2.1 MaterialsP. acnes strain source GDMCC 1.243 and S.aureus strain source GDMCC 1.1220 were purchased from Guangdong Microbial Culture Collection Center. (Guangzhou, China). BHI Broth medium, Tryptic Soy Broth medium, Reinforced Clostridium Agar medium, Tryptic Soy Agar (TSA) medium were purchased from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, China). 2.5L Round Bottom Vertical Anaerobic Culture Bag, 2.5L Anaerobic Gas Generating Bag were purchased from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, China). The AKP enzyme assay kit and ATP content determination kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.2 Strain activation and suspension preparation
Using a sterile syringe, lyophilized bacteria were gently resuspend in liquid culture media in seeding tubes. P.acnes was cultivated in 100 mL of tryptic soy broth for 48 h at 37℃ in an anaerobic gas-generating bag, while S.aureus was cultivated in 100 mL of brain heart infusion growth medium at 37 ℃ for 24 h. For the following assays, the bacterial suspensions were diluted with phosphate-buffered saline (PBS) to appropriate concentrations.
2.3 Preparation of EMQ
MQ was provided and authenticated by Professor Zhuoma Dongzhi (Medical College, Tibet University, China) in August 2020. The certificate sample (No.CBTM-E124) is kept in the Herbarium of Pharmacy Department of Qingdao University of Science &Technology. Pulverized leaves of MQ were extracted twice with 70% EtOH under reflux for 1.5 h each round. The obtained liquid was filtered with filter paper, and the obtained extractive solution was filtered and evaporated in vacuum to obtain extract powder. For antibacterial experiments, ethanol extract was dissolved in dimethyl sulfoxide (DMSO), and then kept at -20℃ for subsequent studies.
2.4 Agar diffusion assay
The antibacterial activity of EMQ was determined based on the agar diffusion method. Briefly, 20 mL of reinforced Clostridium agar medium (for cultivation of P.acnes) or tryptic soy agar medium (for cultivation of S.aureus) were added to Petri dishes, allowed to solidify, and then uniformly coated with 100 µL of the appropriate bacterial suspension. Afterward, a sterilized steel rod was used to punch holes in the test plate, then 80 µL of the bacterial suspensions were injected through the holes and the plates were incubated for 24 h at 37 ℃ for S. aureus or 48 h for P.acnes, which was cultured in an anaerobic gas-generating bag. Finally, the diameter of the inhibition zone (DIZ) was calculated to determine the antibacterial activity of EMQ.
2.5 Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC)
EQM was diluted with liquid medium to concentrations of 0.78–100 mg/mL by the double dilution method. Then, 100 µL of the microbial suspensions at 107 colony-forming units (CFU)/mL were incubated at 37 ℃. The MIC was calculated as the EMQ concentration required to inhibit bacterial growth. Next, 100 µL of the bacterial suspension were collected from each tube without visible bacterial growth and cultured on agar plates at 37 ℃. The MBC was calculated as the concentration of EMQ in the medium that inhibited bacterial growth.
2.6 Bacterial growth curves
Bacterial growth curves were generated to assess the antibacterial activities of EMQ. Briefly, both strains (107 CFU/mL, 0.1 mL) were inoculated into 100 mL of sterile medium containing EMQ at MICs of 0.5 and 1.0. Cultures of P. acnes and S. aureus in aseptic distilled water without EMQ were used as controls. The bacterial suspensions were incubated at 37°C and growth curves were generated from the optical density at 600 nm (OD600) at 0, 2, 4, 6, 8, 10, 12, 14, 24, and 30 h.
2.7 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
SEM was used to investigate alterations to the bacterial cell surface after treatment with EMQ at MICs of 0.5 and 1.0. After 12 h of EMQ treatment, the bacterial cells were pelleted by centrifugation at 4500 rpm for 10 min, then washed twice with PBS, fixed in 2.5% glutaraldehyde for 24 h, and washed twice again with PBS, followed by a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min at each concentration. Afterward, the ethanol was replaced with isoamyl acetate as an intermediate medium. The dehydrated samples were freeze-dried and stored as a powder. Finally, images of all samples were obtained using a field emission scanning electron microscope (JSM-6700F; JEOL, Ltd., Tokyo, Japan).
TEM was used to observe alterations to the cytoplasm and membrane of bacterial cells after treatment with EMQ at MICs 0f 0.5 and 1.0. The bacteria were pretreated as described above for SEM. Finally, images of all samples were obtained using a transmission electron microscope (HT7800; Hitachi High-Technologies Corporation, Tokyo, Japan).
2.8 Extracellular alkaline phosphatase (AKPase) analysis
Bacterial cells were treated with EMQ at MICs of 0.5 and 1.0 for 12 h, then centrifuged for 10 min at 4500 rpm. The supernatants were collected for analysis. An untreated control group was included for comparisons. AKPase activity was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's instructions.
2.9 Measurement of nucleic acid leakage
Bacterial cells were treated with EMQ at MICs of 0.5 and 1.0 and subsequently incubated for 12 h at 37°C. During the incubation period, 1 mL of each culture was collected every 2 h and centrifuged at 12000 rpm for 10 min. Then, the supernatants were collected and the OD260 was measured using a microplate reader.
2.10 Analysis of bacterial proteins
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to assess the effect of EMQ on bacterial proteins. Briefly, bacterial cells (107 CFU/mL) were treated with EMQ at an MIC of 1.0 for 3, 6, 9, and 12 h, respectively, at 37 ℃ while untreated cells were utilized as negative controls. At the indicated times, 2 mL of the cultivation media were collected and centrifuged for 10 min at 4°C. The harvested cell pellets were resuspended in PBS, heated for 5 min at 95°C, and centrifuged. The supernatant was collected for SDS–PAGE with the use of a 5% stacking gel and a 10% separating gel, which were stained with 0.1% Coomassie Brilliant Blue R250 and destained with glacial acetic acid and methanol in distilled water. The decolorized gels were imaged using a Gel Documentation & Analysis Systems (WD-9413B; Beijing Liuyi Biotechnology Co., Ltd., Beijing, China).
2.11 Measurement of intracellular adenosine triphosphatase (ATPase) concentrations
Bacterial cells (107
CFU/mL) were treated with EMQ at MICs of 0.25, 0.5, 1.0, and 2.0 for 12 h at 37°C, while untreated cells were employed as negative controls. The protein extraction method is described in section 2.9
. ATPase activity was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute) in accordance with the manufacturer’s instructions.
2.12 Crystal violet (CV) staining
Biofilm formation was assessed by staining with CV. Bacterial cells (105 CFU/mL) were dispensed into the wells of a 96-well plate and treated with EMQ at MICs of 0.25, 0.5, 1.0, and 2.0 for 24 h at 37 ℃. Untreated cells were included as negative controls. After 24 h, planktonic cells and spent growth media were removed from each well and the plate was washed three times with sterile water. Biofilms were then stained with 150 µL of 0.1% CV for 15 min at room temperature. The plate was washed with sterile water to remove the excess dye and then air-dried. Absorbance of the CV solubilized in 150 µL of 95% ethanol was measured at an OD595 using a microplate reader.
2.13 Effect of EMQ on bacterial adhesion
Bacterial suspensions (100 µL) in the wells of a 96-well plate were cultured overnight in culture medium supplemented with 2% glucose, followed by an equivalent amount of culture medium containing EMQ at MICs of 0.25, 0.5, 1.0, and 2.0. Untreated cells were used as control. After incubation at 37 ℃ for 4 h, the plates were rinsed with sterile water. Then, planktonic cells were removed and the OD600 was measured to determine the number of cells adhering to the wells.
2.14 Statistical analysis
All data are displayed as the mean and standard deviation. The differences between the two groups were analysed by a double-tailed T test. When p < 0.05, there was a significant difference between the groups.