Synergistic Inhibitory Effect and Mechanism of Action of Cinnamaldehyde‐Carvacrol Nanoemulsion on the Staphylococcus aureus and Escherichia coli Mixed Biofilms Formation

TL;DR

This study shows that a nanoemulsion of cinnamaldehyde and carvacrol can effectively inhibit mixed biofilms of Staphylococcus aureus and Escherichia coli, both in lab and mouse models.

Contribution

The novel contribution is the development and testing of a CA/CV nanoemulsion with synergistic biofilm inhibition in a mixed bacterial infection model.

Findings

CA/CV nanoemulsion significantly inhibited mixed biofilm formation at 64 μg/mL by reducing adhesion, motility, and PIA synthesis.

The nanoemulsion inhibited LuxS/AI-2 expression and reduced bacterial burden in a mouse implant infection model.

The synergistic effect of CA and CV was confirmed with a Q value of 2.28.

Abstract

The formation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) mixed biofilms challenges the treatment of bacterial infections. In this study, the synergistic effect of plant essential oils cinnamaldehyde (CA) and carvacrol (CV) on inhibiting S. aureus and E. coli mixed biofilms formation was investigated. The Q value of CA and CV combination on inhibiting mixed biofilms formation was 2.28, suggesting their synergistic effect. Furthermore, CA/CV nanoemulsion was prepared, which further enhanced the inhibitory effect. CA/CV nanoemulsion could significantly inhibit the mixed biofilms formation at 64 μg/mL by reduction of bacterial adhesion and motility, polysaccharide intercellular adhesin (PIA) synthesis, and the inhibition of LuxS/AI‐2 expression. In the S. aureus and E. coli infected implant model of mice, CA/CV NEs demonstrated prominent inhibitory effect on the formation of S. aureus and E. coli biofilms, and simultaneously reduced the bacterial burden.

The study prepared Cinnamaldehyde‐Carvacrol nanoemulsions (CA/CV NEs) through high‐energy emulsification. In a mouse infection model, CA/CV NEs exhibited a synergistic inhibitory effect on the formation of Staphylococcus aureus–Escherichia coli mixed biofilms, reducing PIA, LuxS/AI‐2, adhesion, athletic ability, and extracellular protein.

Figures

(a) FICI determination. (b) The Q value of CA and CV on inhibition S‐E biofilms formation. (c) The size distribution of CA/CV NEs. (d) TEM image of CA/CV NEs. (e) The size changes of CA/CV NEs at 4°C and the corresponding photographs on the 30th day. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Inhibitory effect of CA/CV NEs on bacterial adhesion. (a) The inhibitory effect of NEs with different concentrations on S‐E biofilm mass. (b) The inhibitory effect of different drug on the S‐E biofilm mass. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (c) Photographs of crystal violet staining. (d) SEM images of S‐E. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

NEs inhibited bacterial adhesion. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) Relative adhesion rate, (b) Diffusion inhibition rate, (c) Changes in PIA content, (d) Diffusion results of LB plates, (e) Congo red binding test. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Effects of different groups on LuxS/AI‐2 system. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) Changes in protein content. (b) LuxS mRNA level, (c) AI‐2 production. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Inhibition of CA/CV NEs on S‐E biofilm in vivo. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) Establishment of mouse infection model. (b) Photographs S. aureus and E. coli plate count. (c) SEM images of mixed biofilms after different treatment. (d) Corresponding results of plate count. (e) TNF‐α and IL‐6 level in the serum. (f) H&E stained images of skin tissue. The black arrow referred to the inflammation cells. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

In vivo safety evaluation of CA/CV NEs. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) H&E staining images of major organs, (b) Assessment of blood biochemical indicators. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Full text

Introduction

1

Bacterial biofilms are a three‐dimensional structure of bacteria communities encapsulated by extracellular polymeric substances (EPS) consisting of polysaccharides, proteins, and DNA(Guo et al. 2023; Hartmann et al. 2019). The dense EPS can act as barriers that protect the bacterial cells against the antimicrobial agents and immune evasion, thereby complicating the infection treatment(Flemming et al. 2025). Biofilm‐related infections now represent the majority (65% ~ 80%) of human microbial infections (S. Wang et al. 2023). As common pathogens in clinic, S. aureus and E. coli are often identified simultaneously in various disease, such as skin and urinary tract infections (Esposito et al. 2020; Hatlen and Miller 2021). S. aureus and E. coli mixed infections were frequently happened in the clinical. Additionally, they potentially form mixed biofilms through inter‐specific interactions(Caufield et al. 2017). Owing to the synergistic accumulation of EPS components and cross‐regulation of signaling molecules, the community structure of mixed biofilms exhibits much higher stability and drug resistance than single‐species biofilms, leading to increased difficulty in treatment (Mgomi et al. 2022; Pacheco et al. 2021). Therefore, a strategy for effectively inhibiting in the formation of S. aureus and E. coli mixed biofilms is needed.

Antibiotics are commonly employed to combat bacterial biofilm infections. Nevertheless, excessive antibiotic usage accelerates bacterial resistance development(Maurizi et al. 2024). In recent studies, plant‐derived essential oils have garnered significant research interest owing to their demonstrated biocompatibility and potent antibacterial properties, offering a potential alternative to conventional antibiotics for treating biofilm infections (Cecchini et al. 2021). Among them, the inhibitory effect of cinnamaldehyde (CA) and carvacrol (CV) on the formation of S. aureus or E. coli biofilms has been reported, which show potential in combating S. aureus and E. coli biofilms (Burt et al. 2007; Falsafi et al. 2015; Usai and Di Sotto 2023; Xu et al. 2022). However, utilizing essential oils to combat biofilm infections may be challenging. These plant‐based essential oils possess the drawbacks of poor water solubility and volatility, which reduces their efficacy and limited their antibacterial application in high dosage (Movahedi et al. 2024).

Nanoemulsions enhance the solubility and stability of hydrophobic antimicrobials, offering effective solutions for the application challenges associated with essential oils(Rajasekaran et al. 2024). Additionally, combining antimicrobial agents has demonstrated a significant boost in antibacterial efficacy (Liu et al. 2022). This synergistic approach enables low‐dose combinations to achieve inhibition levels comparable to those of high‐concentration single agents (Baym et al. 2016). Consequently, harnessing synergy represents a promising strategy to minimize dosage requirements while simultaneously enhancing the antibacterial activity of essential oils.

Therefore, in this study, the synergistic effect of CA and CV on inhibiting the formation of S. aureus and E. coli biofilms was first determined. Based on the above results, CA and CV nanoemulsions (CA/CV NEs) were prepared. Furthermore, the inhibitory effect on the mixed biofilms were measured and the synergistic interactions of CA and CV were further studied in vitro. In Vivo studies demonstrated that CA/CV NEs could effectively inhibit the formation of S. aureus and E. coli mixed biofilms on the implant with negligible toxicity. In summary, essential oil‐based nanoemulsions represent a promising strategy for prophylactic and therapeutic intervention against polymicrobial biofilm‐associated infections.

Materials and Methods

2

Materials

2.1

Cinnamaldehyde (CA), carvacrol (CV), Tween 80, phenol, and glutaraldehyde were purchased from Aladdin Biochemical Technology Co. Ltd. (China). Crystal violet was obtained from Maclin Biochemical Technology Co. Ltd. (China). Glucose was purchased from Ruijinte Chemical Co. Ltd. (China). Mueller Hinton Broth (MH), Luria‐Bertani Broth (LB), Mannitol Salt Agar, MacConkey Agar were purchased from Qingdao Hope Bio‐Technology Co. Ltd. (China). The BCA kit was purchased from Solarbio Science & Technology Co. Ltd. (China). The Bacterial RNA Extraction kit and RevertAid RT kit were purchased from Mei5 Biotechnology Co. Ltd. The mouse ELISA kit was purchased from Enzyme‐linked Biotechnology Co. Ltd. (China). The Urea Assay Kit (BUN), Creatinine Assay kit, Alanine aminotransferase assay kit, and Aspartate aminotransferase Assay kit were purchased from Nanjing Jiancheng Biotechnology Research Institute Co. Ltd. All the other reagents were used as received. BALB/c female mice (20–25 g, 6–8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co. Ltd.

Bacterial Strains

2.2

Staphylococcus aureus (S. aureus) ATCC 29213 and Escherichia coli (E. coli) ATCC 25922 were purchased from the American Type Culture Collection (ATCC) and were cultured in MH Broth at 37°C.

Biofilm Formation Study

2.3

S. aureus and E. coli in the logarithmic growth phase were diluted to 1 × 10^7^ CFU·mL^−1^. Then, the bacterial suspension was mixed in a volume ratio of 1:1 to obtain mixed suspension of S. aureus and E. coli (S‐E). Afterwards, 200 μL of bacterial suspensions were added to 96‐well plate and incubated at 37°C. After incubation for 6, 12, 18, and 24 h, the medium was discarded and the unattached planktonic bacteria were gently removed with phosphate buffered saline (PBS). The biofilms in the 96‐well plate were stained with crystal violet (0.1%) for 15 min and then rinsed with PBS for three times. To quantify the biomass of the biofilms, crystal violet was dissolved into glacial acetic acid (33%, 200 μL), and the absorbance at 600 nm was measured using a microplate reader. The formation of biofilms was determined by the following formula:

$$[eqn]$$

ODc refers to the Optical Density Cutoff, negative control refers to the OD value of pure culture medium. OD ≤ ODc = no biofilms producer; ODc < OD ≤ 2 × ODc = weak biofilm producer; 2 × ODc< OD ≤ 4 × ODc = moderate biofilm producer; 4 × ODc < OD = strong biofilm producer(Stepanović et al. 2007).

Antibacterial Activity of CA and CV

2.4

MICs and MBCs Determination

2.4.1

The minimum inhibitory concentrations (MICs) of CA and CV against S‐E were determined by microdilution method. Briefly, bacterial suspension (180 μL, 1 × 10^6^ CFU·mL^−1^) was added into the 96‐well microplate. Serial dilutions of CA and CV (2 ‐ 1024 μg/mL) were added and co‐incubated at 37°C for 24 h. The minimum drug concentration of the drugs that completely inhibited visible bacterial growth was determined as the MIC (Sader et al. 2019).

Next, take 100 μL of the solution from the aseptic culture wells, spread it onto different plates and incubate for 24 h. Among them, S‐E was, respectively, coated on Mannitol Salt Agar plates and MacConkey Agar plates. S. aureus was isolated on Mannitol Salt Agar plates, and E. coli was isolated on McConkey plates. The lowest concentrations with no more than five colonies were determined as the minimum bactericidal concentration (MBC) (T. Wang et al. 2024).

FICI Determination

2.4.2

Checkerboard testing was utilized to calculate fractional inhibitory concentration (FICI) indices, following established protocols. Briefly, bacterial suspension (1 × 10^6^ CFU·mL^−1^) was added to the first row 96‐well microplate. CA was added to the first row, and then diluted successively along the ordinate. Subsequently, CV was added to the last column and diluted along the abscissa. After incubating at 37°C for 24 h, the MICs were recorded as the lowest concentration of drug inhibiting visible growth. The FICI was calculated according to the following formula:

$$[eqn]$$

MIC_a_ or MIC_b_ is the MIC of compound a or b alone, respectively. MIC_ab_ is the MIC of compound a in combination with b, and MIC_ba_ is the MIC of compound b in combination with a. The synergy of additive was defined according to standard criteria (FICI ≤ 0.5 was defined as synergy; 0.5 < FICI ≤ 1 was defined as additive; 1 < FICI ≤ 4 was defined as unrelated; FICI > 4 was defined as antagonism) (Gómara and Ramón‐García 2019).

The Inhibitory Effect on Biofilm Formation

2.4.3

180 µL of bacterial suspension (1 × 10^7^ CFU·mL^−1^) was added to 96‐well plates. Subsequently, different concentrations of CA, CV, or the CA and CV mixture (CA:CV = 1:1, 2:1, 4:1 and 1:2) were added, and the final concentration of drugs in 96‐well plates was 128 μg/mL. After culturing at 37°C for 24 h, the supernatant in each well was removed. After rinsed with PBS, the biofilm was fixed with methanol, and subsequently stained with crystal violet for 30 min. Following three PBS washes and 2 h drying at 37°C, samples were treated with 200 μL of 33% acetic acid before measuring OD₆₀₀ using a microplate reader. The bacterial suspension without any treatment was used as the control. The inhibition rate of different drugs on the formation of S‐E biofilm was calculated according to the formula as follows:

$$[eqn]$$

Based on the above results, three synergistic inhibitory effect was determined by calculating Q value using Zhengjun Jin method (Jin 1980). The Q value was determined as follows:

$$[eqn]$$

E_A_ and E_B_ represent the inhibitory effects of A and B alone, respectively. E_A+B_ is the potency of A and B in combination, where A and B alone are used at concentrations equal to those of A and B in combination. The synergy or additive was defined according to standard criteria (0.55 < Q ≤ 0.85 defined as antagonistic effect; 0.85 < Q ≤ 1.15 defined as additive effect; 1.15 < Q ≤ 20 defined as synergistic effect).

Preparation and Characterization of CA/CV NEs

2.5

CA/CV NEs were prepared by high energy emulsification as previously reported (Nie et al. 2023). Tween 80 was dissolved in distilled water (8%, v/v). Then, CA and CV mixture (mass ratio of 4:1) was added dropwise to the aqueous phase (6%, m/v) with stirring to obtain a crude emulsion. The obtained crude emulsion was further homogenized with a high speed homogenizing emulsifier (RCD‐1A, Changzhou Yuexin) at 10,000 rpm for 6 min at room temperature to prepare CA/CV NEs. The morphology of the obtained CA/CV NEs was observed by transmission electron microscope (TEM, H‐7650, Hitachi, Japan). The hydrodynamic diameter and zeta potential were determined by Zeta Sizer Nano (NANO ZS90, Malvern, UK). The content of CV and CA in the nanoemulsion was determined by high performance liquid chromatography instrument (Milford, MA, USA). The mobile phase was composed of acetonitrile and water (v/v = 45:55) and the flow rate was 1.0 mL/min. The injection volume of the samples was 20 μL. Each sample was conducted in triplicate.

To determine the stability of the CA/CV NEs, the stock NEs and diluted NEs were stored at 4°C. The morphology of NEs was photographed everyday, and the particle size was measured on the 1st, 3th, 5th, 7th, 15th, and 30th day, respectively.

Antibacterial Activity of CA/CV NEs

2.6

The MIC and MBC of CA/CV NEs against S‐E were determined as described in the 2.4.1. Additionally, the effect of CA/CV NEs on the bacterial growth was determined. Bacteria were cultured to exponential phase and diluted to a concentration of 1 × 10^6^ CFU·mL^−1^. Different concentrations of CA/CV NEs (1/8 MIC ‐ MIC) were added in the bacterial suspension and incubated at 37°C. Growth curves were established under the wavelength of OD_600 nm_ with time interval of 0, 2, 4, 8, 12, and 24 h (Bai et al. 2022).

Biofilm Inhibition of CA/CV NEs

2.7

Crystal Violet Staining

2.7.1

Bacterial suspensions were diluted to the concentration of 1 × 10^7^ CFU·mL^−1^ and then added to 96‐well microplate. Then, different concentrations of CA/CV NEs were added, and the final concentration of CA/CV NEs was 1/2 MIC, 1/4 MIC, and 1/8MIC, respectively. After incubation at 37°C for 24 h, the biofilms in the 96‐well plate were stained with crystal violet (0.1%) for 15 min. Then, stained biofilms were rinsed with PBS three times. To quantify the biomass of the biofilms, crystal violet was dissolved into glacial acetic acid (33%, 200 μL), and the absorbance at 600 nm was measured using a microplate reader (Cui et al. 2024).

SEM Measurement

2.7.2

2.7 mL of bacterial suspensions (1 × 10^7^ CFU·mL^−1^) were added to the 24‐well plate with sterile silicon wafer, which was exposed to ultraviolet light for 1 h before use. Then, 300 μL of CA/CV NEs was added and the final concentration was 1/2 MIC (64 μg/mL). After incubation at 37°C for 24 h, the culture medium was discarded and the biofilms were rinsed three times with sterile PBS to remove planktonic bacteria. Afterwards, the biofilms on the silicon wafer were fixed with glutaraldehyde overnight, dehydrated with ethanol gradient and then observed by SEM (SU8010, Hitachi) after being sprayed with gold.

Determination of Adhesion Ability of Bacteria

2.7.3

Bacterial suspensions (1 × 10^7^ CFU·mL^−1^) were diluted and then added to 96‐well microplate. Then, CA, CV, CA/CV, and CA/CV NEs were added with the final concentration of 1/2 MIC (64 μg/mL) and co‐cultured until OD_600_ nm ≈ 0.6. The bacterial suspension was centrifuged (5000 rpm, 5 min) and then rinsed three times with PBS. The bacteria were resuspended in the BHI medium and adjusted to OD_600 nm_ = 1. Then, 1 mL of the bacterial suspension was added to the 24‐well plate and incubated at 37°C for 2 h. Then, the effect of the drug on the bacterial adhesion rate was determined by crystal violet staining. For biomass quantification, 200 μL of 33% glacial acetic acid‐dissolved crystal violet was used, with OD_600 nm_ measured via a microplate reader.

Measurement of Colony Spreading Ability

2.7.4

2.7 mL of bacterial suspensions (1 × 10^7^ CFU·mL^−1^) were added to the 24‐well plate. Then, 300 μL of CA, CV, CA/CV, CA/CV NEs were added and the final concentration was 1/2 MIC (64 μg/mL). After incubation at 37°C for 24 h, 10 μL of the bacterial suspensions was inoculated onto LB Agar plates containing 1.5% agar and dried at room temperature for 20 min, followed by incubation at 37°C for 48 h. The effects of different drugs on the motility of S‐E were determined by measuring the diameter of the colony growth area (Araujo Neto et al. 2020). To ensure an objective and reproducible measurement, the maximum diameter was measured along three distinct axes using a calibrated scale, and the average of these three measurements was calculated and reported as the final spreading diameter.

Determination of PIA Content

2.7.5

2.7 mL of bacterial suspensions (1 × 10^7^ CFU·mL^−1^) were added to the 24‐well plate. Then, 300 μL of CA, CV, CA/CV, CA/CV NEs were added and the final concentration was 1/2 MIC (64 μg/mL). After incubation at 37°C for 24 h, 10 μL of the bacterial suspensions was inoculated onto Congo red plates and dried at room temperature for 20 min, followed by incubation at 37°C for 24 h. The color change of the plates was observed.

Additionally, the bacterial suspension was rinsed three times with PBS and resuspended after incubation with different drugs at 37°C for 24 h. The extracellular matrix of the biofilm was extracted by ultrasound (40 Hz, 20 min), centrifuged (8000 rpm, 5 min), and the supernatant was dialyzed for 24 h and freeze‐dried to obtain the product. The contents of PIA in the extracellular matrix were determined (Mirzaei et al. 2021)

Determination of Extracellular Protein Content

2.7.6

The BCA standard curve was established according to the BCA kit manual (Hamida et al. 2020), and then the purified extracellular matrix (1 mg/mL) was taken to determine the protein concentrations of different groups.

Quantitative RT‐PCR Analysis

2.7.7

Bacterial suspensions (1 × 10^7^ CFU·mL^−1^) were diluted and then added to sterile test tubes. Then, different drugs, including CA, CV, CA/CV, and CA/CV NEs, with the final concentration of 1/2 MIC (64 μg/mL) were added. After incubation at 37°C for 24 h, 2 mL of the bacterial suspension was centrifuged (5000 rpm, 5 min) and rinsed with sterile PBS three times. RNA was extracted from bacteria and the content was determined. The same amount of RNA was reverse transcribed using the RevertAid RT kit. Quantitative real‐time PCR was performed in a final reaction system (10 μL) containing Fast Start DNA Master SYBR Green Mix (5 μL), forward‐primer (0.2 μL), reverse‐primer (0.2 μL), cDNA template (1 μL), and nuclease‐free water (3.6 μL). The qRT‐PCR was performed using a qPCR Detection System (Bio‐Rad Laboratories, Inc, cfx96) under the following conditions: pre‐denaturation at 95°Cfor 1 min, followed by 40 cycles of 95°C for 5 s, 55°C for 30 s, 72°C for 30–60 s with a final hold at 4°C. The effect of different drugs on the expression of the LuxS gene (Table S1) was calculated based on the CT value (Demirci et al. 2018).

AI‐2 Generation Detection

2.7.8

Bacterial suspensions (1 × 10^7^ CFU·mL^−1^) were diluted and then added to 96‐well microplate. Then, CA/CV NEs with the final concentration of 1/2 MIC (64 μg/mL) were added. After incubation at 37°C for 4 h, 1 mL of the bacterial suspension was taken and then centrifuged. Collected the supernatant and then mixed with the BB170 bacterial liquid, BB170 is an AI‐2‐responsive reporter strain that converts undetectable AI‐2 signals into quantifiable luminescent output, enabling precise quantification of AI‐2. After cultured at 30°C for 5 h, the effects of different groups of drugs on the generation of AI‐2 by S‐E were determined using multimode microplate reader (Li et al. 2021).

In Vivo Mouse Studies

2.8

The disposable intravenous infusion needle infusion tube was cut into 5 mm length, which was used as the carrier of S‐E biofilm. Female BALB/c mice were anesthetized by intraperitoneal injection of Tribromoethanol (20 μL/10 g) solution. After shaving the skin on the back of the mice and disinfecting it with iodine, the infusion tube was implanted subcutaneously through suture surgery. After the wound healed, 50 μL of S. aureus solution was injected into the sterile catheter site, as well as 50 μL of 3 × 10^9^ CFU·mL^−1^ E. coli (Vila et al. 2021). The animals we used were raised in accordance with the guidelines of the animal welfare and research ethics committee of Northeast Agricultural University (NEAUEC202403153), conducted in the animal room provided by the Animal Hospital of Northeast Agricultural University.

BALB/c female mice implanted with catheters were randomly divided into five groups (n = 5), then 100 μL of drug solution (3 mg/kg) was injected into the subcutaneous area near the mouse catheter, including CA, CV, CA/CV, and CA/CV NEs. Meanwhile, mice that only received PBS treatment were set as the control. After 7 consecutive days of treatment, the mice were euthanized. The implanted catheters were taken out and gently rinsed with sterile PBS. After treatment, SEM was used to observe the inhibitory effect of different groups on S‐E biofilm. At the same time, the biofilm on the catheter was dispersed under ultrasound (40 Hz, 20 min), and 100 μL was spread.

After 7 days of treatment, mice were euthanized (Intraperitoneal injection of 150 mg/kg pentobarbital sodium), fresh blood was kept for 3 h, then centrifuged (3000 rpm, 10 min) to obtain serum. TNF‐α and IL‐6 levels in serum were detected by ELISA kit. The skin of mice was excised and fixed in 4% paraformaldehyde. H&E staining sections were used for histopathological observation.

Safety Evaluation

2.9

Hemolysis Assays

2.9.1

Hemolysis of CA/CV NEs was investigated by sheep blood cells (RBCs). RBCs were centrifuged three times (1000 rpm, 10 min) in sterile saline and re‐suspended to obtain stock solutions. CA/CV NEs at different concentrations (512, 256, 128, 64 μg/mL) were then added to the stock solution and incubated at 37°C for 3 h. Normal saline and distilled water were the negative and positive controls, respectively. Supernatants were collected after centrifugation (1000 rpm, 10 min). The OD values at a detection wavelength of 570 nm were measured by a microplate reader, and hemolysis rates were calculated according to the following formula (Zou et al. 2023).

In Vivo Biosafety Assays

2.9.2

BALB/c female mice (20 ~ 25 g, 6 ~ 8 weeks old) were randomly divided into five groups (n = 5), 25 in total: control, CA, CV, CA/CV, and CA/CV NEs. 5 mice per group can provide relatively stable data to meet basic statistical analysis requirements and avoid unnecessary mouse deaths caused by excessive use. The mice were anesthetized, and the back skin of mice was shaved and disinfected. For different groups, 100 μL of drug solution or nanoemulsion was subcutaneously injected at the dosage of 3 mg/kg for 7 successive days. The mice were euthanized and the blood samples were collected for blood routine examination. Meanwhile, the heart, liver, spleen, lungs, and kidneys were fixed with 4% paraformaldehyde and stained with H&E for histopathological analysis.

Statistical Analysis

2.10

All data were expressed as the mean ± the standard deviation. All experiments were performed with at least three independent biological replicates (n = 3). GraphPad Prism 9.0 software (GraphPad Inc., San Diego, CA, USA) was used to perform statistical analysis (one‐way analysis of variance, ANOVA). The significance level was 0.05, and the data were indicated with *p < 0.05, **p < 0.01, and ***p < 0.001.

Results and Discussion

3

The Synergy of CA and CV

3.1

In vitro mixed S‐E tends to form biofilms after 24 h of co‐incubation (Figure S1). As previously reported, the formation of biofilms complicates the treatment of infection. Thus, the exploration of strategies for effective inhibition of mixed S‐E biofilm formation was of great importance. Based on the effect of CA and CV on inhibiting, the growth of S. aureus and E. coli and the formation of biofilm, the synergistic effect of CA and CV combinations on the S‐E was investigated. It was found that the combination of CA and CV significantly enhanced the antibacterial against S‐E (FICI = 0.3125, Figure 1a), and the antibacterial effect against S‐E was lower than that of CA (256 μg/mL) or CV (256 μg/mL) alone, demonstrating the synergistic antibacterial activity. Meanwhile, the Q value ranged from 1.15 ~ 2.5 with mass ratio (CA/CV) of 1:1, 2:1, 4:1, and 1:2, demonstrating their synergistic effect on inhibiting S‐E biofilm formation (Figure 1b). The biofilm inhibition rate of the combinations could reach approximately 66%, which was much higher than that of CA (19.15 ± 0.78%) and CV (18.75 ± 0.78%) (Table S2). The highest Q value was achieved at the mass ratio of 4:1. Therefore, the combination of CA and CV with a mass ratio of 4:1 (denoted as CA/CV) was selected for subsequent experimental studies.

(a) FICI determination. (b) The Q value of CA and CV on inhibition S‐E biofilms formation. (c) The size distribution of CA/CV NEs. (d) TEM image of CA/CV NEs. (e) The size changes of CA/CV NEs at 4°C and the corresponding photographs on the 30th day. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Preparation and Characterization of CA/CV NEs

3.2

Despite the synergistic effect of CA/CV, the low solubility and high volatility have hindered their clinical application (Song et al. 2019). Nanoemulsion possesses the advantages of high solubility, strong stability, and good permeability. Therefore, CA/CV NEs were prepared by high‐energy emulsification to realize their practical application. The process and formulation of the NEs were optimized by response surface methodology (Table S3) using Tween 80 as the emulsifier (Figures S2,3,4). Based on this, the optimal preparation process of NEs was obtained: Tween 80 ratio of 8% (v/v), oil phase ratio of 6% (m/v), homogenization speed of 10000 rpm, and homogenization time of 6 min. The size of the optimized NEs was 43.3 ± 0.005 nm with polydispersity index (PDI) of 0.183 ± 0.006 (Figure 1c), and the zeta potential was −7.47 ± 0.39 mV, demonstrating their uniformity. In the TEM image, a spherical morphology was observed, and the size was consistent with the dynamic light scattering (DLS) results (Figure 1d). Furthermore, the stability of CA/CV NEs was studied at 4°C. After 30 days, the NEs showed no obvious change in size and appearance, demonstrating good stability (Figure 1e). The CA and CV loading efficacies were 90% and 97.5%, and the loading contents were 43.2 mg/mL and 11.7 mg/mL as measured by HPLC (Figure S5a–c).

Inhibitory Effect of CA/CV NEs on Bacterial Biofilm Formation

3.3

The MIC and MBC of CA/CV NEs against S‐E were first evaluated. Compared with free CA/CV combination, the MIC and MBC of CA/CV NEs against S‐E were decreased to128 μg/mL. It can be seen from the time‐growth curve (Figure S6) that the NEs had slight effect on the growth of S‐E within the concentration range of 1/8 ~ 1/2 MIC (16 ~ 64 μg/mL), which was not statistically significant. The results suggested that the NEs could further increase the antibacterial activity of CA/CV. Furthermore, the inhibitory effect of CA/CV NEs on the S‐E mixed biofilm formation was further determined. As shown in Figure 2a, CA/CV NEs inhibited the S‐E mixed biofilm formation in a concentration‐dependent manner. Even at a low concentration of 32 μg/mL (1/4 MIC), CA/CV NEs displayed a significant inhibitory effect, achieving an inhibitory rate of 17.3%. For comparison, crystal violet staining was further performed at the CA/CV concentration of 64 μg/mL. As shown in the Figure 2c, dense and deep purple‐blue staining was observed in the control group, indicating the formation of a thick and robust biofilm with high bacterial biomass. In contrast, CA/CV and CA/CV NEs markedly reduced the staining intensity. Quantitative analysis showed that the biofilm inhibitory rate of CA was only 3.36 ± 1.84%, and CA/CV could inhibit 34.54 ± 2.63% of the biofilms owing to their synergistic effect. Remarkably, the inhibitory rate further increased to 40.24 ± 0.69% after CA/CV NEs treatment. Additionally, the morphology of S‐E with different treatment was examined using SEM. As shown in Figure 2d, thick S‐E mixed biofilms were observed in the control group, indicating the formation of mature mixed biofilms. After CA or CV treatment, the biofilm became thin and disintegrated, but obvious bacterial aggregation could still be observed. However, only slight bacterial aggregation was observed in the CA/CV NEs group, and the number of residual S. aureus was much greater than that of E. coli. The enhanced inhibitory effect of CA/CV NEs should be attributed to the increased bacterial uptake of NEs.

Inhibitory effect of CA/CV NEs on bacterial adhesion. (a) The inhibitory effect of NEs with different concentrations on S‐E biofilm mass. (b) The inhibitory effect of different drug on the S‐E biofilm mass. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (c) Photographs of crystal violet staining. (d) SEM images of S‐E. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

CA/CV NEs Inhibited Bacterial Adhesion

3.4

Bacterial adhesion is the first stage in the bacterial biofilm formation process and in order to clarify the mechanism by which CA/CV NEs inhibits S‐E mixed biofilms formation, the anti‐adhesion activity of CA/CV NEs was firstly investigated. As shown in Figure 3a, CV did not affect the adhesion of S‐E, and slight inhibition effect was observed in the CA group. In contrast, CA/CV and CA/CV NEs significantly inhibited the adhesion of S‐E owing to the synergistic effect between CA and CV. Remarkably, the relative inhibition rate reached 41.83 ± 0.78% in the CA/CV NEs group. The mobility of bacteria is beneficial to bacterial colonization and can promote the formation of biofilm. The LB Agar plate results in Figure 3d show that the NEs can significantly inhibit the diffusion of the mixed S‐E. Then, the diameter of the microbiota was further quantified, compared with the control group, the bacterial diffusion distance decreased and the relative inhibition rate was approximately 16%. The results (Figure 3b) demonstrated that CA/CV NEs could significantly inhibit the mobility of S‐E by nearly 16%, thereby reducing the bacterial diffusion ability and subsequently inhibiting biofilm formation.

NEs inhibited bacterial adhesion. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) Relative adhesion rate, (b) Diffusion inhibition rate, (c) Changes in PIA content, (d) Diffusion results of LB plates, (e) Congo red binding test. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Bacterial biofilm formation involves complex molecular processes requiring coordinated expression of numerous protein‐coding genes. Polysaccharide intercellular adhesin (PIA), a key mediator of cell‐cell adhesion, is an essential component of biofilms.(Lerch et al. 2019). PIA facilitates bacterial adhesion by electrostatically interacting with polyanionic teichoic acids on S. aureus cell surfaces (Formosa‐Dague et al. 2016). To assess the effect of CA/CV NEs on PIA production, S‐E strains were cultured on Congo red agar plates for 24 h. PIA production was indicated by black colonies, while its absence resulted in light red colonies. After inoculation for 24 h, black colony color indicated positive PIA results, and light red color indicated negative PIA results. As shown in Figure 3e, the color of the colony was black in both the control and CV groups. After CA treatment, the color of the colony became slightly lighter, indicating that CA inhibited the formation of PIA in the S‐E biofilm. By contrast, the color of the colony in the CA/CV and CA/CV NEs group became lighter than that in the CA group. Additionally, the amount of produced PIA was quantitatively analyzed. The PIA concentration of CA/CV NEs group (40 ± 0.81 μg/mL) was significantly lower than that of the control group (55 ± 0.82 μg/mL) and CA/CV group (46 ± 0.82 μg/mL), which was consistent with the Congo red assay (Figure 3c). The results demonstrated that the combination of CA/CV, especially in the form of NEs, could significantly inhibit the synthesis of PIA in the S‐E biofilms. However, this study only observed phenotypic changes in PIA production and did not elucidate the specific mechanism of CA/CV NEs in inhibiting PIA synthesis at the molecular level, such as by detecting the expression of the icaA. Overall, CA/CV NEs could reduce biofilm formation by inhibiting the adhesion process.

CA/CV NEs Affects EPS Production Through LuxS/AI‐2 System for Anti‐Biofilm Activity

3.5

Extracellular proteins are the main components of biofilm matrix (Campoccia et al. 2023). Then, we monitored changes in the amounts of extracellular protein after different treatments (Figure 4a). Compared with CA (68.85 ± 1.08 μg/mL) or CV (74.08 ± 1.42 μg/mL) alone, CA/CV and CA/CV NEs could significantly reduced the secretion of extracellular protein, and the concentration was reduced to 50.87 ± 1.60 and 42.06 ± 1.80 μg/mL, respectively. The results demonstrated that CA/CV NEs could effectively reduce the extracellular protein of biofilm.

Effects of different groups on LuxS/AI‐2 system. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) Changes in protein content. (b) LuxS mRNA level, (c) AI‐2 production. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

The autoinducer‐2 (AI‐2) quorum‐sensing system operates universally across gram‐positive and gram‐negative bacteria and (Mayer et al. 2023). Previous studies have shown that AI‐2 significantly promotes biofilm formation in both S. aureus and E. coli (Gopishetty et al. 2009). Therefore, the effect of CA/CV NEs on the LuxS gene expression, a key enzyme in AI‐2 synthesis, in S‐E mixed biofilms using qRT‐PCR. As shown in Figure 4b, LuxS expression was significantly down‐regulated in the CA/CV and CA/CV NEs groups compared with the CA or CV alone groups. This down‐regulation led to a reduction in AI‐2 production. Consistent with the gene expression results, the AI‐2 content decreased by 53.97  ±  1.59% after CA/CV NEs treatment, a much greater reduction than observed in other groups (Figure 4c). The results indicated that CA/CV NEs could reduce the AI‐2 production by down regulating LuxS.

In Vivo Inhibitory Effect of CA/CV NEs on S‐E Biofilm Formation

3.6

Medical implants constitute critical components of clinical care, yet their functionality is frequently compromised by biofilm‐associated infections stemming from bacterial colonization on device surfaces (Kim et al. 2022). Clinical data showed that approximately 58.3% of medical implant‐related infections are related to S. aureus and E. coli, of which biofilm‐related infections accounted for more than 70% (Kobayashi et al. 2024). Based on the in vitro results, the in vivo inhibitory effect of CA/CV NEs on the S‐E mixed biofilm formation was evaluated by a mouse implant S‐E infection model (Figure 5a). Mice were subcutaneously implanted with sterile medical catheters and then S‐E suspension was injected. For all the groups, PBS, free CA, free CV, mixture of CA and CV or CA/CV NEs were subcutaneously injected in the infection site of mice once a day for 7 consecutive days. The mice infected with S‐E while only received PBS treatment were set as control group. The implants were removed on the 8th day, photographed and then observed with SEM. For quantitative analysis, the number of bacteria on the surface of catheters was assessed by plate counting method. In order to distinguish S. aureus and E. coli, Mannitol Salt Agar and MacConkey Agar were selected for isolation of S. aureus and E. coli, respectively. As shown in the Figure 5b, the colony number of S. aureus and E. coli significantly decreased after treatment with CA/CV NEs. Further quantitative results showed that there was no significant difference in the bacterial count between CA (CV) group and model group (Figure 5d). Of note, the number of S. aureus was much more than that of E. coli in the control group. The results suggested that S. aureus was the dominant strains in the mixed biofilm formation. For S. aureus, the bacterial counts of CA/CV and CA/CV NEs groups decreased by 1.26 ± 0.03 logs and 1.75 ± 0.10 logs, respectively. While the E. coli counts decreased by 0.76 ± 0.23 logs and 1.39 ± 0.25 logs in the CA/CV and CA/CV NEs groups, respectively. Subsequently, the formation of biofilm on the catheter surface was observed using SEM (Figure 5c). In the control group, dense and thick biofilm was observed on the surface of catheter, and the majority of them was S. aureus. In contrast, only a small amount of bacterial aggregation was observed in the CA/CV NEs group. All the results demonstrated that CA/CV NEs could effectively inhibit S‐E biofilm formation in vivo.

Inhibition of CA/CV NEs on S‐E biofilm in vivo. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) Establishment of mouse infection model. (b) Photographs S. aureus and E. coli plate count. (c) SEM images of mixed biofilms after different treatment. (d) Corresponding results of plate count. (e) TNF‐α and IL‐6 level in the serum. (f) H&E stained images of skin tissue. The black arrow referred to the inflammation cells. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

Bacterial infection is usually accompanied with serious inflammation response. In order to further explore the impact of CA/CV NEs on the mixed S‐E infection, the pro‐inflammation factors in the serum of mice were measured. As shown in Figure 5e, the contents of TNF‐α and IL‐6 were significantly reduced. Meanwhile, as shown in the H&E staining image of the surrounding skin tissue (Figure 5f), inflammatory cell infiltration was significantly reduced in the CA/CV NEs, suggesting an alleviation of inflammation. The implanted infection model offers advantages such as ease of operation, relatively low cost, and good reproducibility. However, it also presents several important limitations for scientific research, including significant structural differences between mouse skin and human skin, as well as marked variations in immune responses to biofilm infection. Additionally, due to the high mobility of mice, catheters are prone to displacement or detachment during experiments, which may affect the results. Therefore, in future studies, it is necessary to consider and control these influencing factors as comprehensively as possible.

Safety Evaluation of CA/CV NEs

3.7

The hemolytic activity of CA/CV NEs was assessed using distilled water as the positive control and normal saline as the negative control. As shown in Figure. S8, the hemolysis rate was below 5% at the concentration (128 μg/mL and 64 μg/mL) used in the treatment. These findings indicated that CA/CV NEs possess good blood compatibility at the used dosage.

Hematological safety evaluation showed (Figure.S9) that there was no significant difference in white blood cell count (WBC), hematocrit (HCT), and platelet count (PLT) between CA/CV NEs group mice and control group (p > 0.05), which was consistent with recent safety study reports of nanopharmaceuticals (Fraga et al. 2021). Histopathological analysis showed (Figure 6a) that no significant pathological changes (H&E staining) were observed in major organs such as heart, liver, spleen, lung and kidney, which was consistent with the safety of reported nano‐delivery system based on plant‐derived antibacterial components (Khan et al. 2019).

In vivo safety evaluation of CA/CV NEs. The concentration of CA and CV in the NEs was 64 and 16 μg/mL, respectively. (a) H&E staining images of major organs, (b) Assessment of blood biochemical indicators. Data are presented as mean ± SD from three independent biological replicates, each measured in triplicate.

For safety assessment of liver and kidney, key organs of drug metabolism (Figure 6b), we tested BUN, Cr, AST, and ALT. The data showed that the above biochemical parameters of mice in CA/CV NEs group were always in normal physiological range, which was similar to the liver and kidney toxicity evaluation model of nano‐drugs established by Kiouas team in 2023 (Kiouas et al. 2023), indicating that CA/CV NEs did not cause damage to liver and kidney of mice. All the results demonstrated that CA/CV NEs possessed good in vivo safety during the wound therapeutic period. Although the potential long‐term safety profile of the CA/CV NEs did not address, it is worth noting that all the components of CA/CV NEs, including CA, CV, and Tween 80 are generally recognized as safe and widely used in food and pharmaceutical industries. Nevertheless, the NEs might lead to different in vivo fate from their free forms, thus a comprehensive long‐term safety assessment remains a critical prerequisite for future clinical translation of this nanoemulsion system.

Conclusion

4

In summary, CA/CV NEs with synergistic inhibitory effects on the formation of S‐E mixed biofilms were constructed and optimized. Compared with free CA/CV, the inhibitory activity on mixed biofilms was significantly enhanced. Additionally, CA/CV NEs inhibited mixed biofilm formation by anti‐adhesion of bacteria, reducing PIA and extracellular protein production, inhibiting the LuxS/AI‐2. In an implant infection model, CA/CV NEs could effectively inhibit the formation of S‐E biofilm and simultaneously reduce the bacterial burden. This study provides a novel approach for leveraging the active components of traditional Chinese medicine in clinical settings, offering a fresh perspective for combating mixed biofilm‐associated infections.

Author Contributions

Siqi He: conceptualization, experiment, writing – original draft. Yuxin Wang: methodology, software. Wenli Wang: investigation, methodology. Zhiyun Zhang and Yanhua Li: project administration, supervision, writing – review and final editing. All authors have read and agreed to the published version of the manuscript.

Ethics Statement

The animals in this study were raised in accordance with the guidelines of the Animal Welfare and Research Ethics Committee of Northeast Agricultural University (NEAUEC202403153).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: S. aureus and E. coli mixed biofilm formation. Figure S2: Response surface and contour plots of the influence of four factors on the particle Size of NEs. Figure S3: Response surface and contour plots of the influence of four factors on PDI of NEs. Figure S4: Response surface and contour plots of the influence of four factors on Zeta of NEs. Figure S5: Determination of CA/CV NEs content. Figure S6: Time growth curve of CA/CV NEs against S‐E bacteria. Figure S7: Standard curve. Figure S8: Hemolysis of sheep blood cells after co‐incubation with CA/CV NEs. Figure S9: Blood routine test. Table S1: Oligonucleotides used in this study. Table S2: Inhibition rate of CA and CV on S‐E biofilm. Table S3: Test design and results.