Strains and culture conditions
Porphyromonas gingivalis (Pg) (ATCC 33277), Staphylococcus aureus (ATCC 29213), and Prevotella intermedius (Pi) (ATCC 25611) were purchased from ATCC. Cultures of Pg and Pi were grown overnight in brain heart infusion broth (BHI) (Difco, USA) under anaerobic conditions (80% N2, 10% CO2, and 10% H2) at 37°C in an anaerobic chamber. S. aureus was grown overnight in BHI in a microaerophilic environment (5% CO2). Then, the bacterial suspensions were centrifuged at 2000 rpm/min for 5 min, and the final concentrations of the cultures were adjusted to 0.5×105 CFU/mL using a spectrophotometer.
Peptide
Pln 149 (YSLQM GATAI KQVKK LFKKK GG) was synthesized by Shanghai Apeptide Co. Ltd (Shanghai, China). The peptide was purified by high-performance liquid chromatography, and its identity was verified by SDS-PAGE. The purity of Pln 149 (>95%) and mass were confirmed by electrospray ionization mass spectrometry.
Minimum inhibitory and minimum bactericidal concentration
The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of Pln 149 were determined for all the strains using a modified version of the Clinical Laboratory and Standard Institute (CLSI) broth microdilution method as described previously8. Bacterial suspensions were prepared to a final concentration of 5×105 CFU/mL in BHI and added to wells in a sterile 96-well microtiter plate containing two-fold dilutions of Pln 149. The blood plate was incubated at 37ºC for 24 h. The MIC was set as the lowest concentration of peptides that reduced bacterial growth by ≥90%, while the MBC was set as the lowest concentration of peptides that reduced bacterial growth by >99.99% after the enumeration of viable bacteria by plate counts compared to bacteria grown without antimicrobial peptides.
Biofilm formation
Biofilm formation was assessed using a previously published method9. Pg and Pi culture were incubated in BHI broth and grown under an anaerobic aerophilic condition for 24 h at 37°C. The cells were washed three times with a saline solution and then adjusted with saline to 0.5×105 CFU/mL. Individual sterile Petri dishes were filled with 10 mL of BHI broth supplemented with 1% sucrose, and a sterile saliva-coated titanium plate was added to each one. Individual or mixed 100 µL Pg and Pi suspensions were mixed in the dishes with 200 µL of Pln 149 suspension. As negative and positive controls, PBS and tinidazole suspensions were used, respectively. All the Petri titanium plates were incubated in anaerobic conditions (80% N2, 10% CO2, and 10% H2) at 37°C in an anaerobic chamber.
The analysis of biofilm formation on titanium by a quantitative real-time polymerase chain reaction
During the 24 hours of biofilm formation, the titanium pieces were placed in 1 mL of PBS buffer and vortexed for 90 seconds. The culture was then diluted in PBS to 10-3. Finally, bacterial RNA from 100µl of the suspension was prepared and analyzed by previously described methods. Real-time PCR was performed in triplicate in a 10µl reaction volume containing 1×SYBR Green Master Mix (DBI, China), 100 nM specific primer, and 50 ng template DNA on an ABI PRISM 7900HT system (Applied Biosystems Inc, USA) in 384-well PCR plates, the qPCR Primers used in this study are shown in Table 2.
Laser confocal microscopy
After a total of 24 h of biofilm formation, titanium was washed with 10 mL of phosphate-buffered saline (PBS) to remove unattached cells. The coverglasses were then fixed with 2.5% glutaraldehyde at 4°C for 1 h. After fixation, the coverglasses were stained with a 0.01% acridine orange solution and washed three times with PBS. Any resulting biofilm was observed under a Nikon 80i microscope equipped with an argon laser with an excitation wavelength of 488 nm (green fluorescence). All the images were captured and saved using Nis-Elements AR software (Nikon, Tokyo, Japan).
Electron microscopy
Scanning electron microscopy (SEM) was used to visualize the damage to bacteria caused by Pln 149. The Pg and Pi mixed biofilms were formed on titanium and washed with PBS. The biofilms were then treated with Pln 149 for 24 h, followed by fixation in 2.5% glutaraldehyde in 0.1-M phosphate buffer at pH 7.3. The dried samples were examined under the scanning electron microscope (Tokyo, Japan). Digital images were taken under the 3000 magnified visual field at 5kv.
Planar lipid bilayer preparation and electrical measurements
The whole‐cell patch-clamp technique was employed at room temperature (23–25°C) to measure ionic currents. Pipettes were pulled from glass capillaries that exhibited a resistance of 1–3 MΩ when filled with the pipette solution. The currents were measured in two sets of solutions, one designed to isolate the Na+ current and the other to measure K+ currents, as described below.
The primary cultured mouse dorsal root ganglion neurons (DRGs) were isolated and cultured according to the standard method. After 24 h, the whole-cell voltage-gated sodium and potassium currents were recorded and plotted the current-voltage curves. When sodium and potassium channel currents were recorded, standard extracellular fluid (NaCl 140 mmol/L, KCl 15mmol/L, CaCl2 2 mmol/L, MgC12 1 mmol/L, HEPES 10 mmol/L, Glucose 10 mmol/L) and electrode inner liquid (K-gluconate 140 mmol/L, KCl 5mmol/L, MgC12 2 mmol/L, EGTA 10 mmol/L, K2-ATP 2 mmol/L, GTP 0. 1 mmol/L), The cells were clamped at -80 mV and given a step voltage of 100 ms, +10 mV, and gradually depolarised from -80mV to +60 mV. Statistical values are given as means ± 95% confidence limits.
MTT assay
The cytotoxicity of Pln 149 (125 µg/ml) to bone marrow stromal cell (BMSC) was assessed using an MTT cell proliferation kit (Roche Applied Science)10. The BMSC were incubated at 37°C under 5% CO2 for 1, 2, and 3 days after cell inoculation as described previously. A 50-mL volume of MTT working solution was added to each well, and the mixture was incubated for another 4 h. Purple crystal formazan was observed around cells at ×40 magnification under a microscope. The cell medium was carefully removed, and then 100 mL of dimethyl sulfoxide was added to each well to dissolve formazan. After 15 minutes of incubation at 37°C to completely dissolve formazan, the absorbance at 490 nm was measured on an enzyme-linked immunosorbent assay (ELISA) plate reader, and the results were expressed as optical density (OD) values.
Drug resistance test
S. aureus was grown overnight in BHI in a microaerophilic environment (5% CO2). The Disc diffusion test was used for the common antibiotics sensitivity test of Pln 149 and Cefuroxime 11. Then S. aureus was added in sublethal concentrations of Pln 149 and Cefuroxime (both 70µg/ml) prepared by the BHI method in a microaerophilic environment for 12 h. The Disc diffusion test was tested after 0, 12, 24 and 48 times of culturing in the methods mentioned above. The inhibition zone diameters were determined with filter paper.
Statistical analysis
The data were analyzed using GraphPad Prism 5.0. One-way ANOVA and Tukey multiple comparison tests were used for comparisons between different treatments. A P-value<0.05 was considered statistically significant.