1.1 Experimental materials and reagents
PAO1 used in this research was purchased from ACTT. RT-PCT related extraction, reverse transcription and amplification reagents were purchased from TAKARA (Japan). Eucalyptus leaves were collected from Hengxian, Guangxi, and the volatile oil was extracted and completed by the First Affiliated Hospital of Guangxi University of Chinese Medicine. Scanning electron microscope (SEM, EDAX-AMETEX) was provided by Guangxi Medical University. RT-PCR detection, bacterial biofilm Model construction and MIC tests were completed by the Laboratory of the the First Affiliated Hospital of Guangxi University of Chinese Medicine.
1.2 GC-MS Analysis of Eucalyptus Leaf Oil
1.2.1 Material 6890A Gas Chromatograph-5973N Mass Spectrometer (Agilent, USA).
1.2.2 GC-MS analysis conditions
1.2.2.1 Gas chromatographic condition Column: 1) Agilent HP-5MS capillary column (30m×0.25nm×0.25μm). 2) Heating program: Initial temperature was 60℃, warm to 70℃ at 2℃/min and hold for 10min. Raised the temperature to 140℃ at 3℃/min and raised to 250 ℃ at 5 ℃/min, and then kept for 10 minutes. 3) Carrier gas: He. 4) Flow rate: 1ml/min. 5) Injection volume: 1.0 μL. 6) Split ratio: 50: 1.
1.2.2.2 Mass spectrometry conditions: 1) Ionization mode: EI. 2) Ionization energy was 70eV, inlet temperature was 250 ℃ and ion source temperature was 230 ℃. 3) Column flow rate: 1.0ml/min.
1.2.2.3 Analysis methods
The Eucalyptus leaf oil was taken and analyzed by GC-MS. The mass spectrum was searched in NIST98 standard mass spectrum database (HPMSD Chem-Station) to confirm each chromatographic peak.
1.3 MIC test
Because the volatile oil of Eucalyptus leaves is insoluble in water, the co-solvent Tween-80 is used for co-solubilization, and the ratio of volatile oil and co-solvent is 5: 1000. Take 10 μL 0.5 MCF of PAO1 bacterial suspension and inoculate it into 1 mL of LB medium, and then add soluble volatile oil to configure a gradient mixed suspension with drug concentration of 10% to 50%, and incubate in a CO2 incubator for 24 hours, and then transfer to the blood plate to observe the growth of bacteria.
1.4 protein fingerprint analysis
Mass spectrometer (Micyoflex LT/SH, BD, US) was used for testing and FlexAnalysis (Bruck, US) for analysis. Standard MBT method, usual for typical laboratory standard samples, used for validation and specification issues. MS/Parent Mode: On. Initial Laser Power: 30%, and maximal laser power: 40%. Allow Only: 80 satisfactory shots per raster spot. Matrix Blaster: Fire initially 10 shots with a laser power of 40%.
1.5 BF Construction and SEM observation [10-12]
1.5.1 Biofilm model construction: Take 20mL of LB medium into a 50mL centrifuge tube, inoculate 1mL of 0.5 McFarley bacterial suspension, put a sterile gastric tube(1cm) into the bacterial suspension, and place it in a shaker at an angle of 45 degrees. After shaking for 24 hours, after that a bacterial film was formed on the surface of the sterile gastric tube, which is a biofilm model.
1.5.2 Preparation before SEM observation: 1) Biological specimens were fixed with 3% glutaraldehyde for 2 hours, 2) 0.1 mol/L PBS buffer solution was immersed and washed 3 times for 10 min each, 3) Osmium tetroxide was fixed for 1 hour, 4) 0. 1mol/L PBS buffer solution for 3 times and 10min each time, 5) Ethanol from 50%, 70%, 80%, 90%, 100% (three times) soak and dehydrate for 10min each time, 6) 100% pure six Methyldisilazane was immersed three times for ten minutes each time, put into a vacuum dryer and vacuum dried, 7) Paste the sample to the sample holder, put it on the IB3 ion sputtering instrument and observe it under an electron microscope.
1.6 RT-PCR detection
After constructing the biofilm model according to the above steps, the gastric tube was gently washed with sterile PBS and the bacteria on the inner and outer surfaces of the gastric tube were collected with a spatula. After enrichment, the sample RNA was extracted using the TAKARA genome extraction kit and reversed. After 15 minutes of recording, use RT-PCR detector (Redstone SLAN-96P, Shanghai, China) to test the LasI expression.
1.7 Statistical methods
SPSS 19.0 and GraphPad were used for statistical analysis and picture processing. Measurement data were analyzed by t test. Sample means were expressed in the form of mean ± standard deviation. The test level α = 0.05. When the P value is less than 0.05, it means The difference was statistically significant.