The Influence Of Azithromycin On The Quorum Sensing System And The Formation Of Bacterial Biofilm Of Pseudomonas Aeruginosa

Objective: Study the biology characteristics as well as the biofilm morphology of PAO1 and PAO-JP2, and identify the sensitivity of PAO1 and PAO-JP2 in planktonic or in bacterial biofilms to different kinds of antibiotics. Investigate the influence of QS system to the pathogenicity and drug resistance of Pseudomonas aeruginosa. Compare the biofilm morphology between PAO1 and PAO-JP2 which were cultured with diffenent dosages of Azithromycin (AZM), and research the effect of QS system to the formation of bacterial biofilm with the adding of AZM. Method: The colonial morphology and bacteria after Gram's stain of PAO1 and PAO-JP2 were identified by using the optical microscope;Their biochemistry was identified by Vitek analysis;The morphology of PAO1 and PAO-JP2 was observed by Atomic force microscopy(AFM);The modified plate culture method was used to establish biofilm model in vitro;The morphology of PAO1 and PAO-JP2 biofilms was observed by the optical microscope after being stained by AgNO₃, SEM, CSLM;The minimal inhibitory concentrations (MICs) of 20 antibiotics to PAO1 and PAO-JP2 were determined by agar plate dilution method;The minimal biofilm eradication concentration (MBEC) was determined by micro-dilution method;The viable counts of bacteria in PAO1 and PAO-JP2 biofilms were detected after treated with Azithromycin and Ciprofloxacin by MTT method;PAO1 and PAO-JP2 biofilms were observed by the optical microscopy after being stained by AgNO₃, SEM, CSLM after treated with different dosages of Azithromycin;PAO1 and PAO-JP2 biofilms were analysised by computer image analysis system after being stained by AgNO₃.Results: The colony colour of PAO1 was reseda while it was light yellow of PAO-JP2. And the colony of PAO1 was moister than that of PAO-JP2. Both of them were red bacillus after Gram's stain. Both of them revealed negative on β-lactamase by β-lactamase assay. There were some differences on Urea and Lysine according to the two strains by Vitek analysis. The length of PAO1 wasfound longer than that of PAO-JP2 by AFM.Microscope graphs of PAOl and PAO-JP2 biofilms showed that cluster bacilli surrounded by dense black stained substance. And the black stained substance on PAO-JP2 biofilm was more than that on PAOl biofilm. The SEM graghs showed that bacteria were both bacillus surrounded by accidented material and the bacteria were connected each other by mucoid substance to form biofilms. There were more bacteria in disorder on PAO-JP2 biofilm. The CSLM graphs showed the three-dimensional morphous of PAOl and PAO-JP2 biofilms. The biofilm formed by PAOl was thicker than that of PAO-JP2. The cell adhesion of PAOl and PAO-JP2 were both diffusible adhesion, and the number of PAOl adhering to cells was larger than that of PAO-JP2.The MICs of 20 antibiotics to PAO-JP2 were almost lower than that to PAOl. Azithromycin enhanced the penetration of Ciprofloxacin to PAOl and PAO-JP2 through biofilms. PAOl and PAO-JP2 biofilms were gradually cleared with the increasing dosages of Azithromycin, and the number of bacteria was decreased, however, the effection to PAOl was more obvious than that to PAO-JP2 at the same dosage of Azithromycin.Conclusions: There were obvious differences between PAOl and PAO-JP2 in biology characteristics in vitro. The differential inhibitory effects of antibacterials to the planktonic or in BBF were also found, which suggested that QS system participated in a series of biology variation and lead to the differences in pathogenicity and drug resistance as well as biofilm morphology. The effect of sub-MICs of AZM that PAOl or PAO-JP2 exposured to when culturing biofilms showed that QS system could actively inhibitor the formation of Pseudomonas aeruginosa bacterial biofilm.