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. 2026 Jul 14;16(37):40291–40306. doi: 10.1039/d6ra01688j

Synergistic bactericidal mechanism of carvacrol and ε-polylysine against Staphylococcus aureus: dysregulation of cellular homeostasis and metabolism

Xiangyu Zang a, Chen Du b, Shuai Wan a, Shengtao Ma a, Yingjie Feng a, Xu Zhao a, Jianchun Yu a, Zihao Wu b, Qianjin Liu a,✉, Jingnan Zhang b,✉
PMCID: PMC13367284  PMID: 42453950

Abstract

This study aims to explore the synergistic bactericidal interaction of carvacrol (CAR) combined with ε-polylysine (ε-PL) against Staphylococcus aureus and its underlying mechanism. The combination of CAR (12.50 µg mL−1) and ε-PL (7.66 µg mL−1) exhibited synergistic bactericidal and antibiofilm activities, primarily attributed to combined destruction of the bacterial cell membrane, as evidenced by increased membrane permeability, time-dependent depolarization, and reduced cell surface hydrophobicity after 3 h of treatment. Membrane damage facilitated intracellular accumulation of CAR, which rapidly triggered energy depletion and early metabolic disturbances. Using metabolomics, the combination after 0.5 h was found to primarily perturb three key pathways, including tryptophan metabolism, cofactor biosynthesis (thiamine, biotin, nicotinamide), and nucleotide metabolism. These early metabolic changes, along with ATP decline, occurred prior to full membrane depolarization, oxidative stress, and pH imbalance at 3 h. Ultimately, the synergy caused irreversible energy depletion, membrane disruption, and severe oxidative damage. Furthermore, the combination reduced S. aureus counts by approximately 1.0 log10 CFU g−1 in raw beef under refrigeration, supporting its practical potential. This study for the first time constructs a complete temporal mechanism chain from initial membrane damage and rapid metabolic disorder to downstream cellular dysfunction for the CAR-ε-PL synergy, providing a novel theoretical basis for developing natural antimicrobial strategies in food preservation.

Early-time-point metabolomics and physiological assays demonstrate that CAR and ε-PL synergistically kill S. aureus by first depleting ATP, then disrupting core metabolic pathways, and finally causing membrane depolarization and oxidative damage.graphic file with name d6ra01688j-ga.jpg

1. Introduction

Chemical synthetic preservatives are widely used in the food industry, while their abuse, residues, and other problems could be harmful to human health. With the improvement of health awareness in recent years, people have become more interested in natural green foods, which puts forward higher requirements for food preservatives. More studies are focusing on the development of natural preservatives derived from plants, animals, and microorganisms.1

Carvacrol (CAR), a plant-derived phenolic monoterpene, has recently obtained wide interest.2 CAR has broad-spectrum antimicrobial activity, great antioxidant activity and high safety, and has been classified as generally recognized as safe (GRAS).3 ε-polylysine (ε-PL) is a natural cationic antibacterial peptide produced by the fermentation of Streptomyces albulus.4 ε-PL has been approved as a food preservative for meat, dairy products, soft drinks, and fruits in many countries due to its great antimicrobial effect, good water solubility, high heat stability and safety.1,5,6 CAR and ε-PL are two promising natural preservatives, while their practical application is limited by the off-odor of high-dose CAR and bitter taste of ε-PL.1,7

Combination application has become an effective strategy to alleviate these limitations and enhance antibacterial efficacy via synergistic effects. Preliminary studies have confirmed the synergistic antibacterial effect of CAR and ε-PL against various pathogens and spoilage bacteria.8–10 Gao et al. demonstrated that CAR and ε-PL induced membrane damage of Staphylococcus aureus through increasing membrane permeability and releasing intracellular contents.9 Additionally, some studies have developed nanoemulsions or composite films based on this combination to improve stability and application feasibility, verifying their potential in food preservation.11,12 However, existing mechanistic studies only stay at the cell membrane damage level, documenting phenomena such as membrane permeability increase and morphological deformation but failing to explore the intracellular physiological and metabolic cascades triggered by membrane damage.9,12

S. aureus is one of the most common human pathogens in various foods due to its strong resistance to the external environment and low nutrient requirements. The consumption of food contaminated with S. aureus may cause gastrointestinal symptoms such as diarrhea, vomiting, and even induce sepsis.13 Therefore, this work selected S. aureus as the target microorganism to assess the combined antibacterial and antibiofilm effects between CAR and ε-PL. Investigations into alterations in cell physiological traits, such as morphology, surface hydrophobicity, membrane function, pH and redox homeostasis, and energy metabolism, were conducted to elucidate the synergistic mechanism. Furthermore, non-targeted metabolomics was employed for the first time to gain deeper mechanistic insights into this synergy. To verify the practical application potential of this combination, its antibacterial efficacy was also evaluated in a raw beef matrix under refrigerated storage. These findings provide a novel theoretical basis for developing natural preservatives, particularly for meat products, and highlight the potential of this synergistic combination for food preservation.

2. Materials and methods

2.1. Bacteria strain and culture conditions

S. aureus (ATCC 6538) employed in the present study was acquired from the China Industrial Microbial Culture Preservation and Management Center (CICC) (Beijing, China). S. aureus was inoculated into tryptic soy broth (TSB, Hopebio, Qingdao, China) and cultivated at 37 °C with shaking (150 rpm min−1) until the optical density at 600 nm (OD600) reached 0.8. The resulting pellet was acquired by centrifugation and resuspended in sterile saline (0.85% NaCl, w/v) to obtain the S. aureus suspension with 1 × 106 colony forming units (CFU) mL−1.

2.2. Preparation of CAR and ε-PL solutions

CAR (98%) (Shanghai Yuanye Bio-Technology Co. Ltd, Shanghai, China) stored away from light was firstly dissolved in Tween 80 (1 : 1, v/v), and subsequently 2-fold serially diluted in sterile saline, resulting in 7.66–980.00 µg mL−1 of the CAR dilutions. ε-PL (Zhengzhou Benafo Bioengineering Co. Ltd, Zhengzhou, China) was oscillated dissolved in sterile saline to obtain ε-PL solutions with the concentrations of 6.25–800.00 µg mL−1.

2.3. Determination of MICs

The MICs of CAR and ε-PL against S. aureus were determined using a microbroth dilution method.14 Briefly, 100 µL of each CAR or ε-PL dilution and 100 µL of bacterial suspension (1 × 106 CFU mL−1) were respectively added to each well of a 96-well microplate. The mixtures of 100 µL of each antimicrobial dilution and 100 µL of TSB were used as negative controls, and 200 µL of S. aureus suspension was used as a positive control. The MIC was the lowest concentration of CAR or ε-PL that completely inhibited bacterial growth after cultivation at 37 °C for 24 h.

2.4. Synergism testing

The synergistic antibacterial activity of CAR and ε-PL was estimated by fractional inhibitory concentration index (FICI) through checkerboard method.13 Firstly, CAR solution at 2 × MIC was serially diluted along the ordinate of the 96-well microtiter plates, and ε-PL at 2 × MIC was serially diluted along the abscissa. Subsequently, each well containing an antimicrobial dilution (100 µL) was inoculated with 100 µL of an inoculum of S. aureus (106 CFU mL−1), and the microtiter plate was cultivated at 37 °C for 24 h. The resulting checkerboard contains each combination of two antimicrobials. The FICI was calculated as follows:2.4.

The interpretations of FICI were defined as synergistic, FICI ≤ 0.5; additive, 0.5 < FICI ≤ 1.0; irrelevant, 1.0 < FICI ≤ 4.0; and antagonistic, FICI > 4.0.

2.5. Time-kill assay

S. aureus suspension was exposed to CAR and ε-PL alone at 1/2 × MIC or their combination (both at 1/2 × MIC or 1/4 × MIC) at 37 °C for 0, 3, 6, 9 and 12 h, respectively. At the indicated time point, appropriate diluents were evenly spread on tryptic soy agar (TSA, Hopebio, Qingdao, China) plates. Colony counting was conducted after incubation at 37 °C for 24 h. Bacterial suspension without any antimicrobial was used as a control. The experiment was repeated three times for each treatment group.

2.6. Assessment of bacterial biofilm-forming ability

The biofilm-forming ability of S. aureus untreated or treated by individual and combined CAR and ε-PL was evaluated by the microtiter plate biofilm assay.15 Firstly, 100 µL of bacterial suspension and 100 µL of an antimicrobial agent were respectively added to each well of a 96-well microtiter plate and mixed slightly. The plate was then covered and incubated at 37 °C for 24, 48, and 72 h, respectively. At indicated time point, the solution in each well was removed successively. Each well of the plate was washed thrice using sterile saline. Subsequently, 200 µL of methanol (99%) was added to each well to fix the bacterial biofilm for 15 min and then discarded. After air-drying for 45 min at room temperature, each well of the plate was stained using 200 µL of 0.1% (w/v) crystal violet for 10 min in the dark and then washed thrice with sterile saline. Lastly, 200 µL of 33% (v/v) acetic acid was added to each well. The absorbance of the solution from each well was measured by a Spark® 20M multimode microplate reader (Tecan Group, Männedorf, Zürich, Switzerland) at 620 nm, which represented the bacterial biofilm production.

2.7. Assessment of cell surface hydrophobicity

Cell surface hydrophobicity (CSH) of S. aureus was determined using bacterial adherence to hydrocarbons (BATH) test.16S. aureus was treated by single CAR or ε-PL (at 1/2 × MIC) and their combination (both at 1/2 × MIC or 1/4 × MIC) at 37 °C for 3 h, respectively. Treated bacterial suspension (3 mL) was thoroughly blended with hexadecane (0.4 mL) and allowed to stand for 15 min to complete the phase separation. The OD600 (A1) of the aqueous phase was measured. CSH was calculated using the following formula:CSH (%) = (A0 − A1)/A0 × 100%where A0 is the initial absorbance of cell suspension.

2.8. Field-emission scanning electron microscope (FE-SEM) observation

Cell morphological changes of S. aureus were observed after exposure to single CAR or ε-PL at 1/2 × MIC and their combination (both at 1/4 × MIC) for 3 h.13S. aureus pellets with or without treatment were washed twice with 0.01 mol L−1 sterile phosphate buffer (PBS) and fixed in a glutaraldehyde solution (2.5%, v/v) for 4 h at 4 °C. Fixed cells were washed thrice with 0.01 mol L−1 PBS, dehydrated using gradient-diluted ethanol solutions (10–100%, v/v), and then replaced with isoamyl acetate. The dehydrated S. aureus cells were dried using a CO2 critical point dryer (Autosamdri-815, Tousimis Research Corp., Rockville, MD, USA), sputter-coated with gold using a vacuum evaporator (HUS-5GB, Hitachi, Tokyo, Japan), and finally observed by a FE-SEM (Merlin Compact, Carl Zeiss, Oberkochen, Germany).

2.9. Measurement of membrane permeability and membrane potential (ΔΨ)

Propidium iodide (PI) was used to examine cell membrane permeabilization of CAR, ε-PL, and their combination.17 Briefly, S. aureus cells were stained by the PI probe (final concentration of 3.0 µg mL−1) for 30 min at 37 °C in the dark. Fluorescence intensity of PI in bacterial cells was detected using a multimode microplate reader. The excitation wavelength (Ex) and emission wavelength (Em) was 485 and 635 nm, respectively. The ratio of fluorescence intensity of PI in the treated sample to that in the untreated sample was used to express the cell membrane permeability. Fluorescence intensity of PI-stained S. aureus suspension was also examined using the Cytofex S flow cytometer (Beckman Coulter International Trade, Shanghai, China). FlowJo V10 (FlowJo, Ashland, OR, USA) was used to analyze the data. Debris and dead cells were first removed by forward scatter area (FSC-A) versus side scatter area (SSC-A) gating. After that, bacterial cells were stringently gated via forward scatter width (FSC-W) versus forward scatter height (FSC-H) plot to discriminate doublets and aggregated cells. Positively PI-stained bacterial cells were gated with a control signal from the PE channel, and a total of 10 000 events were acquired per condition.

The ΔΨ was evaluated according to the method reported by Niu et al.14 After exposure to single or combined CAR and ε-PL, S. aureus cells were stained by the DiBAC4(3) probe (final concentration of 2.5 µg mL−1) at 37 °C for 30 min. Fluorescence intensity of DiBAC4(3) in bacterial cells was detected by a multimode microplate reader with Ex/Em at 488/525 nm. The relative fluorescence of DiBAC4(3) was used to represent the ΔΨ, which was calculated in the same way as the membrane permeability.

2.10. Determination of intracellular reactive oxygen species (ROS) level

Intracellular ROS content was estimated by a DCF-DA dye that can enter cells and be oxidized by ROS to dichlorofluorescein (DCF).16S. aureus cells blended with DCFH-DA solution (the final concentration was 2.5 µg mL−1) were cultured at 37 °C for 30 min. After treatment with single or combined CAR and ε-PL, fluorescence intensity of DCF in bacterial cells suspended in sterile saline was detected by a multimode microplate reader at Ex/Em = 488/525 nm. The relative fluorescence intensity of DCF was used to express the intracellular ROS level, which was calculated in the same way as the membrane permeability.

2.11. Determination of activities of intracellular adenosine triphosphatases (ATPases) and intracellular adenosine triphosphate (ATP) content

Intracellular Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities of untreated or antimicrobial-treated S. aureus were determined by measuring inorganic phosphorus (Pi) released from ATP following instructions of manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The ATP content in S. aureus was measured using an ATP detection assay kit (Beyotime Biotech. Inc., Shanghai, China). The luminescence signal was recorded using a multimode microplate reader, and intracellular ATP content was expressed as the percentage relative to the untreated control (set to 100% at each corresponding time point).

2.12. Determination of intracellular pH

Intracellular pH of S. aureus cells was assessed according to the previously described method reported by Yang et al.18 Untreated S. aureus cells were first collected and immediately loaded with the BCECF-AM probe (final concentration was 10.00 µmol L−1) at 37 °C for 30 min. Afterwards, bacterial cells were washed with 0.01 mol L−1 PBS and suspended in 10.00 mmol L−1 sterile glucose solution. After culture at 37 °C for 30 min, S. aureus cells were treated by single or combined CAR and ε-PL, respectively. The BCECF fluorescence intensity in S. aureus cells was measured using a multimode microplate reader and a flow cytometer at Ex/Em = 488/535 nm. BCECF positivity was analyzed using the FITC channel.

2.13. Intracellular protein analysis

Untreated or antimicrobial-treated S. aureus cell pellets were mixed with lysozyme solution (10 mg mL−1) and TE buffer solution and then were lysed using an ultrasonic cell crusher at 300 W (JY98-IIIDN, Ningbo Scientz Biotechnology Co. Ltd, Ningbo, China). Each lysed sample was used to measure the intracellular protein content by the bicinchoninic acid (BCA) method (Sangon Biotech Co. Ltd, Shanghai, China). Meanwhile, cell pellet after each treatment was mixed with lauryl sodium sulfate (SDS) loading buffer and boiled for 10 min. Supernatants containing soluble proteins were collected by centrifugation for SDS-PAGE.

2.14. Non-targeted metabolomic analysis

S. aureus cells were treated with a combination of CAR and ε-PL (each at 1/4 × MIC) in TSB at 37 °C for 0.5 h. To verify cell viability at the time of sampling, an aliquot of the culture was taken for plate counting on TSA. Cells were then harvested by centrifugation (8000×g, 4 °C, 4 min) and washed twice with ice-cold sterile PBS (0.01 mol L−1) prior to metabolite extraction. Intracellular metabolites were extracted by adding 300 µL of 80% methanol aqueous solution to the cell pellets. The mixture was vortexed, rapidly frozen in liquid nitrogen for 5 min, and then thawed on ice. After vortexing (30 s) and sonication (6 min), the supernatant was collected following centrifugation (5000 rpm, 4 °C, 1 min) and freeze-dried for subsequent analysis. A pooled quality control (QC) sample was prepared by combining equal volumes of each experimental sample.

Metabolite profiling was performed using an LC-MS/MS system (Vanquish UHPLC coupled with a Q Exactive HF mass spectrometer; Thermo Fisher Scientific In. China). Separation was achieved on a Hypersil Gold C18 column (100 × 2.1 mm, 1.9 µm) maintained at 40 °C with a flow rate of 0.2 mL min−1. The mobile phase consisted of 0.1% formic acid in water (A) and methanol (B). A gradient elution was applied as follows: 2% B from 0 to 1.5 min, then linearly increased to 85% B at 3 min, further increased to 100% B at 10 min, held at 100% B for 0.1 min, and then returned to 2% B from 10.1 to 12 min. Mass spectrometry was operated in both positive and negative ionization modes with a scan range of m/z 100–1500. Source parameters were: spray voltage, 3.5 kV; capillary temperature, 320 °C; sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10 L min−1.16

Raw data were processed using the XCMS package for peak detection, alignment, and integration. Differential metabolites were identified based on a variable importance in projection (VIP) > 1, p < 0.05, and a fold change (FC) > 2.0 or < 0.50. The orthogonal partial least squares discriminant analysis (OPLS-DA), heatmap analysis, and Kyoto Encyclopedia of Genes and Genomes database (KEGG) analysis were performed using the NovoMagic platform (https://magic-plus.novogene.com).

2.15. Application of CAR and ε-PL for raw beef preservation

Raw beef was obtained from a local market (Zhengzhou, Henan, China) and immediately transported to the laboratory. The beef was cut into uniform pieces (10 g each). The samples were randomly divided into ten groups. For five groups, the beef pieces were sterilized by UV irradiation (30 min per side) to eliminate background microflora. Subsequently, each piece was immersed in sterile saline (control) or antimicrobial solutions (CAR at 1 × MIC, ε-PL at 1 × MIC, 1/2 × MIC CAR + 1/2 × MIC ε-PL, or 1 × MIC CAR + 1 × MIC ε-PL) for 15 min. After immersion, the surface was air-dried, and 50 µL of S. aureus suspension (approximately 104 CFU g−1) was evenly spread onto each beef piece. The inoculated samples were then placed in sterile culture dishes and stored at 4 °C for 6 days. The remaining five groups (without UV sterilization) were treated with the same antimicrobial solutions for 15 min, then directly placed in sterile culture dishes and stored at 4 °C for 6 days to evaluate the effect on background microflora. At 0, 1, 2, 4, and 6 days, samples were homogenized, serially diluted, and plated on plate count agar (PCA) for total viable counts (TVC) and on TSA for S. aureus counts. Plates were incubated at 37 °C for 48 h, and results were expressed as log10 CFU g−1.

2.16. Statistical analysis

Each experiment was independently repeated thrice in triplicate. All data were presented as the mean ± standard deviation (SD). After pre-evaluating, the normality and homogeneity of the variance of data, one-way analysis of variance (ANOVA) and Ducan's multiple comparative analysis were conducted with SPSS statistics software version 25 (IBM SPSS Inc., Chicago, USA). Results were statistically significant at p < 0.05.

3. Results and discussion

3.1. Synergistic antibacterial effect of CAR and ε-PL against S. aureus

The MICs of CAR and ε-PL against S. aureus were 30.63 and 50 µg mL−1, respectively. CAR and ε-PL displayed great inhibitory effects on S. aureus growth. The fractional inhibitory concentration index (FICI) value of the combination of CAR and ε-PL was 0.50, indicating that there was a synergistic interaction between CAR and ε-PL against S. aureus. When used in combination, MICs of both antimicrobials were reduced to 1/4 × MIC (12.50 and 7.66 µg mL−1, respectively). As shown in Fig. 1, the number of S. aureus decreased to the lowest level after treatment with CAR + ε-PL at 1/4 × MIC for 6 h and then increased slowly, which was still much lower than that of individual treatment with CAR or ε-PL at 1/2 × MIC after 12 h. After being treated by CAR + ε-PL at 1/2 × MIC for 6 h, the S. aureus population was lower than the detection limit (<10 CFU mL−1), and no bacterial growth was observed after prolonged treatment time. These results demonstrated that combined use of CAR and ε-PL significantly enhanced the antibacterial efficiency on S. aureus, which align with previous study of Gao et al.9

Fig. 1. Time-kill curves of single and combined CAR and ε-PL against S. aureus. Error bars show standard deviations (SDs) (n = 3). Different lowercase letters indicate statistically significant differences in different treatments (p < 0.05).

Fig. 1

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3.2. Combined effect of CAR and ε-PL on the biofilm formation and CSH of S. aureus

A biofilm is a multicellular community of microbes attached to biotic or abiotic surface in which microbial cells adapt to their environment.19 Bacterial biofilms have good resistance to disinfectants, antibiotics, and phagocytosis.20 As shown in Fig. 2A, either CAR or ε-PL alone (1/2 × MIC) was able to prevent S. aureus biofilm formation, which was in line with previous reports.21 After culture for 48 h, CAR + ε-PL at 1/4 × MIC or 1/2 × MIC inhibited the biofilm formation of S. aureus by 76.12% and 80.57% (p < 0.05), respectively, compared with that of each individual treatment (<60%). With prolonging cultivation time to 72 h, the formation of S. aureus aggregation with or without treatment significantly increased except for combination treatment both at 1/2 × MIC. These results reveal that ε-PL and CAR had a significant synergistic impact on S. aureus biofilm prevention and exhibited a dose dependent manner.

Fig. 2. The biofilm-forming ability (A) and cell surface hydrophobicity (B) of S. aureus after exposure to single or combined CAR and ε-PL. Error bars represent SDs (n = 3). Different lowercase letters indicate statistically significant differences in different treatments (p < 0.05).

Fig. 2

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The CSH of bacteria is closely related to the adhesion ability, which is particularly important for bacteria to exert the toxic effect. Generally, the higher the hydrophobicity of the strain, the stronger its adhesion ability.16 As shown in Fig. 2B single or combined CAR and ε-PL treatment significantly reduced the CSH of S. aureus (p < 0.05). After being treated by CAR or ε-PL alone at 1/2 × MIC for 3 h, the CSH of S. aureus was decreased by 11.34% and 10.63%, respectively. CAR + ε-PL at 1/4 × MIC or 1/2 × MIC remarkably decreased the CSH of S. aureus by 22.88% and 30.09%, respectively, significantly lower than that of individual treatment (p < 0.05) (Fig. 2B). The decreasing trend of CSH is consistent with that of bacterial biofilm-forming ability of S. aureus after individual or combined treatment with CAR and ε-PL (Fig. 2A).

Previous study has reported that ε-PL significantly lowered hydrophobicity and extracellular polymeric substances (EPS) production of L. monocytogenes to inhibit the biofilm formation.22 Moreover, due to the amphiphilicity of CAR, its relative hydrophilicity may allow CAR to diffuse through the polar polysaccharide matrix of the biofilm, while the hydrophobicity of CAR may result in interactions with the cell membrane, affecting its structure and function.23 The results in this study suggest that CAR + ε-PL can enhance the interaction between antimicrobial agents and microbial cell membranes, resulting in decreased CSH and bacterial adhesion, which could be the reason for prevention of S. aureus biofilm formation.

3.3. Combined effect of CAR and ε-PL on cell morphology of S. aureus

Fig. 3 shows the FE-SEM images of untreated and antimicrobials-treated S. aureus cells. S. aureus cells without treatment were typically spherical with a complete membrane structure (Fig. 3A). After being treated by CAR at 1/2 × MIC for 3 h, cell surfaces of some cells were wrinkled and collapsed, and cell membranes were obviously damaged (Fig. 3B). Morphological alteration of S. aureus induced by ε-PL at 1/2 × MIC for 3 h were like those treated by CAR (Fig. 3C), except that the number of deformed cells was low and most of them were still spherical. However, after combined treatment with CAR and ε-PL both at 1/4 × MIC, cell morphological changes of S. aureus became more serious, and the number of damaged bacteria greatly increased (Fig. 3D). Similarly, S. aureus treated by ε-PL combined with d-limonene nanoemulsion endured a complete collapse of cell morphology.24 The degree of morphological change of S. aureus treated by individual or combined ε-PL and CAR is consistent with the result of bacterial survival (Fig. 1).

Fig. 3. FE-SEM images of S. aureus cells. (A) Control; (B) CAR at 1/2 × MIC; (C) ε-PL at 1/2 × MIC; (D) CAR + ε-PL both at 1/4 × MIC.

Fig. 3

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3.4. Combined effect of CAR and ε-PL on membrane permeability of S. aureus

PI is a commonly used fluorescent nuclear and chromosome counterstain, which binds to DNA by inserting between bases and then exhibits red fluorescence. PI probe can only enter cells with damaged membranes.14 As depicted in Fig. 4A, compared with untreated sample (100%), the PI uptake in bacterial cells after combination treatment both at 1/4 × MIC was pronouncedly increased by 501.27% (p < 0.05), while 74.25% and 32.47% for single treatment with CAR and ε-PL at 1/2 × MIC, respectively. PI uptake in S. aureus cells under different treatment conditions was also verified by using a flow cytometry (Fig. 4B). In the untreated group, the number of PI-negative bacterial cells accounted for 96.02%. After single treatment with CAR or ε-PL at 1/2 × MIC, the number of PI-negative cells decreased to 84.02% and 92.25%, respectively, while less than 70.00% after combination treatment both at 1/4 × MIC or 1/2 × MIC (p < 0.05). This demonstrated that ε-PL in combination with CAR severely destroyed the physical integrity of S. aureus cell membrane.

Fig. 4. Relative fluorescence intensity (A) and flow cytometric analysis (B) of S. aureus cells stained with PI after exposure to single or combined CAR and ε-PL. (a) Control; (b) CAR at 1/2 × MIC; (c) ε-PL at 1/2 × MIC; (d) CAR + ε-PL at 1/4 × MIC; (e) CAR + ε-PL at 1/2 × MIC. Error bars show SDs (n = 3). Different lowercase letters in (A) represent statistically significant differences between the means (p < 0.05).

Fig. 4

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3.5. Combined effect of CAR and ε-PL on membrane potential of S. aureus

Cell membrane potential (ΔΨ) plays a crucial role in multiple physiological processes, such as active transport, intracellular pH regulation, and energy transformation.25 ΔΨ was monitored using DiBAC4(3), which accumulates in depolarized cells and increases fluorescence. As shown in Fig. 5A, after 0.5 h, 1/2 × MIC ε-PL treatment caused no significant change in S. aureus membrane potential (102.3%, p > 0.05), whereas 1/2 × MIC CAR (108.9%), the 1/4 × MIC combination (114.5%) and the 1/2 × MIC combination (139.2%) all significantly increased fluorescence (p < 0.05). At 1 h, ε-PL remained non-significant (107.5%, p > 0.05), while the other groups further increased to 114.2%, 120.6% and 131.0%, respectively (p < 0.05). By 3 h, all treatments caused substantial depolarization, with CAR at 145.6%, ε-PL at 124.5%, the 1/4 × MIC combination at 195.4% and the 1/2 × MIC combination at 257.3%. For the combinations, the increases were statistically significant compared to the control and to each single agent (p < 0.05). These results demonstrate that CAR and its combination with ε-PL depolarize S. aureus in a time-dependent manner, with the strongest synergistic effect observed at 3 h. These results are in line with earlier studies showing that CAR alone dissipates membrane potential.26 However, the synergistic enhancement by the CAR-ε-PL combination has not been previously quantified.

Fig. 5. Relative fluorescence intensity of DiBAC4(3) (A) and DCF (B) in S. aureus exposed to single or combined CAR and ε-PL. Error bars represent SDs (n = 3). Different lowercase letters represent a significant difference at the p < 0.05 level between the mean values.

Fig. 5

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It has been reported that positively charged ε-PL molecules can be embedded in the negatively charged components of bacterial cell wall (such as peptidoglycan and lipoteichoic acid), forming pores in the cell wall.1 Moreover, due to the hydrophobic characteristics, it was suggested that CAR mainly targeted the lipid-containing bacterial plasma membrane, making the membrane more permeable.23 CAR and ε-PL molecules could act simultaneously on the cell membrane and disturb the interactions of components in the bilayer membranes, thereby destructing the cell membrane and resulting in a decline in cell activity.

3.6. Combined effect of CAR and ε-PL on intracellular ROS of S. aureus

Under normal physiological conditions, cells produce a small number of free radicals through cell metabolism, which can be eliminated by antioxidant enzymes and other antioxidants to maintain the cellular redox homeostasis. However, overproduction of free radicals can result in oxidative damage to cells and even cause cell death.27 As shown in Fig. 5B, CAR at 1/2 × MIC induced an increase of 13.35% in the DCF fluorescence intensity relative to the untreated sample (p < 0.05), indicating that CAR induced the increase in ROS production in S. aureus cells, while there was no obvious change in ε-PL (1/2 × MIC) treated sample (p > 0.05). Previous studies have found that CAR and ε-PL could increase the ROS level in bacterial cells.1,28 ε-PL did not raise intracellular ROS level in this study may be due to the low concentration used. However, CAR + ε-PL treatment (both at 1/2 × MIC) elevated the relative fluorescence intensity of DCF in S. aureus cells to 148.53%, which was significantly higher than that of each individual antimicrobial treatment (p < 0.05). ROS accumulation in S. aureus could cause imbalance of redox homeostasis and induce oxidative damage to various cellular components such as DNA, lipids, and proteins.28 The alteration of intracellular ROS levels under the action of various antibacterial agents is in line with the trend of bacterial inactivation (Fig. 1).

3.7. Combined effect of CAR and ε-PL on the ATP content and ATPases activities in S. aureus

To capture the temporal dynamics of energy depletion, we measured intracellular ATP levels at 0.5, 1, and 3 h after treatment. As shown in Fig. 6B, the control group maintained relatively stable ATP levels over time. In contrast, all treatment groups exhibited a time-dependent decline in relative ATP content. At the earliest time point (0.5 h), the combination at 1/2 × MIC each reduced ATP to 10.1% of the control (set as 100%), and the combination at 1/4 × MIC each caused a reduction to 15.0%. Single agent treatments also decreased ATP levels at 0.5 h, with CAR reducing it to 24.7% and ε-PL to 65.7%. By 1 h and 3 h, ATP levels in all treated groups fell further, with the combinations reaching below 10% at 1 h and below 1.5% at 3 h. These results demonstrate that CAR and ε-PL act synergistically to cause rapid and profound ATP depletion in S. aureus, with a significant drop detectable as early as 0.5 h after exposure. The early onset of ATP collapse (0.5 h) clearly precedes the full membrane depolarization observed at 3 h (Fig. 5A). This temporal sequence suggests that energy failure is an early, if not the earliest, event driving subsequent cellular dysfunction. The rapid ATP depletion observed here is consistent with the known protonophore activity of CAR, and the synergistic effect of the combination is stronger than that reported for either compound alone.9,26

Fig. 6. Activities of ATPases (A) and intracellular ATP content (B) in S. aureus after exposure to single or combined CAR and ε-PL. Error bars represent SDs (n = 3). Different lowercase letters represent a significant difference at the p < 0.05 level between the mean values.

Fig. 6

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Both Na+/K+-ATPase and Ca2+/Mg2+-ATPase rely on the energy provided by ATP hydrolysis for their catalytic activity.29 The sharp decline in intracellular ATP content observed after CAR and/or ε-PL treatment would inevitably compromise the function of these ATPases. As presented in Fig. 6A, after 3 h of treatment, Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities were reduced by 34.6% and 28.3% with single CAR at 1/2 × MIC, while ε-PL alone only decreased Na+/K+-ATPase activity (by 28.5%). When CAR and ε-PL were combined (both at 1/4 × MIC or 1/2 × MIC), both ATPase activities were remarkably reduced by more than 57.0% compared with single agents (p < 0.05). The decrease in Na+/K+-ATPase activity is closely associated with the observed cell membrane depolarization (Fig. 5A), as this ATPase is critical for maintaining membrane potential.30 The reduction in Ca2+/Mg2+-ATPase activity would disrupt Ca2+ homeostasis, further contributing to cellular dysfunction.31

The rapid ATP depletion likely results from two synergistic effects: (i) membrane damage (Fig. 4) impairs the electron transport chain and ATP synthase, blocking aerobic ATP synthesis; (ii) increased membrane permeability causes leakage of ATP to the extracellular environment. The consistency among time-kill curves, membrane damage, and ATP dynamics supports that energy disruption is an early and critical event leading to cell death.

3.8. Combined effect of CAR and ε-PL on intracellular pH of S. aureus

Intracellular pH homeostasis is crucial for microbial growth and metabolism, and cell life activities such as nutrient absorption and utilization, protein biosynthesis, glycolysis, and ATP biosynthesis.18,32 BCECF-AM, a fluorescent probe, can be clipped into BCECF by the esterase inside the cell and excited to produce green fluorescence at the appropriate pH value.32 In this study, compared with the untreated sample (100.00%), the relative fluorescence intensity of BCECF in S. aureus cells treated by CAR + ε-PL (both at 1/4 × MIC or 1/2 × MIC) was significantly lowered by 57.45% and 75.11%, respectively (p < 0.05), lower than that in single treatment groups (1/2 × MIC; <40.00%) (Fig. 7A). The change in intracellular pH was also analyzed by using a flow cytometry (Fig. 7B), and the results accord with those obtained by the multimode microplate reader. S. aureus cells emitting green fluorescence accounted for 95.88% in the untreated group, however, it gradually decreased to 84.87%, 86.28%, 44.57% and 27.46% after exposure to individual and combined treatment with CAR and ε-PL, respectively. These results indicate that CAR combined with ε-PL could promote cellular acidification of S. aureus. In the presence of ε-PL, more CAR molecules could penetrate the cell membrane damaged by antimicrobial treatment. The phenol hydroxyl group of CAR molecules could function as a univalent cation exchange carrier to release proton in the cell and bind to K+, resulting in the reduction of the pH gradient across the cytoplasmic membrane. The collapse of the proton motive force that drives ATP biosynthesis can lead to the depletion of the ATP pool and ultimately cell death.7

Fig. 7. Relative fluorescence intensity (A) and flow cytometric analysis (B) of S. aureus stained with BCECF after exposure to CAR, ε-PL, or their combination. (a) Control; (b) CAR treatment at 1/2 × MIC; (c) ε-PL treatment at 1/2 × MIC; (d) CAR + ε-PL (both at 1/4 × MIC) treatment; (e) CAR + ε-PL (both at 1/2 × MIC) treatment. Error bars represent SDs (n = 3). Different lowercase letters in (A) represent a significant difference at the p < 0.05 level between the mean values.

Fig. 7

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3.9. Combined effect of CAR and ε-PL on intracellular protein of S. aureus

As the main undertaker of life activities, protein plays a key role in life. Compared with the untreated sample, there was no significant alteration in the level of intracellular protein of S. aureus treated by CAR or ε-PL alone at 1/2 × MIC (p > 0.05); however, after exposure to both CAR and ε-PL at 1/4 × MIC or 1/2 × MIC, it was significantly reduced by 1.81 and 3.03 mg mL−1 (p < 0.05), respectively (Fig. 8A). As shown in the SDS-PAGE image (Fig. 8B), the protein bands of S. aureus treated by single antimicrobial (1/2 × MIC) were lighter than those in the untreated sample, which is consistent with the quantitative results of proteins. In comparison with untreated and single antimicrobial-treated groups, some of the protein bands of S. aureus after the combined treatment (both at 1/2 × MIC) became lighter or even disappeared, and new bands appeared. This suggest that except for the cellular protein leakage due to cell membrane damage, changes in expression of some proteins in S. aureus treated by CAR in combination with ε-PL may be one of the reasons why intracellular protein content was decreased. Wang et al.33 and Ning et al.34 have proposed that both ε-PL and CAR could bound to bacterial DNA. Combined use of ε-PL and CAR could facilitate the delivery of more CAR and ε-PL into the cytoplasm, alter more genomic DNA structure, and disrupt DNA-dependent biological processes, thereby exerting its antibacterial activity. Protein biosynthesis could also be interfered with the imbalance of intracellular environmental homeostasis of S. aureus.18

Fig. 8. Intracellular protein content (A) and gel electrophoresis images of intracellular soluble proteins (B) of S. aureus. (1) CAR + ε-PL (both at 1/2 × MIC) treatment; (2) CAR + ε-PL (both at 1/4 × MIC) treatment; (3) ε-PL (1/2 × MIC) treatment; (4) CAR (1/2 × MIC) treatment; (5) control. Error bars show SDs (n = 3). Different lowercase letters in (A) represent a significant difference at the p < 0.05 level between the mean values.

Fig. 8

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3.10. Changes in metabolic characteristics of S. aureus treated by CAR + ε-PL

To capture early metabolic events while ensuring cell viability, metabolomics was performed under low-lethality conditions (1/4 × MIC each, 0.5 h). At the same sampling time, viable plate counting showed that the treated cells retained 58% of the initial inoculum (6.04 log10 CFU mL−1vs. 6.28 log10 CFU mL−1), confirming that the majority of cells were still physiologically active (Fig. S1). The untargeted metabolomic analysis was carried out to further explore the antibacterial mechanism of CAR + ε-PL on S. aureus at the metabolomic level. A total of 3683 metabolites were identified in treated and untreated S. aureus cells. According to the PCA and OPLS-DA, the metabolic differences following antimicrobial exposure were analyzed and visualized. As exhibited in the PCA score plot (Fig. 9A), a clear separation between the treatment group and the control reflected a distinct metabolic difference. The OPLS-DA analysis (Fig. 9B) was consistent with that of the PCA analysis, and a high Q2 (>0.90) value validated the statistical reliability of the model (Fig. 9C).35 These results suggest that CAR + ε-PL induces metabolic disorders in S. aureus.

Fig. 9. Multivariate cluster analysis and intracellular metabolite profiles of S. aureus after treatment with or without CAR + ε-PL. (A) PCA score plots; (B) PLS-DA score plots; (C) permutation test map; (D) metabolite difference volcano plots.

Fig. 9

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A volcano plot was generated based on the criteria VIP > 1 in OPLS-DA analysis, fold change > 2.0 or < 0.5, and p < 0.05 in the Student's t-test (Fig. 9D). Compared with the control, a total of 398 significantly altered metabolites were screened in S. aureus cells treated by CAR + ε-PL. There were 218 up-regulated metabolites and 180 down-regulated metabolites. Detailed information about some of the differentially expressed metabolites was listed in Table S1. These results show that the combined treatment disrupted metabolic homeostasis in S. aureus through multiple targets.

The KEGG enrichment analysis was further carried out to categorize the differential metabolites between the combined treatment and the control groups into different metabolic pathways. Functional annotation of differential metabolites based on the KEGG database demonstrated amino acid metabolism and metabolism of cofactors and vitamins as the primary altered pathway, followed by nucleotide metabolism and carbohydrate metabolism (Fig. 10A). These differential metabolites were enriched in 22 metabolic pathways and mainly involved in tryptophan metabolism, biosynthesis of cofactors, and nucleotide metabolism (Fig. 10B).

Fig. 10. The KEGG classification diagram (A) and the enriched KEGG pathways (B) of differential metabolites in S. aureus cells between the control and CAR + ε-PL treatment group.

Fig. 10

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3.10.1. Tryptophan metabolism disruption

The combined treatment of CAR and ε-PL profoundly disrupted tryptophan metabolism in S. aureus. Specifically, serotonin, N-acetylserotonin, and 2,3-pyridinedicarboxylic acid were identified as differential metabolites. Serotonin and N-acetylserotonin are indoleamine derivatives that can modulate bacterial oxidative stress responses and membrane integrity.36,37 Their altered levels suggest a disturbance in the tryptophan-derived signaling pathways, which may affect bacterial adaptation to environmental stresses. More importantly, 2,3-pyridinedicarboxylic acid is a key intermediate in the kynurenine pathway that leads to the synthesis of nicotinamide adenine dinucleotide (NAD+), an essential cofactor for numerous redox reactions and energy metabolism.38 The change in this metabolite indicates that the flux from tryptophan to NAD+ is likely compromised. Given that NAD+ is required for the activity of NADH dehydrogenase (complex I) in the respiratory chain, its deficiency can directly impair electron transfer, decrease ATP production, and promote ROS generation.39,40 This provides a metabolic explanation for the observed increase in intracellular ROS level (Fig. 5B) and the decline in ATP content (Fig. 6B) following CAR + ε-PL treatment. Thus, disruption of tryptophan metabolism not only affects bacterial stress responses but also compromises the core energy-generating machinery.

3.10.2. Biosynthesis of cofactors impairment

The combined treatment also interfered with the biosynthesis of cofactors pathway, as evidenced by changes in thiamine, dethiobiotin (a biotin precursor), 2,3-pyridinedicarboxylic acid, and adenosine monophosphate (AMP). Thiamine (vitamin B1) is the precursor of thiamine pyrophosphate, an essential cofactor for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, two key enzymes that link glycolysis to the tricarboxylic acid (TCA) cycle.41 A reduction in thiamine availability can therefore restrict the entry of pyruvate into the TCA cycle, leading to ATP shortage and accumulation of glycolytic intermediates. Dethiobiotin is a direct precursor of biotin. Biotin serves as a cofactor for carboxylases involved in fatty acid synthesis, gluconeogenesis, and branched-chain amino acid metabolism.42 The alteration in dethiobiotin suggests that biotin production may be impaired, which would further compromise fatty acid biosynthesis and membrane lipid assembly. This aligns with the observed downregulation of fatty acyl compounds and glycerophospholipids in the metabolomic data (Table S1). Furthermore, 2,3-pyridinedicarboxylic acid, as mentioned above, is linked to NAD+ synthesis, while AMP is directly involved in energy charge regulation.43 The concurrent disruption of multiple cofactor biosynthesis pathways (thiamine, biotin, NAD+) creates a severe bottleneck in central carbon metabolism and lipid homeostasis, thereby sensitizing S. aureus to the membrane-damaging action of CAR and ε-PL.

Although the differential metabolites were not enriched in lipid metabolism based on KEGG analysis, specific lipid-related metabolites (acylcarnitines, lysophospholipids, glycerophospholipids) showed marked changes that are mechanistically linked to coenzyme A (CoA) deficiency.44 CoA is required for fatty acid activation; its deficiency reduces acyl-CoA pools, leading to decreased acylcarnitine levels (reflecting impaired β-oxidation) and accumulation of lysophospholipids (due to failure of re-acylation).45,46 These lipid alterations directly compromise membrane integrity, consistent with the observed membrane depolarization and increased permeability.

3.10.3. Nucleotide metabolism and energy stress

In addition to tryptophan and cofactor pathways, nucleotide metabolism was also perturbed, as indicated by changes in AMP and adenine. AMP is a key regulator of cellular energy status; an increase in AMP (or an altered AMP/ATP ratio) typically reflects energy stress and impaired ATP regeneration.47 This observation is consistent with the measured decrease in intracellular ATP level and the inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities (Fig. 6). The accumulation of adenine may result from the degradation of adenine nucleotides under energy-limited conditions.48 Because ATP is required for the phosphorylation of nucleosides to nucleotides, energy depletion directly hinders DNA and RNA synthesis, as previously discussed.49 Therefore, nucleotide metabolism disruption not only reflects energy collapse but also amplifies the metabolic crisis by limiting the availability of precursors for genetic information flow.

3.11. Synergistic bactericidal mechanism of CAR and ε-PL against S. aureus

The metabolomic data revealed that under low-lethality conditions (1/4 × MIC, 0.5 h) the combined treatment rapidly disrupted multiple key metabolic pathways, including amino acid metabolism, cofactor biosynthesis, and nucleotide metabolism. These early metabolic perturbations coincided with rapid ATP depletion (0.5 h) and preceded the progressive energy failure, oxidative stress, and membrane dysfunction, where significant ATP depletion, increased ROS, decreased pH, and reduced membrane integrity were detected. Thus, the early metabolic disturbances are temporally associated with the initiation of energy crisis and ultimately drive the collapse of cellular homeostasis upon prolonged exposure. The synergistic bactericidal effect of CAR and ε-PL is achieved through a multi-target mechanism that triggers rapid metabolic changes, subsequently leading to irreversible energy depletion, oxidative damage, and membrane disruption (Fig. 11).

Fig. 11. Schematic diagram of the synergistic bactericidal mechanism of CAR and ε-PL against S. aureus.

Fig. 11

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3.12. Antibacterial effect of CAR combined with ε-PL in raw beef

To assess the practical potential of CAR combined with ε-PL in a food matrix, a preservation test was conducted using raw beef under refrigerated storage (4 °C). The beef samples were treated with CAR and/or ε-PL, and total aerobic count (TVC) and S. aureus count were determined at 0, 1, 2, 4, and 6 days. As shown in Fig. 12, the untreated control showed rapid microbial growth in both assays, reaching approximately 10.9–11.3 log10 CFU g−1 by day 6. Single treatments with CAR or ε-PL at MIC reduced bacterial counts by 0.6–0.8 log10 CFU mL−1 compared to the control. The combination at 1/2 × MIC each gave intermediate reductions (0.86–1.15 log10 CFU mL−1), while the combination at 1 × MIC each consistently exhibited the strongest inhibitory effect. Specifically, against S. aureus, the combination at 1 × MIC each reduced counts by 1.05-log at day 1 and 1.33-log at day 6 relative to the control; against background microflora, the reductions were 0.95 log and 0.97 log, respectively.

Fig. 12. Changes in total viable counts (TVC) (A) and S. aureus (B) counts in raw beef treated with CAR and/or ε-PL during refrigerated storage (4 °C). Different uppercase letters denote significant differences between treatments at the same storage day (p < 0.05), and different lowercase letters denote significant differences between storage days for the same treatment (p < 0.05).

Fig. 12

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In contrast to the rapid and extensive killing (>5.0 log reduction) observed in liquid culture at 37 °C within 3 h (Fig. 1), the antibacterial efficacy in refrigerated beef was notably attenuated, achieving only ∼1.0 log reduction over 6 days. This discrepancy is likely attributable to the lower temperature, which slows bacterial metabolism and reduces antimicrobial activity, as well as the complex beef matrix (e.g., fat, proteins) that may adsorb or bind CAR and ε-PL, lowering their effective concentrations.50 Nevertheless, the consistent superiority of the combination over individual treatments in beef confirms that the synergistic action is at least partially preserved even in a real food matrix. These results demonstrate that CAR and ε-PL act synergistically to suppress microbial growth in raw beef, including both natural background flora and the specific pathogen S. aureus. The consistent superiority of the combination over individual components supports its potential application as a natural preservative for meat products.

4. Conclusion

This study focused on the synergistic bactericidal mechanism of CAR and ε-PL against S. aureus. By combining metabolomics with physiological measurements, the results revealed that the combination triggered rapid ATP depletion and early disruptions in tryptophan metabolism, cofactor biosynthesis (thiamine, biotin, nicotinamide), and nucleotide metabolism. These events coincided with each other and occurred prior to full membrane depolarization, oxidative stress, and membrane disintegration, thereby accounting for the synergistic antibacterial and antibiofilm effects. This work for the first time constructs a complete temporal mechanism chain from early energy crisis and metabolic reprogramming to downstream cellular dysfunction for the CAR-ε-PL combination. Furthermore, its efficacy was validated in raw beef under refrigeration, where the combination reduced S. aureus counts by approximately 1.0 log10 CFU g−1. These findings provide a novel theoretical basis for developing natural antimicrobial strategies in meat and other food preservation. Future studies employing genetic approaches (e.g., pathway-specific knockouts) or molecular docking could further confirm the direct molecular targets; the modest reduction observed in beef also suggests that formulation optimization may be needed to enhance efficacy, and the impact on food sensory properties remains to be evaluated.

Author contributions

Xiangyu Zang: conceptualization, data curation, formal analysis, writing – original draft; Chen Du: investigation, methodology, visualization; Shuai Wan: data curation, software, formal analysis; Shengtao Ma: investigation, validation; Yingjie Feng: data curation, validation; Xu Zhao: investigation, visualization; Jianchun Yu: writing – review and editing, funding acquisition; Zihao Wu: resources, validation, visualization, project administration; Qianjin Liu and Jingnan Zhang: conceptualization, funding acquisition, supervision, writing – review and editing.

Conflicts of interest

The authors have declared that there is no conflicts of interest.

Supplementary Material

RA-016-D6RA01688J-s001
RA-016-D6RA01688J-s002

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21706244), the Key Research Projects of the Science and Technology Department of Henan Province (No. 252102320187), and the Technical Program of China Tobacco Henan Industrial Co., Ltd (No. AW2024008).

Data availability

All relevant data are available within the manuscript and its supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra01688j.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RA-016-D6RA01688J-s001
RA-016-D6RA01688J-s001.pdf (116.2KB, pdf)
RA-016-D6RA01688J-s002
RA-016-D6RA01688J-s002.pdf (117KB, pdf)

Data Availability Statement

All relevant data are available within the manuscript and its supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra01688j.

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