Effects Of Andrographolide On The Formation Of Pseudomonas Aeruginosa PAO1 Biofilm

ObjectiveObserve the biofilm formation process of Pseudomonas aeruginosa PAO1 which treated with andrographolide(Agp), and detect the correlation between drug and quorum sensing system(QS) on the molecular level for exploring the antibacterial mechanism of Agp, and and provide experimental evidence and treatment theory for further develop and utilize Agp.Method1 Using high performance liquid chromatography (HPLC) to control the purity of experimental drug for ensuring stable quality of Agp.2 using ELISA detection instrument(OD600nm) to detect absorbance of culture which contains different stains Pseudomonas aeruginosa (CMCC10104), Staphylococcus aureus (CMCC26003), Escherichia coli strain (CMCC44102) and Pseudomonas aeruginosa PAO1 which cultivate in the different doses of Agp 100 μg/mL,10 μg/mL,1 μg/mL,0.1 μg/mL,0.01 μg/mL and 0.001 μg/mL, and make growth curve in 24h.3 Using coverslip to cultivate PAO1 BF under different doses of Agp(10 μg/mL, 1 μg/mL,0.1 μg/mL and 0.01 μg/mL), remove the cover slip in 24,48,72 h, using fluorescein isothiocyanate conjugated concanavalin A (FITC-ConA) and pyridine iodide (PI) fluorescent dye double immunofluorescence staining, using confocal laser scanning microscopy (LSCM) differences in morphology and observed morphology and thickness of PAO1 after treated with different dose of Agp using LCMS.4 Applying RT-PCR to detect the quantitative of mRNA of relevant gene(lasR/rh1R and pvdQ) to QS within 24 hours.Result1 the figure shown above demonstrates that the purity of andrographolide was 99.7%, which could meet the test requirements.2 As we can see from the growth curve, the growth curve of the control group is the same as the drug group, we can make a conclude that Agp has no effect on the growth of bacteria.3 LSCM shows that green and red fluorescent signal could be observed at 24h, which indicates that the bacteria begun to gather, small amounts of polysaccharide matrix appears and biofilm begins to form. visible green fluorescence increased gradually, the red signal begins to gather as a small group at 48h, which represents that the biofilm formation has been gradually increasing. Red signal gathers successfully, green signal’s generation as compact structure, which reveal that biofilm has formed. There is no significance difference on aggregation and fluorescence intensity of PA01 BF could be observed between blank control group and blank control group under the microscope, which indicate that solvent DMSO has no obvious influence on biofilm formation. Compared with the blank control group, after administration, the effect of 10 μg/mL group on aggregation and fluorescence intensity of PAO1 BF has improved until 48h, but 0.01 μg/mL～μg/mL group shows obvious difference in 24 h. All of these shows that Agp can inhibits the formation of PAO1 BF.4 The Real-time PCR shows that the Agp could reduce the relative expression of lasR and rh1R at the early stage from 3 h to 9 h, then increased from 9 h to 21 h, and reached the peak at 15 h, and following gradually decrease from 21 h to 24 h. For pvdQ gene,100 μg/mL and 0.01 μg/mL dose of Agp has same effective trend as lasR and rh1R genes, while 1 μg/mL Agp plays a role of inhibition on pvdQ.ConclusionAgp could inhibits the BF formation of PAO1, and it also could affect the expression of PAO1 QS system lasR, rh1R and pvdQ mRNA, which show a law of inhibition at first and increase followed and suppression at last. All of these suggesting that these three gene are not the drug targets which affect the biofilm formation.