Study On The Mechanism Of Antibacterial Effect Of Amlodipine Besylate On Quorum Sensing System Of Pseudomonas Aeruginosa

ObjectiveQuorum sensing system (QSS) is a key regulation system of Pseudomonasaeruginosa (PA) to control its virulence production. The initiation of quorum sensing(QS) is related to the increase of cytosolic free Ca2+concentration ([Ca2+]i).Amlodipine besylate (AML), which is a calcium channel blocker, could block Ca2+influx of PA and might suppress QS of it. This study is to explore the inhibition effectof AML on QSS of PA and its possible mechanism.Methods1Cell cultureThe single colony of PA was activated and isolated by dilution-plate method, andwas subcultivated. The single colony of PA was then inoculated in LB, MH, PTSBliquid culture media.2ProtocolControl group, solvent group (0.5%Dimethyl Sulfoxide, DMSO), A23187group(calcium ionophore,1μg·mL-1), AML group (64,32,16μg·mL-1) were set in cellscultured with normal medium.3Measurements and methodology3.1The viable count of PA in liquid culture media was measured with the method ofplate culture count, and a cfu-OD600standard curve was drawn based on it. Theviable count of PA in liquid culture media was then drawn by photoelectricturbidimetry (λ=600nm)3.2The effect of AML to grow curve of PA was drawn by photoelectric turbidimetry (λ=600nm)3.3The minimun inhibitory concentration (MIC) of AML to PA was determined bydisc-diffusion method3.4The production of proteolylic enzymes was determined by protease assay3.5The production of elastase was determined by elastase assay3.6The production of pyocyanin was determined by pyocyanin assay3.7Fura-2/AM was acted as fluorescence probes for cytosolic Ca2+, and fluorescentmicroscope was used to observe the loading efficiency of Fura-2/AM to PA, anddual-wavelength fluorophotometry was used to determinate the [Ca2+]iof PAResults1The cfu-OD600standard curve of PA during the logarithmic phase is y=0.0797x+0.0094, R2=0.99752The MIC of AML to PA was512μg·mL-13The growth of PA was inhibited by AML (256,128μg·mL-1, P<0.05); AML (64,32,16μg·mL-1) had no effect on the growth of PA; AML (64,32,16μg·mL-1) wereselected to be the drug concentration of assays the rest4The production of pyocyanin was significantly inhibited by AML (64,32,16μg·mL-1, P<0.0l); and the production of proteolylic enzymes and elastase wereinhibited by AML (64,32,16μg·mL-1, P<0.05)5The cultured cells were loaded by Fura-2/AM successfullly; the [Ca2+]iof PA couldbe measured by dual-wavelength fluorophotometry, demonstrated as fluorescenceintensity of340nm/380nm (F340nm/F380nm, abbr.R)6The [Ca2+]iof PA could be decreased by AML (64,32,16μg·mL-1, P<0.05)Conclusion1AML (64,32,16μg·mL-1) inhibited the virulence production of PA2AML could inhibited the QSS of PA by interrupting its calcium signal transductionsystem...