噬菌体PaP3多糖解聚酶对铜绿假单胞菌生物膜的作用研究

Reaction of polysaccharide depolymerase of bacteriophage PaP3 to Pseudomonas aeruginosa biofilms

目的检测重组铜绿假单胞菌噬菌体PaP3多糖解聚酶对铜绿假单胞菌生物膜的降解活性。方法经FITC-ConA/PI荧光双染色后,采用激光扫描共聚焦显微镜观察重组多糖解聚酶处理前后PA3生物膜的形态变化,以测定多糖解聚酶对生物膜的降解活性;测定多糖解聚酶作用前、后铜绿假单胞菌对4种敏感抗生素(乳酸环丙沙星、加替沙星、头孢他啶、硫酸庆大霉素)的MIC、MBC变化情况,从而判断多糖解聚酶对抗生素的协同杀菌作用。结果FITC-ConA/PI荧光双染色结果显示,经重组多糖解聚酶处理后,铜绿假单胞菌生物膜的胞外多糖组分少而分散,包裹在胞外多糖组分里的细菌数量也大大减少,表明多糖解聚酶确实能够特异性的降解生物膜的胞外多糖组分。多糖解聚酶作用后四种抗生素相应的MIC、MBC都出现了不同程度的降低,其中以头孢他啶降低最为明显,表明多糖解聚酶对抗生素具有协同杀菌活性。结论重组噬菌体PaP3多糖解聚酶在体外可以特异性的降解铜绿假单胞菌生物膜的胞外多糖,有利于抗生素通透生物膜,作用于菌体,具有协同抗菌作用。

Objective To detect the degrading activity of bacteriophage PaP3 polysaccharide depolymerase to Pseudomonas aeruginosa biofilms. Methods The morphologic changes of FITC-ConA/PI labeled Pseudomonas aeruginosa biofdms was observed before and after cocultivation with recombinant polysaccharide depolymerase fusion protein. Then the changes in sensitivities of Pseudomonas aeruginosa PA3 to four antibiotics after co-cultivation with polysaccharide depolymerase were contrasted by microdosis doubling dilution. Results By labeling the biofdms of Pseudomonas aeruginosa PA3 and treating them with polysaccharide depolymerase fusion protein, we discovered that there were less labeled green extracellular polysaccharide （EPS） and fewer amount of bacteria wrapped in EPS compared with that of control group. Polysaccharide depolymerase induced the decrease in minimal inhibitory concentration （MIC） and minimal bactericidal concentration （MBC） of all the four antibiotics, especially for ceftazidime, which indicated polysaccharide depolymerase has antibiotic activity. These results demonstrated that polysaccharide depolymerase could specifically degrade the EPS. Conclusion The recombinant PaP3 polysaccharide depolymerase protein has the ability to degrade bacterial extracellular polysaccharide in vitro and can be used to weaken the drug resistance of bacterial biofdm.