1.1 Experimental materials and reagents
In this research, PA was obtained from clinical patients. RT-PCR–related extraction, reverse transcription, and amplification reagents were purchased from TAKARA (Japan). Eucalyptus leaves were collected from Hengxian, Guangxi, and the First Affiliated Hospital of Guangxi University of Chinese Medicine extracted and completed the volatile oil. Scanning electron microscope (SEM, EDAX-AMETEK) was provided by Guangxi Medical University. Moreover, RT-PCR detection, bacterial biofilm model construction, and MIC tests were completed by the laboratory of the abovementioned hospital.
1.2 GC-MS analysis of eucalyptus leaf oil
1.2.1 Material 6890A Gas Chromatograph-5973N Mass Spectrometer (GC-MS; Agilent, USA).
1.2.2 GC-MS analysis conditions
188.8.131.52 Gas chromatographic condition
Column: 1) Agilent HP-5MS capillary column (30 m × 0.25 nm × 0.25 μm). 2) Heating program: Initial temperature was 60 ℃, which was then increased to 70 ℃ at 2 ℃/min and held for 10 min. The temperature was raised to 140 ℃ at 3 ℃/min, further elevated to 250 ℃ at 5 ℃/min, and then maintained for 10 min. 3) Carrier gas: He. 4) Flow rate: 1 mL/min. 5) Injection volume: 1.0 μL. 6) Split ratio: 50:1.
184.108.40.206 Mass spectrometry conditions
1) Ionization mode: Electron ionization. 2) Ionization energy was 70 eV, inlet temperature was 250 ℃, and ion source temperature was 230 ℃. 3) Column flow rate: 1.0 mL/min.
220.127.116.11 Analysis methods
After extraction, the eucalyptus leaf oil was analyzed by GC-MS. To confirm each chromatographic peak, we searched the mass spectrum in NIST98 standard mass spectrum database (HPMSD ChemStation).
1.3 MIC test
Considering that the volatile oil of eucalyptus leaves is insoluble in water, cosolubilization was performed using the cosolvent Tween-80. The ratio of volatile oil and cosolvent is 5:1000. We extracted 10 μL of 0.5 MCF of PAO1 bacterial suspension and inoculated it into 1 mL of LB medium. Then, we added soluble volatile oil to configure a gradient-mixed suspension with a drug concentration of 10% to 50% in volume. Next, we incubated it in a CO2 incubator for 24 h and transferred to the blood plate to examine bacterial growth.
1.4 Protein fingerprint analysis
The protein fingerprint of PA in each group was tested and analyzed by mass spectrometry (Micyoflex LT/SH, BD, US) and FlexAnalysis (Bruck, US), respectively. For validation and specification issues, we used the standard MBT method, which is commonly employed for typical laboratory standard samples. MS/Parent mode: On. Initial laser power: 30%, and maximal laser power: 40%. Allow only: 80 satisfactory shots per raster spot. Matrix blaster: Initially, 10 shots were fired, with a laser power of 40%.
1.5 Biofilm construction and SEM observation[10-12]
1.5.1 Biofilm model construction: We poured 20 mL of LB medium into a 50 mL centrifuge tube, inoculated 1 mL of 0.5 McFarley bacterial suspension, placed a sterile gastric tube (1 cm) into the bacterial suspension, and positioned it in a shaker at 45° angle. After shaking for 24 h, a bacterial film was formed on the surface of the sterile gastric tube, indicating the biofilm model.
1.5.2 Preparation before SEM observation: 1) Biological specimens were fixed with 3% glutaraldehyde for 2 h. 2) We immersed 0.1 mol/L PBS buffer solution and washed it thrice for 10 min each. 3) Osmium tetroxide was fixed for 1 h. 4) We again immersed 0. 1 mol/L PBS buffer solution and washed thrice for 10 min each. 5) We soaked 50%, 70%, 80%, 90%, and 100% of ethanol thrice and dehydrated for 10 min each, 6) We immersed 100% pure hexamethyldisilazane thrice for 10 min each and then vacuum-dried. 7) The sample was pasted to the sample holder, placed on the IB3 ion sputtering instrument, and subsequently observed under an electron microscope.
1.6 RT-PCR detection
After constructing the biofilm model, we gently washed the gastric tube with sterile PBS and sonicated the bacteria on the inner and outer surfaces of the gastric tube to obtain the biofilm. After enrichment, the sample RNA was extracted using the TAKARA genome extraction kit and then reversed. After 15 min of recording, the LasI expression (6 cases per group) was tested using an RT-PCR detector (Redstone SLAN-96P, Shanghai, China).
1.7 Statistical methods
For statistical analysis and picture processing, we used the SPSS 19.0 and GraphPad, respectively. Measurement data were analyzed by t test. Sample means were expressed as mean ± standard deviation. The test level was α = 0.05. Furthermore, P < 0.05 indicated a statistically significant difference.