The S. aureus BNCC 186335 and USA300-sfgfp strains were purchased from Forhigh Biotech. The S. aureus strain was cultivated in LB medium (18 g/L Nutrient Broth (NB), Solarbio) for 24 h at 37°C and 180 r/min in a shaker incubator. A microplate reader (Tecan, Infinite M2000) was used to measure OD₆₀₀. ILs (C₂MIMCl, C₃MIMCl, C₄MIMCl, C₆MIMCl, C₈MIMCl, C₁₂MIMCl, C₁₆MIMCl, ≥99%) were prepared by Shanghai Cheng Jie Chemical Co., Ltd. Then, the ILs were diluted in sterile, distilled water. Cy5 NHS Ester (Cy5, SE) was purchased from Beijing Fanbo Biochemicals Co., Ltd. Then, 4% of the paraformaldehyde fix solution was purchased from Solarbio.

Iconic liquids were diluted in sterile, distilled water following the corresponding concentration gradient. Then, 50 μL of bacteria solutions and different ILs were added to 5 ml of LB medium. Afterward, mixed solutions were hatched at 37°C and 180 r/min in a shaker incubator for 24 h. Before measuring OD₆₀₀, the solution was fully shaken to distribute S. aureus and ensure the accuracy of the measurement. Finally, 100 μL of solutions were removed from each sample and placed into 96-well plates to measure OD₆₀₀. To avoid human or machine error, every OD₆₀₀ value was repeated at least three times. EC50s were determined from OD₆₀₀ by GraphPad Prism8. The activity of S. aureus cultured with ILs was observed by a confocal microscope, and the fluorescence was quantified.

Then, 200 μL of IL was added to 10 mL of solid LB medium (18 g/L NB, 1L water, and 15 g agarose), which was melted to liquid in advance and put in 10-cm plates. Then, 100 μL of bacterial suspension (OD_{600 =} 0.15) with further dilution was spread on solid LB plates and cultured at 37°C for 48 h. The CFUs were counted after being cultured, and the cultures were photographed with a camera.

C₃MIMCl or C₁₂MIMCl was added to S. aureus (OD₆₀₀ = 0.15) and mixed with the ROS Assay Kit (DCFH-DA), and the mixed solution was placed in a 96-well plate for 30 min at 37^oC. Then, the ROS value was detected with the microplate reader at the excitation wavelength of 488 nm and the emission wavelength of 525 nm. Following the above experimental procedure, 200 μL of the mixture was added to a confocal dish, and fluorescence was photographed using confocal laser scanning microscopy (Nikon Corporation, A1, CLSM) at the excitation wavelength of 488 nm with green as the pseudo color.

Staphylococcus aureus (OD₆₀₀ = 0.15) was cultured in a liquid LB medium with ILs within 3 h at 37°C. Then, the bacteria were washed with PBS three times and fixed in 4% of the paraformaldehyde fix solution at 4°C overnight. The next day, bacteria were washed with PBS three times and treated with ethanol gradient dehydration at different concentrations (30, 40, 50, 60, 70, 80, 90, 95, and 100%). Bacteria were successfully mixed with different ethanol solution concentrations, were left to stand, were dehydrated for 15 min, and were collected by centrifugation. Finally, the bacteria dehydrated with 100% ethanol onto the tin foil was dropped and dried at 37°C for 2 h. The morphological changes of S. aureus treated with ILs were characterized by scanning electron microscopy (SEM, HITACHI, SU8020).

S. aureus was cultured with ILs, centrifuged (8000 r/min, 8 min), washed with water (3 times), and fixed with glutaraldehyde solution (PH = 7.4) overnight. They were taken in cross-sections and stained for further transmission electron microscopy (TEM, HITACHI, H-7650B) observation.

S. aureus (OD₆₀₀ = 0.25) was treated with C₃MIMCl and C₁₂MIMCl (the final concentrations: 0.01 mM, 0.04 mM, and 0.16 mM) in liquid LB medium at 37°C for 3 h with gentle shaking. Then, S. aureus was washed with PBS three times and sterilized with water one time a day. The obtained bacteria were dispersed in 1 mL of water. The uniform suspension was measured for zeta potential using a Zetasizer Nano ZS (Malvern Instruments Ltd., Zetasizer Nano ZS90). As a control group, the bacteria were treated with an LB medium containing corresponding volumes of water at the same conditions. At the same time, we used Cy5, SE labeled C₁₂MIMCl to interact with S. aureus and CLSM to observe the interaction between IL and bacteria.

BALB/c mice (6–8 weeks old, female) were obtained from Vital River Laboratories (Beijing, China). The Institutional Animal Care and Use Committees approved the animal protocol of the Institute of Process Engineering, Chinese Academy of Sciences (approval ID: IPEAECA 2022103). This study was performed in strict accordance with the Regulations for the Care and Use of Laboratory Animals and the Guideline for the Ethical Review of Animals.

For the skin abscess model, mice were divided into three groups (controls, infected group, and ILs). Mice were anesthetized with pentobarbital sodium (1%) and inoculated with 100 μl of PBS containing 10¹³ cfu/L S. aureus or sterile PBS in the right flank by subcutaneous injection. After abscess formation, 50 μl of C₁₂MIMCl (2 mM) was injected daily at the abscess site for 3–4 days. We observed the abscess healing continuously. Skin biopsy samples collected on day 11 were fixed and prepared for histopathological evaluation. The tissues were stained with hematoxylin and eosin (H&E) and 4′,6-diamidino-2-phenylindole (DAPI) and visualized by the scanner (3DHISTECH, P250 FLASH).

The antibacterial activities of a series of imidazole chloride ILs against S. aureus were determined. To evaluate different ILs' antimicrobial activity against S. aureus, different concentrations of six ILs were used to measure EC50 (Figure 1A). The EC50 values in the LB medium of C₂MIMCl, C₃MIMCl, C₄MIMCl, C₆MIMCl, C₈MIMCl, C₁₂MIMCl, C₁₆MIMCl were 76.7, 28.7, 18.2, 3.5, 0.3, 1.9 x 10⁻³, and 0.5 x 10⁻³ mM for S. aureus, respectively. Imidazolium chloride ILs with substituents of 12 and 16 carbon lengths had strong antibacterial effects on S. aureus. The toxicity of S. aureus increased with the length of the cation side chain. It may be due to the increased hydrophobicity. With an increase in C_nMIMCl concentration, the inhibition rate of S. aureus increased. For example, when the concentration of C₁₂MIMCl was 0.0015, 0.0020, 0.0025, and 0.0030 mM, the inhibition rates of S. aureus were 23.2, 60.5, 83.1, and 87.7%, respectively (Figure 1B). It showed that the toxicity of C_nMIMCl varied for each concentration. S. aureus transfected with a green fluorescent protein (GFP) was incubated with C₃MIMCl or C₁₂MIMCl and observed by CLSM. There was no difference in GFP fluorescence intensity between the C₃MIMCl group and the control group, while S. aureus GFP fluorescence intensity in the C₁₂MIMCl group was significantly reduced (Figures 1C, D).

To further verify the toxicity of C_nMIMCl, the less toxic C₃MIMCl and the more toxic C₁₂MIMCl were selected for plate coating experiments (Figure 2). Compared with the control group, there was no bacterial inhibition when the concentration of C₃MIMCl was 0.0040 mM. The inhibition rates were 65.8 ± 0.8%, 89.1 ± 5.1%, and 94.6 ± 4.7% when the C₃MIMCl concentration was 26 mM, 32 mM, and 40 mM, respectively. C₁₂MIMCl showed a significant inhibitory effect on S. aureus compared with C₃MIMCl. When the concentrations of C₁₂MIMCl were 0.0026 mM, 0.0032 mM, and 0.0040 mM, the inhibition rates were 9.7 ± 3.9%, 23.0 ± 5.1%, and 98.4 ± 0.8%, respectively. With ~95% inhibition of S. aureus, the concentration used for C₃MIMCl is about 10⁴ times higher than that used for C₁₂MIMCl.

The toxicity of C_nMIMCl to S. aureus was proportional to the length of the imidazolidinyl side chain. At the same time, the longer the alkyl side chain length of the imidazole, the greater the hydrophobicity of C_nMIMCl. Therefore, the toxicity of C_nMIMCl to S. aureus increased with the enhancement of hydrophobicity. It is well known that phospholipid is one of the main components of the cell membrane, and the outer surface of the cell membrane is electronegative. The ILs were easily adsorbed to the bacterial surface due to the positive charge of the cationic part, and then, the ILs interacted with the phospholipid bilayer due to hydrophobicity. Therefore, C₃MIMCl with a short carbon chain and C₁₂MIMCl with a long carbon chain were selected for the subsequent research.

To further investigate the mechanisms leading to the inhibitory and destructive effects of C₁₂MIMCl on S. aureus, as well as the significant reduction of the viability of the exposed bacteria induced by ILs, the release of reactive oxygen species (ROS) within S. aureus was explored. ROS is a general term for a class of molecules with oxidative activity produced by cells during energy metabolism under aerobic conditions. In bacteria, excess ROS can damage nucleic acids, proteins, and lipids (Brynildsen et al., 2013). Moreover, it can also lead to bacterial oxidative stress and inhibit bacterial metabolism (Ning et al., 2019; Zhao et al., 2022). Bacterial death occurs when intracellular ROS exceeds the cell's ability to detoxify and repair. Therefore, it is necessary to determine the amount of ROS in bacteria to reveal the mechanism of IL acting on bacteria. Figure 3 shows CLSM images of ROS (green). No significant changes in ROS fluorescence values were observed with 0.16 mM C₃MIMCl compared with the control group. It was found that C₁₂MIMCl could significantly increase ROS in bacteria compared with C₃MIMCl at the same concentration, and the ROS gradually increased with an increase in C₁₂MIMCl concentration. Therefore, C₁₂MIMCl could induce ROS release intracellularly.

The cell membrane mainly consists of phospholipids with a negative charge on the surface and hydrophobicity. ILs are amphiphilic, and long cationic side chains can be inserted into the phospholipid bilayer, causing cell membrane disorder (Kaur et al., 2020; Liu et al., 2021). According to the above experimental contents, C₁₂MIMCl could be adsorbed on the surface of S. aureus by electrostatic action. Afterward, C₁₂MIMCl was inserted into cell membranes driven by hydrophobicity. This led to an oxidative stress reaction in bacteria, that is, the rapid production of a large amount of ROS. At the same time, it changed the permeability of the bacterial cell membrane, which could change the bacterial osmotic pressure, destroying the cell membrane and allowing cytoplasm to flow out of the broken cell membrane. Figure 7 depicts a plausible antibacterial mechanism for interacting C₁₂MIMCl with S. aureus.