High performance liquid chromatography analysis
Matrine and oxymatrine were the main contents of SFAs and were analyzed by HPLC methods. The standard control sample of matrine and oxymatrine were both purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), the chromatographic analyses were implemented on the Agilent UPLC system (Shimadzu, Japan), and the Agilent XDB C18 5μm (4.6×150mm) was used for the chromatographic separation. Mobile phase consisted of solvent A (10% water) and B (90% methanol solution), and the flow rate was set to 1 ml/min with the injection volume was 4 μL. A sample of 1g SFAs was put into a 100ml volumetric flask with constant volume of methanol to obtain 10mg/ml test solution, the control sample matrine and oxymatrine were used as the the referenced sample. The operating parameters were as follows: both the reference and test samples were prepared into different gradient solutions with the same concentration. The reference substance and the test substance solution were injected respectively at the same time to obtain the peak area of the different component. Since the response value factor (F) of each component is unchanged, therefore, the concentration of test sample can be calculated according to F value. F is calculated as follows: F= reference concentration/reference peak area = test concentration/test solution peak area. So the oxymatrine content =[(concentration× dilution ratio)/test product weight sample volume] ×100%. Results are presented as mean ± SD of the mean of at least triplicates.
Isolation and culture of G.vaginalis strains
The strains used in this study were isolated from vaginal secretions of BV patients in the Gynecology Clinic as per the following procedure. The vaginal secretions were obtained from one-third of the vaginal wall using a sterile cotton swab and applied to a Casman agar plate (Beijing AOBOX Biotechnology Co., Ltd.).Then the agar plate was placed into an aerobic bag and incubated with 5% CO2 at 37°C for 48 h. Round, needle-like, and translucent single colonies were selected and applied in line to a new Casman agar plate followed by anaerobic culture for 24-48 h. After three generations of pure cultures, single colonies were collected, mixed evenly with 30% glycerol, and stored at -80°C.
Identification of G.vaginalisstrains
The clinically isolated strains were identified using colony polymerase chain reaction (PCR) and 16s rRNA gene sequencing. 16S rRNA gene hypervariable V1–V3 region was amplified using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTA GACTT-3′) .The protocol used was as follows: total volume of the PCR mixture was 50 μL, and the reaction conditions included pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, then annealing and extension at 72°C for 80 s. The cycle was repeated 33 times. The PCR amplification products were sent to Beijing SinoGenoMax Co., Ltd. for sequencing, and the sequences of the splicing results were compared with the bacterial 16s rRNA gene sequences in GenBank data library for identification and confirmation of G.vaginalis. Bacteroides fragilis ATCC 25285 was used as a control for the tests carried out under anaerobic conditions.
Antimicrobial susceptibilitytesting
SFAs (NMPN Z20050058) was provided by Guiyang Xintian Pharmaceutical Co., Ltd. (Guizhou, China) and metronidazole (purity: 99.97%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). SFAs was dissolved in ethanol with concentration of 400 mg/mL and metronidazole dissolved by sterile distilled water with 2.56 mg/mL concentration.. The antimicrobial susceptibility activity was detected by the microdilution broth method as described by Sutyak Noll et al with minor modifications [11]. Briefly, the antimicrobials were diluted (a series of 2-fold dilutions) with an appropriate volume of fresh BHI broth (Beijing AOBOX Biotechnology Co., Ltd.) in 96-well culture plate. The final concentration of the SFA was 0.039-20 mg/mL and metronidazole was 0.125-128 μg/mL. An equal proportion of ethanol solution at the highest final concentration of 5% and sterile distilled water were served as the negative control, respectively. Sterile distilled water served as the negative control. G.vaginalis and control were cultured until they achieved the logarithmic phase and was diluted in BHI to the final 5 × 106 CFU/mL. From the diluted bacterial cells, 100 μL was transferred in the wells containing predetermined concentrations of antimicrobials. The inoculated plate was placed into an anaerobic chamber and incubated under 5% CO2 at 37°C for 48 h. The lowest antibiotic concentration yielding marked reduction to no growth was read as the Minimum Inhibitory Concentration (MIC). According to the 2012 Clinical and Laboratory Standards Institute guidelines for anaerobic drug sensitivity testing, metronidazole MIC was evaluated as follows, MIC ≤8 μg/mL: sensitive to metronidazole, MIC =16 μg/m: moderate sensitivity, and MIC ≥32 μg/mL: resistant to metronidazole.
As previous described, after the plate was inoculated for 48h, 100 μL of the bacterial solution was separately taken from the non-turbidity and control wells, and then were evenly applied to a drug-free solid medium, followed by anaerobic culture for 48 h. The drug concentration in the well where the total bacterial count declined by 99.9% or more was compared against the control well to reveal the minimum bactericidal concentration (MBC).
Bacterial biofilm formation assay
To develop the biofilm formation level of G.vaginalis clinical strains, a starting inoculum of 5 × 106 CFU/mL of prepared bacterial suspension in the BHI broth was planted in 96-well culture plate. The microplate was incubated anaerobically for 48 h. Crystal violet staining (Beijing AOBOX Biotechnology Co., Ltd.) was used to quantify the total amount of biofilm biomass [12]. After the incubation period, each well was gently washed twice with 200μL of PBS to remove the non-adhered bacteria, and dried for 15 min. The dried biofilm was then stained with 200 μl crystal violet (1%,w/v, Sigma) and incubated for 30min. Finally, the well was rinsed with PBS three times and decolorized with 95% alcohol 200μL for 5min. Then the liquid was moved to a new microtitre plate and absorbance at 595nm was measured by a microplate reader (Bio-Rad Laboratories, Hercules,CA, USA). The ability of biofilm formation was evaluated according to OD value, which which was defined as three standard deviations (SD) above the mean OD of the blank control (ODc), OD ≥4 × ODc indicates strong biofilm producer[13].
Determination of the minimal biofilm inhibitory concentration (MBIC)
The inhibitory concentration of SFAs against G.vaginalis biofilm formation was determined using the above described. The logarithmic-phase bacteria were cultured with antimicrobial-containing medium and anaerobically cultured for 48 h. The G.vaginalis biofilm was detected by violet solution as previously described, and the minimum biofilm inhibition concentration (MBIC) was defined as the lowest concentration of an antibiotic that completely inhibited the growth of microorganisms compared with control.
Observation of biofilm microstructure using scanning electron microscopy
The morphology and structure of biofilms changes were described by scanning electron microscopy examination. Sterile cover glass was placed at the bottom of each well of a 24-well plate, and bacteria were inoculated into each well glass, followed by anaerobic culture for 48 h to form biofilm. Then the cover glasses were removed, washed three times with PBS, fixed with 2% cold glutaraldehyde for 15 h, vacuum-dried for 72 h, and plated with gold. The morphological features of biofilm was observed using a scanning electron microscope (HITACHI S-3400, Japan).
Statistical analysis
GraphPad Prism 8 software was used for statistical analysis. Non-parametric Mann-Whimey U test was used for comparison because the data did not conform to normal distribution. P < 0.05 indicated a statistically significant difference.