The antibacterial activity and mechanism of imidazole chloride ionic liquids on Staphylococcus aureus

Abstract

Ionic liquids (ILs) have garnered increasing attention in the biomedical field due to their unique properties. Although significant research has been conducted in recent years, there is still a lack of understanding of the potential applications of ILs in the biomedical field and the underlying principles. To identify the antibacterial activity and mechanism of ILs on bacteria, we evaluated the antimicrobial potency of imidazole chloride ILs (C_nMIMCl) on Staphylococcus aureus (S. aureus). The toxicity of ILs was positively correlated to the length of the imidazolidinyl side chain. We selected C₁₂MIMCl to study the mechanism of S. aureus. Through the simultaneous change in the internal and external parts of S. aureus, C₁₂MIMCl caused the death of the bacteria. The production of large amounts of reactive oxygen species (ROS) within the internal parts stimulated oxidative stress, inhibited bacterial metabolism, and led to bacterial death. The external cell membrane could be destroyed, causing the cytoplasm to flow out and the whole cell to be fragmented. The antibacterial effect of C₁₂MIMCl on skin abscesses was further verified in vivo in mice.

Keywords: Staphylococcus aureus; antibacterial activity; ionic liquids; mechanism; skin abscess.

Figure 1

Toxicity test of C_nMIMCl against Staphylococcus aureus. (A) EC50 values of Staphylococcus aureus incubated with C_nMIMCl for 24 h. (B) Effects of C₁₂MIMCl on Staphylococcus aureus at different concentrations. (C) Quantitative analysis of bacterial activity. (D) CLSM images of bacterial activity. Data in (A–C) represent the mean ± s.d. Statistical significance was calculated via two-tailed unpaired Student's t-test (A–C). Ns means no significant difference, ^*P<0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 2

Bacterial colony photos of Staphylococcus aureus incubated with various concentrations of C₃MIMCl and C₁₂MIMCl for 48 h.

Figure 3

Oxidative stress of Staphylococcus aureus. (A) CLSM images of ROS release from bacteria in control. (B–E) CLSM images of ROS release from bacteria in the presence of 0.16 mM C₃MIMCl (B), 0.01 mM C₁₂MIMCl (C), 0.04 mM C₁₂MIMCl (D), 0.16 mM C₁₂MIMCl (E). (F) Quantitative analysis of ROS release by a microplate reader. Data in (F) represent the mean ± s.d. Statistical significance of (F) was calculated via two-tailed unpaired Student's t-test. Ns means no significant difference, **P < 0.01.

Figure 4

SEM characterization of Staphylococcus aureus morphologies and membrane integrities after 3h co-culture with water (control group), 0.16 mM C₃MIMCl or different concentrations of C₁₂MIMCl (0.003 mM and 0.16 mM).

Figure 5

The changes of Staphylococcus aureus surface. (A) Zeta potentials of Staphylococcus aureus co-cultured with C₃MIMCl or C₁₂MIMCl in PBS for 3h. (B) Picture of Staphylococcus aureus incubated with Cy5, SE-C₁₂MIMCl. (C–E) CLSM images of C₁₂MIMCl interacting with Staphylococcus aureus. Green: Staphylococcus aureus transfected with GFP (C), Red: Cy5, SE-C₁₂MIMCl (D), merged (E). Data in (A) represent the mean ± s.d. Statistical significance of (A) was calculated via two-tailed unpaired Student's t-test. Ns means no significant difference, ****P < 0.0001.

Figure 6

TEM images of Staphylococcus aureus within 3 h treatment with 0.16 mM of C₁₂MIMCl. (A-D): The destruction process of Staphylococcus aureus by C₁₂MIMCl.

Figure 7

Plausible antibacterial mechanism for interaction between C₁₂MIMCl and Staphylococcus aureus.

Figure 8

Changes of skin abscess in mice after Staphylococcus aureus infection and C₁₂MIMCl treatment.

Figure 9

Histopathology of skin abscess and fluorescent scan in mice after Staphylococcus aureus infection and C₁₂MIMCl treatment. (A) Histopathology of the control group (PBS). (B) Histopathology of skin abscess after Staphylococcus aureus injection. (C) Histopathology of C₁₂MIMCl treatment in skin abscess. (D) Fluorescent scan of the control group (PBS). (E) Fluorescent scan of skin abscess after Staphylococcus aureus injection. (F) Fluorescent scan of C₁₂MIMCl treatment in skin abscess. DAPI (blue), GFP-Staphylococcus aureus (green).