Synergistic Killing of Staphylococcus aureus by Mefloquine and 18β-Glycyrrhetinic Acid

The combination MIC was designated as the minimal concentration with no detectable bacterial growth post-incubation. The fractional inhibitory concentration (FIC) index was calculated using the corresponding formula to characterize the interaction mode between the two agents. The interaction was categorized based on the FIC value: synergism (FIC ≤ 0.5), additive effect (0.5 < FIC ≤ 1), indifference (1 < FIC ≤ 2), and antagonism (FIC > 2).FIC=A（MIC of combination）A(MIC of alone)+B(MIC of combination)B(MIC of alone)3.

Time–Kill AssayThe time–kill assay was used to verify the synergistic effect of 18β-GA and Mef. ATCC 29213 andMRSA were diluted in BHI at the concentration of 5 × 10⁵ CFU/mL and then treated with 16 ug/mL18β-GA, 8 ug/mL Mef, and 16 ug/mL 18β-GA+8 ug/mL Mef, DMSO were incubated as control. After 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 24 hours of incubation, the OD₆₀₀ values of bacterial suspension were measured to plot curves.

Simultaneously, 100 μL of bacterial suspension was serially diluted, spread onto BHI plates, and incubated at 37°C for 16-20 hours for colony counting to quantify viable bacteria.4. Detection of Bacterial Biofilm Formation CapacityThe effect of the drug on MRSA biofilm formation was detected using the crystal violet staining method. The logarithmic-phase bacterial suspension was diluted to 5×10⁵ CFU/mL and then treated with 16 ug/mL 18β-GA, 8 ug/mL Mef, and 16 ug/mL 18β-GA+8 ug/mL Mef, DMSO were incubated as control.

After the 96-well plate was incubated at 37°C for 20 h, the supernatant was discarded and gently washed three times with PBS. Each well was then filled with 200 μL of 0.1% crystal violet solution and incubated at room temperature in the dark for 15 min. The staining solution was discarded, and the wells were washed three times with PBS.

Afterward, 200 μL of 95% ethanol was added to each well, and the plate was shaken at room temperature for 10 min for decolorization. The absorbance at 590 nm was measured using a microplate reader (Bio-RAD, iMark).5. Reactive oxygen species (ROS) GenerationThe level of ROS was detected using a Cell Reactive Oxygen Species Detection Kit (Jiangsu KeyGEN BioTECH Corp.,Ltd).

MRSA in the logarithmic phase was diluted to 5×10⁵ CFU/mL, followed by treated with 18β-GA and Mef at 37°C for 2 h. Subsequently, the bacterial were centrifuged at 5000 rpm for 5 min, and then washed three times with PBS. The fluorescent probe was added into bacterial and incubated in the dark at 37°C for 30 min.

After incubation, the sample was centrifuged at 5000 rpm for 5 min, the supernatant was discarded and washed three times with PBS. Finally, 350 μL of PBS was added to resuspend the bacteria, and a fluorescence microplate reader (BIOTEK, Synergy2) was used to measure the fluorescence intensity (excitation wavelength of 485 nm; emission wavelength of 528 nm).6. Bacterial Morphological Observation (Scanning Electron Microscopy, SEM)Staphylococcus aureus in the logarithmic growth phase was diluted to 5×10⁵ CFU/mL and undergo different treatments at 37°C for 2 h.

A 100 μL aliquot of bacterial suspension from each experimental group was taken, serially diluted, and spread onto solid BHI agar plates, which were then incubated overnight at 37°C. Sterile cover slips were placed over the bacterial colonies on the agar plates, and the bacteria were incubated overnight to allow bacterial adhesion to the cover slip surfaces. The cover slips were removed and placed in 2.5% glutaraldehyde fixative for 30 min at room temperature.

They were then washed three times with 0.1 M PBS for 15 min each. Subsequently, the samples were fixed with 1% osmium tetroxide in the dark at room temperature for 1–2 h, followed by three 15 min rinses with PBS. Dehydration was performed using a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%) and isoamyl acetate for 15min each.

The dehydrated samples underwent critical point drying, were sputter coated with gold, and were then observed and photographed using a scanning electron microscope (Hitachi-SU8100, Japan).7.Bacterial adhesion and invasion assays7.1Cell adhesion assayCell culture and radiation exposure293T and Hela cells were cultured in Dulbecco’s modified Eagle’s High glucose (DMEM-H, Gbico) supplemented with 10% fetal bovine serum (FBS, Gbico), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gbico).

Cells were maintained in a humidified incubator (37 ℃, 5% CO2, HeracellTM 150i, Thermo Scientific). Before bacterial adhesion, MRSA was diluted to 5×10⁵ CFU/mL, and incubated with different concentrations of 18β-GA and Mef at 37 °C for 2 h. 293T and HeLa cells(2×10⁵ cells/well) received 200 μL of bacterial suspension (MOI =50), while the control groups treated with DMSO. After being cocultured in a 5% CO₂, 37 °C incubator for 2 h, the plates were gently washed three times with precooled PBS.

Each well was treated with 0.25% trypsin-EDTA to detach adherent cells. The cell suspension was serially diluted, and spread onto TSB agar plates, followed by incubation at 37 °C for 18 h. Colony counts within the 30–300 CFU range were recorded to calculate the bacterial adhesion per cell (CFU/mL).7.2 Cell Invasion AssayThe bacterial pretreatment and cell culture were the same as those in Section 7.1.

In the experimental group, 200 μL of bacterial suspension (MOI =50) was added and incubated under 5% CO₂ at 37°C for 3 h. The supernatant was discarded, and the cells were washed three times with precooled PBS. Each well was then supplemented with DMEM containing 200 μg/mL gentamicin and cultured for 2 h.

After three PBS washes, 1 mL of 0.1% Triton X-100 lysis buffer was added to each well and left at room temperature for 5 min, followed by vortexing for 1 min to lyse the cells and release the intracellular invasive bacteria. The cell lysate was serially diluted, and 100 μL of the diluted solution was spread onto TSB agar plates. After incubation at 37 °C for 18 h, the number of colonies within the 30–300 CFU range was counted to quantify bacterial invasive ability.8.

Transcriptome Sequencing and Bioinformatics AnalysisStaphylococcus aureus from different drug treatment groups and the control group were collected, washed with PBS, and enriched by centrifugation at 4000 rpm and then sent to Shanghai Majorbio Biopharm Technology Co., Ltd. (Majorbio) for prokaryotic transcriptome sequencing. The raw sequencing data were subjected to quality control and filtering, followed by sequence assembly.

The assembled unigene sequences were submitted to public databases for functional annotation, including the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Bacterial resistance genes and virulence genes were annotated using the Comprehensive Antibiotic Resistance Database (CARD) and the Virulence Factors of Pathogenic Bacteria Database (VFDB), respectively. Differential gene expression analysis was performed using DESeq2 software, with |log₂FC| ≥ 1 and padjust < 0.05 as the criteria to screen for differentially expressed genes (DEGs).9.

Skin Infection ModelThe hair on the dorsal skin of mice was removed using depilatory cream. After sterilization of the injection site, mice were subcutaneously (s.c.) inoculated with 100 μL of Staphylococcus aureus suspension at a concentration of 5×108 CFU/mL. At 6 hours post-infection (hpi), 100 μL of the combined solution containing Mef (5 mg/kg) and 18β-GA (10 mg/kg) was administered subcutaneously at a site 0.5 mm adjacent to the infected lesion.

Subsequently, the same combination therapy was administered subcutaneously once daily for 7 day. On day 7 post-infection, the length and width of the lesions were measured. The lesion area was calculated by multiplying the length by the width (expressed in mm2).

Mice were then euthanized. To determine the bacterial burden, skin lesions, liver, spleen, and lungs were excised and weighed. Each 0.1 g tissue sample was homogenized using a high-throughput tissue homogenizer in 0.3% Triton solution.

The homogenates were serially diluted with PBS, plated onto LB agar plates, and colony-forming units (CFU) were counted. All animal experiments were conducted following the institutional and national guidelines and approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University ( 202602T013).10. Enzyme-Linked Immunosorbent Assay (ELISA)Skin lesions were collected and weighted.

Each 0.1 g tissue sample was lysed in RIPA lysis buffer (WB3100, NCM Biotech) supplemented with a protease inhibitor cocktail (VP1012, VicMed). The lysate was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The concentrations of IL-1β and IL-6 in the supernatants were measured using a non-coating mouse ELISA kit purchased from Invitrogen (USA).11.

Hematoxylin and Eosin (H&E) StainingPrepared paraffin sections were deparaffinized and rehydrated. Hematoxylin staining was performed, followed by differentiation and bluing to clearly visualize cell nuclei. The sections were then dehydrated in graded ethanol and stained with eosin to demonstrate cytoplasmic structures.

Subsequently, the sections were dehydrated in graded ethanol, cleared in xylene, and mounted with neutral balsam. All sections were observed and imaged under an upright optical microscope (Nikon Eclipse E100, Japan) equipped with an image acquisition system (Nikon DS-U3, Japan).12. Statistical AnalysisAll experimental data were statistically analyzed using GraphPad Prism 9.0 software, with at least three independent biological replicates used for each sample.

The experimental results are expressed as the mean ± standard deviation (mean ± SD). Comparisons among multiple groups were performed using one-way ANOVA combined with multiple comparison tests while the comparison between two groups was conducted using the unpaired t-test. The criteria for determining statistical significance were as follows: P < 0.05 indicated statistically significant differences, whereas P ≥ 0.05 indicated no statistically significant differences.