The Effects Of M331 On Pseudomonas Aeruginosa And The Influence On The Formation Of Its Bacterial Biofilm

Objective:To study the biological characteristics as well as the biofilm morphology of PA1 and PA27853, and identify the sensitivity of PA1 and PA27853 in planktonic or in bacterial biofilms to different kinds of antibiotics. To investigate the influence of different concentration of M331 and the combination of M331 and Roxithromycin on the formation of Pseudomonas aeruginosa biofilm. And to examine the cytotoxicity of M331.Method:The bacteria were purified by coloning them and then the colony forming unit was used to the bacteria counting. The modified plate culture method was used to establish biofilm model in vitro; The morphology of PA1 and PA27853 biofilms was observed by both optical microscopy and SEM. The minimal inhibitory concentrations(MICs) of M331, gentamycin, Carbenicillin, Polymyxin B, and Roxithromycin and of the combination of M331 and Roxithromycin, Piperacillin and tazobactam were determined by micro-dilution method. The minimal biofilm eradication concentration(MBEC) was also determined by micro-dilution method. The viable counts of bacteria in PA1 and PA27853 biofilms were carried out by MTT method after treated with different concentration of M331 or the combination of M331 and Roxithromycin. PA1 and PA27853 biofilms were observed by the optical microscopy after being stained by AgNO3, and by SEM after treated with different dosages of M331 and the combination of M331 and Roxithromycin. The cytotoxicity of M331 were monitered by the of MTT method.Results:Microscopy of PA1 and PA27853 biofilms showed that cluster bacilli is surrounded by dense black-stained substance. The SEM results showed that bacteria were either the bacillus surrounded by accidented substance or the bacteria connected to each other by mucoid substance to form biofilms.The cell adhension of PAl and PA27853 were ubiquitous, and PA1-adhered cell outnumbered that of PA27853. The MTT assay results imply that M331 have no cytotoxicity. of PA27853. The MTT assay results imply that M331 have no cytotoxicity.The MICs of M331 were lower than that of gentamycin and Polymyxin B, on the other hand, higher than Carbenicillin. Roxithromycin enhanced the penetration of M331 to PA1 and PA27853 strain through biofilms. PA1 and PA27853 biofilms were gradually cleared with the increased dosages of M331 or the combination of M331 and Roxithromycin.Conclusion:Previous research work have been done to illustrate that a strain of actinomycetes desingated M331 which extracted from the soil in Guizhou Province has therapeutic effects on Pseudomonas aeruginosa, and macrolides can enhance the its biological effects on Pseudomonas aeruginosa and its biofilms. From this investigation, we provide evidence that Sub-MIC levels of Roxithromycin and M331 substantially inhibited the structure and architecture of the biofilm, and reduced the bacteria count accordingly.