Experimental Study On The Regulation Effect Of Baicalin On The QS System Of Pseudomonas Aeruginosa

Objective 1.The formation of Pseudomonas aeruginosa biofilm and the observation of the intervention role of baicalin at different concentrations on BF of Pseudomonas aeruginosa were identified by EP tube crystal violet staining method,SEM and TEM other different experimental methods.2.The fluorescence quantitative PCR was used to observe whether there were significant differences in the m RNA expression levels of baicalin in las R,rhl R,and pvd Q genes at different concentrations.The correlation between the intervention of baicalin on BF of Pseudomonas aeruginosa and QS system was analyzed,and the mechanism of baicalin intervention on BF of Pseudomonas a eruginosa at gene level was investigated.Method 1.Based on pseudomonas aeruginosa as the research object,the biofilm formation of Pseudomonas aeruginosa was identified by EP tube crystal violet staining,and the biofilm formation status of Pseudomonas aeruginosa were observed after the culture conditions of Pseudomonas aeruginosa were 72 h,96 h,120 h,144 h and 168 h(That is,seventh days),respectively.2.Scanning electron microscope(SEM method)was used in the experiment.The effects of the baicalin on the biofilm formation status of Pseudomonas aeruginosa cultured to the 7th day under the concentrations of 15 mg/ ml,7.5 mg/ml and 3.75 mg / ml were observed under the microscope.3.Transmission electron microscopy(TEM method)was used in the experiment.The effects of the liquid medicine of baicalin on the biofilm formation status of Pseudomonas aeruginosa cultured to the 7th day under the concentrations of 15 mg/ml,7.5 mg/ml and 3.75 mg / ml were observed under the microscope.4.96 hole plate quantitative determination was used in the experiment.The thickness of the biofilm in Pseudomonas aeruginosa within 144 h under 8 different concentrations of baicalin by 15,10,5,2.5,1.25,0.625,0.312,0.156 mg / ml was measured.5.Real-time fluorescence quantitative PCR was used in the experiment.The effect of baicalin at different concentrations on the expression level of m RNA in human colony sensing system was observed and the expression of m RNA was analyzed after the action of Las RG Rhl R and Pvd Q,which were closely related to the formation of biofilm.And its relevance.was analyzed.Result1.EP tube crystal violet staining method showed that P Pseudomonas aeruginosa had the most obvious purple staining on the wall of EP tube about 5-7 days of culture,indicating that the biofilm formation of Pseudomonas aeruginosa was positive.2.The results of Scanning electron microscopy(SEM method)showed that in the high-dose and medium-dose groups of baicalin,Pseudomonas aeruginosa did not obviously show a large patch of biofilm after 7 days of culture,and there was only a small amount of mucous-like substance between the cells of the bacteria.Under the microscope,the bacteria distributed in the form of long rods,and the background was clear.Under the microscope,most of the bacteria were similar to the floating state and are dispersedly scattered on the tin paper carrier,and only some bacteria exhibited aggregated and proliferative states attached to the tin paper carrier.No obvious mucous-like biofilm structure was found.In baicalin low-dose group and blank control group,it was observed that mature and dense organisms adhered unevenly adhered to the tin paper sheet surface at 30000 magnification,and Pseudomonas aeruginosa was wrapped in thick mucoid substan ce.Only a small number of bacteria were not completely outside.3.The results of Transmission electron microscopy(TEM method)showed that the background of baicalin in high-dose group and middle-dose group was clear under negative staining condition and the bacterial flagells were seen.The vast majority of Pseudomonas aeruginosa growed dispersively.and the edge of bacteria was clear.The bacterial biofilm was broken and decomposed under the action of middle-dose baicalin,and there was no obvious large s heet biofilm structure,indicating that most Pseudomonas aeruginosa were isolated in liquid medium.In low-dose group and the blank control group of the baicalin,the bacterial morphology was full and dispersed unevenly,the bacteria were wrapped by irregul ar mature organism membrane,the aggregated state of fibrous substances connected between the cells of the bacteria.Irregular mature biofilm structures were observed around the bacteria,such as fragmented,tower-shaped or mushroom-shaped,indicating that bacteria were not directly dissociated in liquid medium.4.The results of 96 Hole plate quantitative detection showed that baicalin i n 15,10,5,2.5,1.25,0.625,0.312,the growth of Pseudomonas aeruginosa biofilm could be affected by 0.156 mg / ml within 144 h,and baicalin at the dose of 0.625 mg/ml or more could affect the growth of Pseudomonas aeruginosa biofilm..The thickness of the biofilm was significantly thinner than that of the blank control group at 24,48,72,96,120,144 h,and there was a with statistical significance between the two groups(P<0.05).However,C ompared with the control group.there was no significant difference in the late growth of biofilm between baicalin at the dose of 0.312 mg/ml and 0.156 mg / ml(P>0.05).5.The results of real-time fluorescence quantitative PCR showed that baicalin could decrease the expression of las R and pvd Q genes at high,middle and low doses compared with the control group(P<0.05).The m RNA expression of rhl R gene in high-dose and medium-dose of baicalin was significantly lower than that in blank control group,and the difference was significant(P<0.05).Compared with the control group The m RNA expression of rhl R gene at low-dose of baicalin was not significantly decreased(P>0.05).Conclusion Baicalin can play a significant role in the formation and matu ration of Pseudomonas aeruginosa biofilm.Baicalin can decompose the large sticky sheets of biofilm and reduce the thickness of biofilm.Different concentration s of baicalin could reduce the m RNA expression of las R,rhl R gene and pvd Q gene,which were closely related to biofilm formation in Pseudomonas aeruginosa population sensing system.These results suggest that baicalin may interfere with the biofilm formation and maturation of Pseudomonas aeruginosa.