Wild and cultivative O. vulgare L. grow in Yexian County of Henan province, China, located at latitude: 33.62^o N, longitude: 113.35^o E. These plants were all collected in a single day in the September. In this study, cultivative O. vulgare L. were divided into white flower and purple flower, and extracted separately. Oregano essential oil was obtained by hydrodistillation process using Clevenger type apparatus for 4 h. The obtained oils were further isolated using a separatory funnel and dried with anhydrous sodium sulfate for further analysis.

The determination of chemical composition content in oils and chromatographic separation was performed on Agilent 8,890–7250 GC-QTOF (Agilent Technologies, Santa Clara, CA). An HP- 5MS UI (15 m × 0.25 mm × 0.25 μm) was employed and the column temperature was maintained at 180°C. Sample (1 µL) was injected at a split mode of 20:1 and helium was used as gas carrier at 1.0 mL/min. Multi-mode injection (MMI) temperature was set to 300°C, and the temperature programming was as follows: the temperature remained at 45°C for 1 min, linearly increasing to 300°C at 10 ^oC/min rates and holding for 10 min. Electron ionization was 70 eV, with a mass range of 40–500 amu. Ion source and quadrupole temperature were set to 230^oC and 150^oC, respectively. The identification of the compounds was carried out by comparing their mass spectra with those of the NIST-MS and WILEY-MS library, considering a quality coincidence >85% (Özogul et al., 2022).

Standard strains of Gram-positive and negative bacteria were used in this study, including S. aureus ATCC 25923 (S. aureus), methicillin-resistant S. aureus BNCC 337371 (MRSA), methicillin-resistant Staphylococcus epidermidis ATCC 51625 (MRSE), Enterococcus faecalis ATCC51299 (E. faecalis), E. faecium ATCC 35667 (E. faecium), Streptococcus agalactiae ATCC 27956 (S. agalactiae), E. coli ATCC 25922 (E. coli) and Salmonella typhimurium SL1344 (S. typhimurium). The clinical isolates of MRSA (56 strains) were isolated from dairy farms in four different provinces in China. These strains were routinely cultured in Mueller Hinton broth (MHB) and agar or Blood Agar.

Seventy-five specific pathogen-free Balb/c mice weighing 18–20 g (six-week-old) were purchased from the Laboratory Animal Center of Lanzhou University, and the animal studies were carried out in accordance with the ethical principles of animal research and approved by the Ethics Committee of Laboratory Animal Center of Lanzhou University (No. SCXK 2024-0007). Animals were kept in clean, stainless steel cages with free access to food and water under 23°C conditions, with a constant 12 h light-dark cycle. Animals were used for the experiments after being acclimatized for 7 days in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020).

The MICs of essential oils extracted from wild oregano (OEO-1), cultivated oregano with white flower (OEO-2) and cultivated oregano with purple flower (OEO-3), the commercial OEO (OEO-4, purchased from Aromaaz International Private Limited, New Delhi, India), as well as carvacrol and thymol, were determined by micro-dilution technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical Laboratory Standards Institute, 2012). Briefly, the above mentioned oil and compounds were dissolved in 20% DMSO to a concentration of 128 mg/mL, respectively. Subsequently, 100 μL solutions were diluted twofold with 100 μL MHB to provide 11 serial dilutions. The logarithmic phases of strains, including standard strain and 56 strains of clinical MRSA were diluted to 10⁵–10⁶ CFU/mL in MHB. Then, 100 μL volumes of the tested strains were added to the serial dilutions. The mixtures were then incubated at 37°C for 18–24 h. The MIC was determined by optical density (OD) measurements at a wavelength of 492 nm as the lowest concentration that resulted in no bacterial growth. The same procedure was repeated three times.

After determining the MICs of selected antibiotics (tobramycin, chloramphenicol, ampicillin, polymyxin, tiamulin, vancomycin and cefepime) against MRSA and E. coli using broth dilution method, the synergistic effects of carvacrol/thymol with these antibiotics were determined by the checkerboard method as described in previous works (Zhang et al., 2022; Zhang et al., 2024). The strains were cultured and diluted with fresh MH medium to 1 × 10⁶ CFU/mL. The diluted bacterial suspension (100 µL) was mixed with 50 µL thymol/carvacrol and 50 µL antibiotics with two-fold serially diluted concentrations using 96-well plate. The plate was then sealed and placed in a constant temperature incubator at 37°C for 20 h. The single drug MIC and the MIC values of the best combination effect (MICA combined with MICB) were recorded. The fractional inhibitory concentration index (FICI) was calculated by the following equation: FICI = FICA + FICB, in which FICA = MIC of carvacrol or thymol in combination/MIC of thymol or carvacrol alone; FIC B = MIC of antibiotic in combination/MIC antibiotic alone. The synergistic effects of thymol/carvacrol was categorized as synergistic when the FICI was ≤0.5, additive when 0.5 < FICI ≤1, indifferent when 1 < FICI ≤4, and antagonistic when FICI >4 (Leber, 2016).

Escherichia coli ATCC 25922 and MRSA BNCC 337371 were cultured in MHB at 37°C for 8 h with shaking and diluted to approximately 1 × 10⁶ CFU/mL. Test compounds (with the final concentrations of 1 × MIC and 6 × MIC) were inoculated with the aliquots of bacteria resuspended in fresh media. After specified time intervals (0, 2, 4, 6, 8, 12 and 24 h), cultures incubated at 37°C with 200 rpm shaking were serially diluted by 10-fold in 0.9% saline. The resulted serial dilutions (10 μL) were plated on sterile MH agar plates and incubated at 37 °C for 24 h. The viable colonies were counted and represented as log₁₀ (CFU/mL). The same procedure was repeated in triplicate on different days.

MRSA BNCC 337371 were incubated in the MH broth medium at 37°C, with a shaking speed of 200 rpm for 16 h. Carvacrol, tobramycin, and carvacrol combining with tobramycin (4 × MIC) were added to culture for 4 h. The precipitate was collected by centrifuging at 8,000 rpm for 5 min. The obtained sample was then washed twice with PBS. After centrifuging at 8,000 rpm for 5 min, bacteria were fixed using 2.5% pentanediol. The cells were subsequently washed with 0.1 M PBS and dehydrated with 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol. The bacterial morphology was finally analyzed using SEM.

The fluorescent dyes 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were used to further explore the effect of compounds on cell membrane permeability. Carvacrol, tobramycin, and carvacrol combining with tobramycin (4 × MIC) were added to MRSA (BNCC 337371) suspension, and negative control only used PBS (pH = 7.2–7.4) without any drug. After incubation at 37°C and centrifuging at 200 rpm for 1 h, DAPI (10 μg/mL) and PI (20 μg/mL) were mixed in suspension in the dark, and then the suspension was cultured at 0°C for 20 min. Finally, the results were recorded on a fluorescence microscopy.

The SPF Balb/c mice were randomly divided into five groups and rendered neutropenic upon treatment with 150 mg/kg cyclophosphamide intraperitoneally for 4 days and with 100 mg/kg for 1 day prior to inoculation, respectively. The neutropenic mice were then intraperitoneally injected with a 0.5 mL MRSA inoculum containing 1 × 10⁸ CFU/mL. After 1 h of infection, the mice in four groups were intravenously administered tobramycin dissolved in saline and carvacrol mixed with tobramycin (weight ratio = 10:1) which was dissolved in 0.5 mL of vehicle (comprising DMSO:Tween-80:sterile water in a ratio of 5:5:90) at doses of 30 and 10 mg/kg body weight, respectively. Mice in the rest group were used as control and only intravenously administered with the same volume of vehicle. For the survival rate experiment, animals (n = 10) were monitored twice daily for symptoms and mortality. Survival at 7 days after infection was used as the endpoint. To determine MRSA in tissues and histological analyses, treated mice (n = 5) at 3 days after infection were anesthetized with excess CO₂ and euthanized via exsanguination. Organ samples (kidneys, liver and lungs) from surviving mice and those that died during the treatment were prepared for the tissue staining experiment.

Statistical analysis was conducted using IBM SPSS Statistics for Windows version 24.0 (SPSS Inc., Chicago, USA). One-way analysis of variance was utilized to analyze the data, followed by Dunnett’s posthoc tests when applicable (IBM SPSS, 2012). A P-value of <0.05 was considered statistically significant, and a P-value of <0.01 was considered highly significant.

After extraction from wild and cultivated O. vulgare L., essential oils were obtained varying from 3.58% to 4.12% yielding with no significantly difference. Dozens of chemical compounds that represented over 99% of its total composition were identified via GC-MS (Figure 1). Monoterpenes and sesquiterpenes were proved to be the most abundant bioactive components, in which thymol and carvacrol comprised 16.75% and 21.98% content, respectively.

Medicinal plants have been recognized as the major sources for the prevention and treatment of infectious diseases. To confirm the essential oils harboring antimicrobial activities that were extracted from cultivated oregano and wild oregano, we determined their MICs against six Gram-positive and two Gram-negative strains. The MICs results are presented in Table 1. All the tested OEOs exhibited moderate antibacterial efficacy against Gram-positive and negative strains, with MIC range from 0.25–8 mg/mL. Essential oils extracted from oregano with white flower, whether it came from cultivated or wild, displayed the similar MICs (0.5–1 mg/mL). To our surprise, except against MRSA and S. agalactiae, the MICs of essential oil that extracted from purple flower oregano (OEO-3), with MICs range from 2–8 mg/mL, showed markedly lower antibacterial activities than that of essential oil extracted from white flower oregano (OEO-1 and OEO-2). In general, the commercial oil (OEO-4) possessed the higher antibacterial activities than the other oils. The antibacterial activities of carvacrol against the test strains were apparently higher than that of thymol, especially against S. aureus, E. faecium and S. agalactiae, with MIC values of ≤0.04 mg/mL.

For further validating the antibacterial activities of OEOs, we continued to test MIC against the 56 clinical isolates of MRSA. The inhibition rate of OEOs, carvacrol and thymol against these isolates that reflected their antibacterial activities were showed in Figure 2, and the MIC data were showed in Supplementary Table S1. The results revealed that the antibacterial activities of four essential oils or the two bioactive components against the clinical isolates agreed well with that against the standard MRSA. OEO-1 and OEO-2 showed the similar activities against all these strains with MIC with 0.25–2 mg/mL, slightly higher than that of OEO-3 (MICs were 0.5–4 μg/mL) but much lower than that of OEO-4 (MICs were 0.125–1 μg/mL). At the 0.5 μg/mL, OEO-4 inhibited 83.93% strains, while OEO-1 and OEO-2 inhibited 28.57% strains. However, OEO-3 showed the lowest antibacterial activity with only 19.64% strains inhibited at this concentration (Figure 2A). The MICs values of carvacrol and thymol were 0.01–0.16 mg/mL and 0.02–0.64 μg/mL, respectively. Carvacrol displayed higher antibacterial activities than that of thymol within the collections of clinical isolates (Figure 2B).

Given the potent in vitro antibacterial activity of carvacrol and thymol, we subsequently used checkerboard method to evaluate their synergistic effects and seven classes of common antibiotics, including tobramycin (aminoglycoside antibiotics), chloramphenicol (chloramphenicol antibiotics), ampicillin (β-lactam antibiotic), cefepime (cephalosporin antibiotic), vancomycin (glycopeptide antibiotics), polymyxin (polypeptide antibiotics) and tiamulin (pleuromutilins), against E. coli and MRSA, respectively. As shown in Figure 3 and Supplementary Figure S1, carvacrol displayed significantly synergistic effects for the selected seven antibiotics, with FICI values from 0.1875 to 0.5 for E. coli and from 0.1875 to 0.375 for MRSA, respectively. Notably, carvacrol dramatically enhanced the antimicrobial activity of tobramycin (FICI = 0.25 against E. coli and 0.125 against MRSA), polymyxin (FICI = 0.1875 against E. coli and MRSA) and vancomycin (FICI = 0.1875 against E. coli and 0.25 against MRSA). However, thymol displayed reduced synergistic effects for antibiotics, with FICI values from 0.5 to 1 for E. coli and from 0.125 to 1 for MRSA, respectively. These results revealed the strong potential of carvacrol for increasing the susceptibility of E. coli and MRSA to antibiotics as an antimicrobial adjuvant.

Based on the results from synergistic effects study, we chose 1.25 μg/mL carvacrol and 0.125 μg/mL tobramycin as the best synergistic combinations which displayed the ideal antibacterial activity against E. coli and MRSA, and used to further evaluate. To reveal the synergistic effect, we compared the bacterial killing kinetics of carvacrol, tobramycin and carvacrol combining with tobramycin. The strains were treated with compounds at the concentration of 1 × and 6 × MIC to count the number of bacteria over 24 h (Figure 4). The results showed that1 × MIC of carvacrol, tobramycin, and carvacrol combining with tobramycin slowed bacterial propagation when compared to the control. At the concentration of 6 × MIC, all the tested compounds showed relatively bacteriostatic kinetics against E. coli (Figure 4A) and MRSA (Figure 4B). Carvacrol combining with tobramycin showed the highest antibacterial activities than carvacrol and tobramycin alone, obtaining a 3.62 log reduction in the bacterial counts of E. coli and a 3.70 log reduction in the bacterial counts of MRSA within 8 h. These findings demonstrated that carvacrol combining with tobramycin could quickly kill bacteria via time dependent and can be used to efficiently treat the infections of troublesome nosocomial pathogens, such as E. coli and MRSA.

We performed SEM assay and DAPI/PI staining to confirm whether carvacrol combining with tobramycin exert their antibacterial effects through a membrane-targeting mechanism. The changes of the cell membrane morphology of MRSA after treatment with carvacrol, tobramycin and carvacrol combining with tobramycin were directly observed by SEM (Figure 5A). Apparently, the membranes of MRSA in the control group were morphologically intact with a flat and smooth surface. In contrast, the cell surface of MRSA after carvacrol treatment was perforated with obvious holes on it, while the integrity of the membranes of MRSA was evidently wrinkled and concaved after treatment by tobramycin. However, both perforation and shrinkage of membrane were found in the group treated by carvacrol combining with tobramycin.

We further determined the integrity of bacterial membranes using fluorescent probes DAPI and PI. As shown in Figure 5B, only blue fluorescence were observed in the blank control group, indicating that the bacteria were morphologically intact and not dead. By contrast, both blue and red fluorescence were observed after MRSA treated with carvacrol, tobramycin and carvacrol combining with tobramycin, revealing that these compounds can disrupt cell membranes of MRSA and thus lead to bacterial death.

The above experiments have proved that carvacrol combining with tobramycin showed good antibacterial activities in vitro. For further evaluating its therapeutic effect in vivo, a murine infection model was established by intraperitoneally injection of MRSA.

All mice in the positive group died with typical clinical signs of disease within 3 days postchallenge. Treatment with 30 and 15 mg/kg of tobramycin alone, and carvacrol combining with tobramycin, respectively, displayed dose-dependent protection (Figure 6A). Analysis based on time revealed 70% survival observed in animals treated with 30 mg/kg of carvacrol combining with tobramycin.

For further in vivo evaluation of the synergistic effects of carvacrol, the lung, liver and kidney of the infected mice were examined for their bacterial load after treatment for 3 days. Compared with the control group, treatment with carvacrol combining with tobramycin at doses of 30 and 15 mg/kg significantly (p < 0.05) reduced the bacterial loads in the lungs by 2.93 and 1.36 log₁₀ colony forming units (CFU), respectively. Tobramycin also exhibited significantly (p < 0.05) reductions of 2.23 and 1.99 log₁₀ CFU in the lungs at doses of 30 and 15 mg/kg, respectively. Only the MRSA loads in the liver of the rats treated with carvacrol combining with tobramycin at 30 and 15 mg/kg were all significantly lower (p < 0.05) than that in the control group. In the kidney, two dosages of carvacrol combining with tobramycin, as well as tobramycin at doses of 30 mg/kg, significantly (p < 0.05) reduced the MRSA load (Figure 6B).

Base on the results that carvacrol combining with tobramycin significant reduced the bacterial load in the lungs, we further assessed whether the combining drugs attenuated the damaged tissue caused by MRSA using light microscopy method. Compared to the normal control group, the positive control group exhibited evident histopathological changes, including congestion, alveolar wall thickening, and widespread neutrophil cell infiltration within the alveolar septa and lumen. Mice treated with 15 and 30 mg/kg of carvacrol combining with tobramycin, respectively, cased attenuated histopathological changes in lung tissue when compared to the groups that treated with the same dose of tobramycin alone (Figure 6C).