Therapeutic Potential of Isoallolithocholic Acid in Methicillin-Resistant Staphylococcus aureus Peritoneal Infection

Background

Methicillin-resistant Staphylococcus aureus (MRSA), discovered in 1961, has become one of the most common antibiotic-resistant bacteria worldwide. MRSA infections can lead to fatal sepsis, pneumonia, and high rates of skin and soft tissue infections. Due to its rapid spread and extensive drug resistance, many antibiotics are ineffective against MRSA infections. The World Health Organization (WHO) has classified MRSA as a high-priority multidrug-resistant pathogen. Therefore, exploring new antibacterial drugs or alternative strategies to address MRSA infections has become an urgent priority.

Bile acids (BAs) are steroid-derived natural products that play essential roles in digestion, immunity, and lipid metabolism. Recent studies have found that the bile acid metabolite isoallolithocholic acid (isoallo-LCA) may reduce the risk of infection among centenarians, but its precise mechanism remains unclear. This study aims to investigate the role of isoallo-LCA in MRSA-associated peritoneal infection and its potential therapeutic value.

Source of the Paper

This paper was co-authored by Ying Lu, Jun Du, Shicheng Peng, Ying Wang, and Yongtao Xiao from the Division of Pediatric Gastroenterology and Nutrition at Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, the Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, and the Shanghai Institute of Pediatric Research. The paper was published on December 4, 2024, in The Journal of Antibiotics.

Research Process and Results

1. Evaluation of the Antibacterial Activity of Bile Acids Against MRSA

The study first assessed the antibacterial activity of various bile acids, including isoallo-LCA, against MRSA and other pathogens (such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, and Candida albicans). Experiments were conducted through bacterial growth inhibition assays and minimum inhibitory concentration (MIC) determination.

• Experimental Methods: Different concentrations of bile acids (0-25 μM) were mixed with bacterial suspensions, and bacterial growth was evaluated by measuring optical density (OD600) and colony-forming units (CFU).
• Results: Isoallo-LCA significantly inhibited the growth of MRSA and Staphylococcus aureus, with a minimum inhibitory concentration (MIC90) of 3.0 μM. In contrast, isoallo-LCA showed no significant antibacterial activity against other pathogens (such as Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, and Candida albicans).

2. Effect of Isoallo-LCA on MRSA Biofilms

MRSA often enhances its drug resistance by forming biofilms. The study further evaluated the impact of isoallo-LCA on MRSA biofilm formation and eradication.

• Experimental Methods: The crystal violet staining method was used to assess the inhibitory and eradication effects of isoallo-LCA on MRSA biofilms.
• Results: Isoallo-LCA (10 μM) significantly reduced MRSA biofilm formation and eradicated approximately 50% of mature biofilms.

3. Cell Membrane Permeability Experiments with Isoallo-LCA

To explore the antibacterial mechanism of isoallo-LCA, the study evaluated its effect on MRSA cell membrane permeability using the Sytox Green staining method.

• Experimental Methods: MRSA cells were co-incubated with Sytox Green dye and different concentrations of isoallo-LCA (2-10 μM). Changes in cell membrane permeability were observed using fluorescence microscopy and a fluorescence spectrophotometer.
• Results: Isoallo-LCA rapidly increased the permeability of MRSA cell membranes, leading to impaired membrane integrity.

4. Electron Microscopy Observations

The effects of isoallo-LCA on MRSA cell structure were further observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

• Results: SEM showed no significant damage to the surface of MRSA cells after isoallo-LCA treatment, but TEM revealed significant deformation and detachment of the cell membrane, with bright areas appearing in the cytoplasm.

5. Hemolytic Activity and Cytotoxicity Assessment of Isoallo-LCA

The study also evaluated the toxicity of isoallo-LCA to mammalian cells.

• Experimental Methods: Hemolysis assays and lactate dehydrogenase (LDH) cytotoxicity assays were used to assess the toxicity of isoallo-LCA to human red blood cells and mammalian cells (RAW264.7 and HEK293T).
• Results: Isoallo-LCA exhibited low toxicity (%) to red blood cells and mammalian cells at concentrations up to 100 μM.

6. Efficacy of Isoallo-LCA in a Mouse MRSA Peritoneal Infection Model

Finally, the study evaluated the therapeutic efficacy of isoallo-LCA in a mouse MRSA peritoneal infection model.

• Experimental Methods: Mice were divided into four groups (DMSO control, isoallo-LCA, MRSA-infected, and MRSA-infected + isoallo-LCA). The infection model was established by intraperitoneal injection of MRSA (1.5 × 10^7 CFU), and isoallo-LCA (2 mg/kg) was injected 2 hours post-infection.
• Results: Isoallo-LCA significantly reduced MRSA loads in peritoneal lavage, liver, and spleen, and alleviated MRSA infection-induced peritoneal inflammation and organ damage. Additionally, isoallo-LCA reduced pro-inflammatory cytokine levels (such as IL-6 and IL-1β) in peritoneal lavage while increasing the expression of the anti-inflammatory cytokine IL-10.

Research Conclusions and Significance

This study is the first to reveal the potential therapeutic value of the bile acid metabolite isoallo-LCA in MRSA infections. Isoallo-LCA significantly inhibits MRSA growth and biofilm formation by disrupting the integrity of MRSA cell membranes. In the mouse model, isoallo-LCA effectively alleviated MRSA infection-induced peritoneal inflammation and organ damage. These findings suggest that isoallo-LCA may become a novel therapeutic agent for MRSA infections.

Research Highlights

1. Novel Antibacterial Mechanism: Isoallo-LCA exerts its antibacterial effects by disrupting the integrity of MRSA cell membranes, providing new insights for the development of novel antibacterial drugs.
2. Significant In Vivo Efficacy: In the mouse model, isoallo-LCA significantly reduced MRSA loads and alleviated inflammatory responses, demonstrating its potential application in treating MRSA infections.
3. Low Toxicity: Isoallo-LCA exhibited low toxicity to mammalian cells, indicating its good safety profile.

Research Value

This study not only provides a new candidate drug for the treatment of MRSA infections but also opens up new research directions for the application of bile acid metabolites in the field of antimicrobials. In the future, isoallo-LCA is expected to become an important drug for treating multidrug-resistant bacterial infections.