the medium with 0.01 M HCl. For determination of N, N′-dicyclohexylcarbodiimide (DCCD)-sensitive proton fluxes, cells were preincubated with DCCD (0.1 mM) for 10 min under the same experimental conditions (at 37 ֯C, in pH = 7.5 buffer). DCCD-sensitive proton fluxes were calculated as the difference between total proton flux and flux measured in the presence of DCCD and represents the ATPase-dependent component of proton transport mediated by the F₀F₁-ATPase. J_H⁺ was calculated based on the change in proton concentration, expressed in mmol H⁺/min per 10⁸ cells/mL. Cell numbers were calculated by measuring optical density using a spectrophotometer (SP-LUV752P UV-VIS Spectrophotometer, Bioevopeak, PRC) and converting OD values to CFU using a previously established calibration curve.

Determination of ATPase activity

Membrane vesicles were isolated from E. coli K12 and E. coli pARG-25 strains according to a previously established protocol²⁶. Protein concentrations were determined using the Lowry method²⁷. FoF₁-ATPase activity was evaluated in 100 µg of membrane vesicle protein by quantifying the release of inorganic phosphate (Pi) in a reaction buffer containing 50 mM Tris–HCl (pH 7.5), 1 mM CaCl₂, and 2.5 mM MgSO₄ at 37 °C. The absorbance was measured spectrophotometrically (SP-LUV752P UV-VIS Spectrophotometer, Bioevopeak, PRC). Enzymatic activity was expressed as nmol P_i min⁻¹ mL⁻¹ µg⁻¹ protein. DCCD-sensitive ATPase activity was calculated according the above-described principle under identical incubation conditions.

Chemicals and statistical analysis

All chemicals and reagents were obtained from Sigma-Aldrich Co. Ltd. (Taufkirchen, Germany), Carl Roth GmbH & Co. KG (Karlsruhe, Germany), and VWR International (Pennsylvania, USA).

Experimental data represent the mean ± standard deviation (SD) from three biologically independent replicates. Statistical analyses was performed using a grouped two-way ANOVA using GraphPad Prism version 8.0.3 (GraphPad Software, San Diego, CA, USA). The software is available at: https://www.graphpad.com. Differences were considered statistically significant at p < 0.05, unless stated otherwise. Graphical data visualization was performed using GraphPad Prism and Microsoft Excel 2010.

Results

Plant material and essential oil yield

According to our measurements the T. argyrophyllum EO yield was calculated to be 0.1%, w/w.

T. argyrophyllum EO chemical composition

The results from the quantitative and qualitative analysis of EO constituents are presented in the Table 1.

Table 1.

Chemical composition of T. argyrophyllum EO.

Chemical components	Area Percentage (%)	RRI*
Camphene	17.0	943
Carene	0.50	947
α-Pinene	1.50	948
β-Pinene	0.85	970
β-ocimene	0.55	976
Santolina epoxide	7.50	991
γ-Terpinene	0.70	1047
Eucalyptol	35.0	1059
Camphor	24.0	1121
Trans-Chrysanthenyl acetate	2.90	1131
Verbinol	0.90	1136
Terpinen4-ol	1.80	1137
Davanone	0.80	1138
Bornyl acetate	2.25	1277
β-eudesmol	1.20	1504
Caryophyllene oxide	1.50	1576
Total (%)	98.50

*for HP-5 MS capillary column.

GC–MS analysis of T. argyrophyllum EO led to the identification of 16 chemical constituents, representing 98.5% of the total oil composition. The EO exhibited a terpene-rich profile dominated by monoterpenes and their oxygenated derivatives, consistent with a eucalyptol–camphor chemotype. The most abundant compound was eucalyptol (1,8-cineole), comprising 35.0% of the total content, followed by camphor (24.0%) and camphene (17.0%).

Additional constituents included santolina epoxide (7.5%) and trans-chrysanthenyl acetate (2.9%), along with terpinen-4-ol (1.8%), bornyl acetate (2.25%), and verbinol (0.9%). Trace amounts of α-pinene, β-pinene, and γ-terpinene were also detected. Furthermore, caryophyllene oxide (1.5%) and β-eudesmol (1.2%) were present, contributing to the overall sesquiterpene content. The compositional profile highlights the chemical complexity of T. argyrophyllum EO and confirms the predominance of oxygenated monoterpenes in this species.

Antibacterial activity of T. argyrophyllum based on disk-diffusion and broth dilution assays

The disc diffusion and broth dilution assays revealed that T. argyrophyllum essential oil (EO) exhibited broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacterial strains, as well as selected yeasts. Further tests revealed that the EO demonstrated inhibitory effects against D. hansenii WDC M104 and C. guilliermondii NP-4, indicating its potential antifungal properties. Among the bacterial isolates, S. aureus WDC 5233 and B. subtilis WT-A17 exhibited the highest sensitivity to the EO, with a minimum inhibitory concentration (MIC) of 50 µL/mL (Table 2).

Table 2.

Minimum inhibitory concentration (MIC) values of antibiotics and T. argyrophyllum EO against tested microbial strains.

Microbial strain	MIC of T. argyrophyllum EO (µL/mL)	MIC of kanamycin (mg/mL)	MIC of tetracycline (mg/mL)
D. hansenii WDC M104	25	-	5
C. guilliermondii NP-4	12.5	-	5
S. aureus WDC 5233	50	0.5	0.1
B. subtilis WT-A17	50	0.5	0.1
E. coli K-12	100	5	0.1
E. coli pARG-25	100	5	0.1

Gram-negative E. coli strains, including both the wild-type (K-12) and kanamycin-resistant (pARG-25) variants, exhibited comparatively lower susceptibility to the T. argyrophyllum essential oil, with MIC values reaching 100 µL/mL (Table 2). This reduced sensitivity is likely attributable to the inherent structural characteristics of Gram-negative bacteria, notably the presence of an outer membrane that serves as a selective permeability barrier, restricting the diffusion of hydrophobic compounds such as those present in essential oils.

As it was mentioned, the investigated EO demonstrated inhibitory effect against the kanamycin-resistant E. coli pARG-25 strain, indicating its potential as a natural antimicrobial agent targeting drug-resistant pathogens. Based on these results, further experimental investigations were conducted using the resistant strain, while the non-resistant E. coli K-12 strain— from which the antibiotic-resistant E. coli pARG-25 strain was derived —served as the reference. Subsequent assays confirmed the bacteriostatic nature of T. argyrophyllum EO against the tested bacterial strains.

Antibiotic modulatory activity of T. argyrophyllum EO

Our investigation results suggest that T. argyrophyllum exhibits the antibiotic-modulatory effect on the kanamycin-resistant E. coli (Fig. 1). The 31.25 and 62.5 µL/mL non-inhibitory concentrations of EO were used for the investigation of antibiotic-modulatory properties.

Fig. 1.

Modulatory effect of T. argyrophyllum essential oil at non-inhibitory concentrations on the antibiotic activity against kanamycin resistant E. coli pARG-25 strain. The abbreviations refer to: T+ – cells treated with T. argyrophyllum essential oil, K+ – cells treated with kanamycin. All experiments were independently repeated at least three times (**** p < 0.0001). The MIC values were identical across all replicates.

According to the obtained data, the T. argyrophyllum essential oil at the concentration of 62.5 µL/mL exhibited notable antibiotic-modulating effect with kanamycin, reducing the MIC value of antibiotic fourfold (MF = 4) (Fig. 1B), meanwhile the lower concentration did not show any influence (Fig. 1C).

The interaction between the kanamycin and T. argyrophyllum EO could be described as synergistic, as FICI is calculated to be 0.54.

In case of ampicillin, we did not observe any modulation in the presence of T. argyrophyllum essential oil under the tested conditions.

The influence of T. argyrophyllum EO on Bacterial Colony-Forming ability and growth parameters

To further evaluate the antimicrobial effect of EO, the colony-forming ability evaluation test was applied, quantitatively assessing the impact of EO treatment on bacterial viability.

The obtained results demonstrated that treatment with T. argyrophyllum essential oil led to a reduction of approximately 30% in the colony-forming ability of the non-resistant E. coli K-12 strain (Fig. 2). Similarly, in the kanamycin-resistant E. coli pARG-25 strain, colony formation was suppressed by 30%, a reduction comparable to that observed in the non-resistant control (Fig. 2).

Fig. 2.

The colony-forming units of the E. coli K-12 and E. coli pARG-25 strains under the influence of T. argyrophyllum EO at minimal inhibitory concentration. (T- are the control cells without treatment with T. argyrophyllum EO, T+ -cells treated with T. argyrophyllum EO). The results are means ± SD of three independent experiments implemented in triplicate (*p < 0.05).

In addition, the same concentration of T. argyrophyllum EO exhibited a suppressive influence also on the specific growth rates of both tested bacterial strains. A measurable decrease in growth kinetics was observed following EO exposure, further confirming its bacteriostatic properties (Fig. 3).

Fig. 3.

The specific growth rate (a) and cell doubling time (b) of the E. coli K-12 and E. coli pARG-25 strains under the influence of T. argyrophyllum EO at minimal inhibitory concentration. (T- are the control cells without treatment with T. argyrophyllum EO, T+ -cells treated with T. argyrophyllum EO). The results are means ± SD of three independent experiments carried out in triplicate (** p < 0.01, **** p < 0.0001).

According to our investigations, the specific growth rates in case of E. coli K-12 and E. coli pARG-25 strains, reduced by 1.6-fold, and 1.5-fold, respectively (Fig. 3a). Additionally, the EO prolonged the lag phase in case of E. coli K-12 by 63% and by 52% in case of the antibiotic-resistant E. coli pARG25 (Fig. 3b), affecting the adaptation of bacteria to the growth conditions during the early stages.

The influence of T. argyrophyllum EO on proton fluxes and ATPase activity

The investigations of the effects of T. argyrophyllum EO on some membrane-associated functions in E. coli revealed significant alterations in proton flux dynamics (Fig. 4a). In the untreated E. coli K-12 control strain, the rate of DCCD-sensitive H⁺ fluxes decreased by 2.8-fold (Fig. 4b, T − D+). In the presence of investigated EO alone (T + D−), this parameter was reduced by 2.5-fold compared to the untreated control. This suggests that the EO has similar effects with DCCD by targeting the F_OF₁ domains in F_OF₁ ATPase.

Fig. 4.

The effect of T. argyrophyllum EO on total (a) and DCCD-sensitive (b) H⁺-fluxes through the E. coli K-12 and kanamycin-resistant E. coli pARG-25 cell membranes. T-D- are the control cells without the treatment with EO and DCCD, T-D + are the cells treated only with DCCD, T + D- are the cells treated only with EO and the T + D+ are the cells treated with both T. argyrophyllum EO and DCCD. The results are means ± SD of three independent experiments performed in triplicate (*** p < 0.001, **** p < 0.0001).

In case of the kanamycin-resistant E. coli pARG-25 strain, treatment with the EO resulted in a 1.8-fold reduction in J_H⁺ (Fig. 4a, T + D−). Under DCCD treatment, the control strain exhibited approximately a 4.5-fold decrease in DCCD-sensitive H⁺ flux, while in the resistant strain, exposure to the EO under the same conditions led to a 1.7-fold decrease (Fig. 4b, T+).

These findings suggest that T. argyrophyllum essential oil affects proton translocation across bacterial membranes, with differential impacts observed between wild-type and antibiotic-resistant strain.

Exposure to T. argyrophyllum essential oil resulted in a notable reduction also in total ATPase activity in both tested strains (Fig. 5a). In the E. coli K-12 control strain, total ATPase activity decreased by approximately 1.6-fold following EO treatment (Fig. 5a, T + D−). A comparable reduction was observed in kanamycin-resistant E. coli pARG-25 strain, where ATPase activity was suppressed by approximately 1.5-fold under the same conditions (Fig. 5a, T + D−).

Fig. 5.

The effect of T. argyrophyllum essential oil on Total (a) and DCCD-sensitive (b) ATPase activity through the E. coli K-12 and E. coli pARG-25 strains’ membranes. T-D- are the control cells without treatment with EO or DCCD, T-D + are the cells treated only with DCCD, T + D- are the cells treated only with EO and T + D+ are the cells treated with both T. argyrophyllum EO and DCCD. The results are means ± SD of three independent experiments performed in triplicate (** p < 0.01, *** p < 0.001, **** p < 0.0001).

Further analysis of DCCD-sensitive ATPase activity revealed a 1.3-fold reduction in the E. coli K-12 strain and a 1.2-fold decrease in the E. coli pARG-25 strain upon EO treatment (Fig. 5b, T+).

Interestingly, in the control samples, the E. coli pARG-25 strain exhibited a 1.4-fold higher total ATPase activity compared to the Wt strain (Fig. 5a). Similarly, DCCD-sensitive ATPase activity was elevated by 1.6-fold in the resistant strain (Fig. 5b).

These results suggest that T. argyrophyllum EO exerts a suppressive effect on both total and proton-translocating ATPase activities, with comparable magnitude across antibiotic-sensitive and -resistant strains.

Discussion

The essential oil of T. argyrophyllum characterized in this study exhibits a distinct eucalyptol–camphor chemotype, with eucalyptol (1,8-cineole; 35.0%) and camphor (24.0%) as the dominant constituents. These two bicyclic oxygenated monoterpenes are well known for their potent antimicrobial and anti-inflammatory activities, as previously reported²⁸. The third most abundant compound, camphene (17.0%), a bicyclic monoterpene hydrocarbon, has similarly been associated with antioxidant and antimicrobial properties²⁹, thus reinforcing the bioactive potential of the oil.

The chemical composition observed aligns closely with previous investigations of T. argyrophyllum populations. For instance, it is reported³⁰(a similar dominance of camphor, borneol, and 1,8-cineole in T. argyrophyllum (C. Koch) Tvzel var. argyrophyllum collected from Anatolia, suggesting the prevalence and stability of this chemotype across different regions. Similarly, it was found 1,8-cineole, camphor, and borneol to be major constituents in Tanacetum densum (L.) Heywood ssp. eginense Heywood, further supporting the chemotaxonomic consistency within the species^31,32.

Beyond the major constituents, the presence of other bioactive components such as santolina epoxide (7.5%) and trans-chrysanthenyl acetate (2.9%) adds chemotaxonomic significance. Comparable sesquiterpene profiles have been reported in related species like T. vulgare³³, suggesting conserved biosynthetic pathways across the genus.

Other compounds identified, including terpinen-4-ol (1.8%), bornyl acetate (2.25%), and verbinol (0.9%), mirror the structurally diverse terpenoid signatures characteristic of the Tanacetum genus. Minor constituents such as α-pinene, β-pinene, and γ-terpinene, which contribute to species-specific volatile profiles and ecological interactions, were also detected. Furthermore, sesquiterpene oxides like caryophyllene oxide (1.5%) and β-eudesmol (1.2%) were consistent with previous findings in T. balsamita and T. heterotomum³⁴.

Altogether, the EO composition determined in this work not only confirms the eucalyptol–camphor chemotype but also underscores the phytochemical richness of T. argyrophyllum, suggesting valuable pharmacological and ecological applications.

In our previous studies, the essential oil of T. argyrophyllum, in the line of other substances demonstrated significant antibacterial activities in the investigated concentrations, including effects against antibiotic-resistant E. coli strains³⁵. The current investigation extends these findings by providing some mechanistic insights into the mode of antimicrobial action of the same concentration of this EO.

Initial antimicrobial screening using the disk diffusion method demonstrated the broad-spectrum activity of T. argyrophyllum essential oil (EO) against both bacterial and yeast strains. Given the global urgency of antibiotic resistance, further analyses focused on the EO’s effect against kanamycin-resistant E. coli pARG-25. Quantitative colony-forming unit (CFU) assays revealed a significant reduction in bacterial viability—approximately 30% in the non-resistant E. coli K-12 strain and 33% in the resistant E. coli—indicating comparable inhibitory effects regardless of resistance status. Notably, when combined with kanamycin, the EO was found to reduce the minimum inhibitory concentration (MIC) of the antibiotic by fourfold against the resistant strain. While the EO alone was not fully bactericidal, its capacity to suppress bacterial proliferation and potentiate antibiotic efficacy underscores its antibiotic-modulatory potential. This phenomenon can be explained by the fact that kanamycin, an aminoglycoside antibiotic, relies on energy-dependent uptake across the bacterial cytoplasmic membrane. This process is tightly coupled to the proton motive force and membrane-associated bioenergetic pathways, including ATP synthesis and maintenance of membrane potential. As T. argyrophyllum essential oil significantly perturbs proton flux and ATPase activity disrupting bioenergetic homeostasis and likely altering transmembrane electrochemical gradients and membrane permeability, thereby facilitating enhanced intracellular accumulation of kanamycin³⁶. The observed modulatory effect may therefore arise from essential oil–mediated destabilization of membrane energetics, which potentiates aminoglycoside efficacy through improved antibiotic uptake and/or increased vulnerability of metabolically compromised cells.

In contrast, ampicillin, a β-lactam antibiotic, exhibits its antibacterial activity by binding to penicillin-binding proteins and inhibiting peptidoglycan biosynthesis within the cell wall. This mechanism is largely independent of membrane potential and does not require ATP-driven transport across the cytoplasmic membrane³⁷. Consequently, disruption of membrane bioenergetic processes by T. argyrophyllum essential oil would not be expected to substantially influence ampicillin activity. The absence of a modulatory effect under our experimental conditions therefore likely reflects these fundamental mechanistic differences between energy-dependent aminoglycoside uptake and the energy-independent cell wall–targeting action of β-lactams.

These findings highlight the promise of T. argyrophyllum EO as a natural adjuvant in addressing aminoglycoside-resistant infections.

Moreover, the extension of the lag phase observed in both strains following EO treatment suggests a disruption of early bacterial adaptive mechanisms, potentially by interfering with critical metabolic pathways required for cell division and growth.

To further elucidate the underlying mechanisms, the effects of the EO on proton fluxes and ATPase activity were assessed. Measurements of proton fluxes (J_H⁺) revealed significant decreases following EO treatment in both strains. In E. coli K-12, the DCCD-sensitive J_H⁺ flux rate decreased by 2.8-fold and 2.5-fold when treated with the EO alone. In the kanamycin-resistant E. coli strain, the membrane J_H⁺ flux rate was suppressed 1.8-fold after EO treatment, and DCCD-sensitive fluxes decreased by up to 1.7-fold.

These findings suggest that T. argyrophyllum EO disrupts the proton cycling affecting the energetics of the cell. Disruption of proton gradients is able to impair ATP synthesis, nutrient transport, and other energy-dependent processes, leading to reduced bacterial viability (Fig. 6)³⁸.

Fig. 6.

The action mechanisms of T. argyrophyllum EO on bacterial ATPase and proton fluxes.

Consistently, ATPase activity assay demonstrated that total ATPase activity was reduced by 1.6-fold in the E. coli K-12 strain and by 1.5-fold in the E. coli pARG-25 strain upon EO treatment. Furthermore, DCCD-sensitive ATPase activity was suppressed by 1.3-fold in the wild-type and by 1.2-fold in the resistant strain. It is important that initially the E. coli pARG-25 strain exhibited 1.4-fold higher total ATPase activity and 1.6-fold greater DCCD-sensitive ATPase activity than the wild type strain, likely reflecting an increased energetic demand associated with plasmid-mediated antibiotic resistance³⁶.

DCCD, as a specific inhibitor of the F_OF₁ ATP synthase, prevents proton translocation through the enzyme complex³⁹. Therefore, reductions in DCCD-sensitive ATPase activity indicate that the EO targets membrane-bound ATP synthase, impairing both ATP production and the maintenance of the proton motive force (PMF). Such interference critically hampers bacterial energy metabolism and stress response systems, contributing to the observed antimicrobial effects.

The ability of T. argyrophyllum EO to inhibit both total and proton-translocating ATPase activity in both susceptible and resistant strains highlight its potential as a broad-spectrum antimicrobial agent. Moreover, targeting bacterial membranes and energy metabolism could complement conventional antibiotic therapies, providing a promising strategy against drug-resistant infections.

In summary, T. argyrophyllum EO, rich in eucalyptol and camphor, demonstrates significant antibacterial properties, particularly against antibiotic-resistant E. coli. Its mechanism of action appears to involve disruption of membrane proton flux and ATPase function, leading to impaired energy metabolism and bacterial growth inhibition. These findings suggest that T. argyrophyllum EO represents a promising natural antimicrobial candidate, warranting further pharmacological evaluation and development for clinical and agricultural applications.

Conclusions

The present study demonstrates that the essential oil of T. argyrophyllum, characterized by a eucalyptol–camphor chemotype, possesses significant antimicrobial and antibiotic-modulatory activity against both antibiotic-sensitive and kanamycin-resistant E. coli strains. The EO reduced bacterial viability, prolonged the lag phase, and suppressed key membrane-associated bioenergetic processes, including proton fluxes and ATPase activities which is stated for the first time. These findings suggest that the antibacterial effects of T. argyrophyllum EO are largely mediated through the disruption of membrane integrity and inhibition of energy metabolism. Notably, the essential oil was equally effective against the antibiotic-resistant E. coli pARG-25 strain, highlighting its potential as a natural antimicrobial agent capable of targeting drug-resistant pathogens. Further investigations of in vivo efficacy of this EO are warranted to fully explore its therapeutic potential.

Limitations and Future Perspectives

Although the present study provides evidence for the antibacterial and antibiotic-modulating effects of T. argyrophyllum essential oil, several limitations should be acknowledged. First, despite the data indicate interference with proton flux and ATPase activity, the mechanistic conclusions remain indirect. Direct evidence such as intracellular ATP measurements, membrane potential analyses, and specific binding assays was beyond the scope of the current study.

Importantly, cytotoxicity of the tested essential oil concentrations was not assessed in mammalian cells. Consequently, the safety profile, therapeutic window, and selectivity index remain unknown. While antimicrobial activity was demonstrated, the clinical or topical safety of the effective concentrations cannot yet be determined.

Future studies will therefore include the intracellular ATP quantification and membrane potential measurements which will strengthen the mechanistic framework. Evaluation of cytotoxic effects on relevant mammalian cell lines will also be conducted to determine biocompatibility and to define the therapeutic window. These investigations will provide a more comprehensive assessment of the mechanism of action, safety, and translational potential of T. argyrophyllum essential oil.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the conception and design of study. ST, LM, and AS carried out the investigations and analyzed the data. NS and KT directed the experiments, corrected, and edited the manuscript. All authors read and approved the final manuscript and have participated sufficiently in the work and agreed to be accountable for all aspects of the work and provided approval of the final submitted manuscript.

Funding

The research was supported by the Science Committee of MESCS RA, in the frames of the research projects № 24WS-1F003.

Data availability

Some raw data are provided as supplementary files including specific growth rate raw data, disc-diffusion assay results, and CFU plate images. Other datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.