Study On The Effect Of Berberine Combined With Azithromycin On Alginate,the Main Component Of Pseudomonas Aeruginosa Biofilm

[Objective]To explore the effects of berberine combined with azithromycin on the formation of Pseudomonas aeruginosa PAO1 biofilm,the secretion of the main components of the extracellular matrix in the biofilm,alginate,virulence factors,and the expression of corresponding genes,and reveal the effect of berberine combined with azithromycin on Pseudomonas aeruginosa The influence and mechanism of the biofilm of Pseudomonas aeruginosa provide new treatment ideas and methods for the clinical treatment of Pseudomonas aeruginosa infection[Methods]1.Measure the minimum inhibitory concentration(MIC)and combined drug sensitivity index(FICI)of berberine and azithromycin against Pseudomonas aeruginosa PAO1 by micro broth dilution method and checkerboard titration method;use spectrophotometer to detect different time periods OD600 value of bacterial suspension,draw growth curve to observe the effect of berberine on the growth of PAO1.2.Set different concentrations of berberine group,azithromycin group,combination medication group and blank control group,using the following methods:semi-quantitative crystal violet staining method to detect biofilm content,ELISA method to detect biofilm alginate content,and immunofluorescence technique observation Biofilm alginate content;phenol-sulfuric acid method for determination of PAO1 extracellular polysaccharide content;hydrochloric acid-chloroform method for determination of PAO1 production of pyocyanin content;Congo red method for determination of PAO1 production of elastase Las B content;preparation of specific media to observe PAO1 migration Ability to observe the effect of berberine on the expression of PAO1 biofilm and virulence factors.3.Real-time fluorescent quantitative PCR method was used to detect the effect of berberine on the expression of genes related to the formation of alginate,an important component of PAO1 mucus biofilm.[Results]1.The MIC of berberine against mucoid Pseudomonas aeruginosa PAO1 is greater than 1250 μg/ml,and the MIC of azithromycin is 256 μg/ml.When used in combination,the berberine MIC decreased to 315μg/ml,and the azithromycin MIC decreased to 64 μg/ml,showing a certain synergistic effect;in the growth curve experiment,as the concentration of berberine doubled,the growth inhibitory effect on PAO1 was enhanced,showing a concentration-dose-dependent inhibitory effect.2.The combined use of berberine and azithromycin can significantly reduce the formation of PAO1 biofilm,and the content of biofilm alginate is significantly reduced.At the same time,the results of PAO1 extracellular polysaccharide EPS and biofilm absorbance are consistent,and the difference is statistically significant(P<0.05).The combined application of berberine and azithromycin can significantly inhibit the secretion of PAO1 virulence factors(Pyocyanin,Elastase Las B),and the difference is statistically significant(P<0.05).At the same time,the combined use of berberine and azithromycin can reduce the cluster exercise ability of PAO1,and the difference is statistically significant.(P<0.05).3.RT-PCR results showed that the relative expression of berberine combined with azithromycin on alginate synthesis-related regulatory genes alg D,alg R,and alg G was significantly lower than that of the untreated group,and the difference was statistically significant(P<0.05).[Conclusion]Berberine combined with azithromycin has a synergistic antibacterial effect on Pseudomonas aeruginosa PAO1;when used in combination,it can significantly reduce the formation of PAO1 biofilm,reduce the secretion of virulence factors and its migrating ability in the culture medium;against Pseudomonas aeruginosa.The expression of related genes of alginate,an important component of mucus phenotype,has a significant inhibitory effect,providing new treatment ideas and methods for the clinical treatment of PA infection.