铜绿假单胞菌噬菌体的分离鉴定及其控制生物被膜的应用研究

目的 分离鉴定铜绿假单胞菌烈性噬菌体,研究噬菌体控制宿主菌形成牛物被膜的效率.方法 以铜绿假单胞菌临床菌株为指示菌,从不同环境样品中分离噬菌体;采用限制性内切酶图谱分析和宿主范围测定方法,对分离的噬菌体进行分类;利用透射电子显微镜对分离噬菌体进行形态学研究;以TJC729为指示菌,开展噬菌体控制牛物被膜形成的应用研究.结果 分别以14株铜绿假单胞菌临床分离菌株为指示菌,共分离得到13株烈性噬菌体,命名为C1～C13.利用限制性内切酶EcoR Ⅰ分析结果表明,13株噬菌体基因组均为双链DNA,并可被分成8组;宿主谱测定结果显示,c1和C13、C6和C7、C9和C11分别具有相同的宿主范围,其余7株噬菌体的宿主范围各不相同.随机挑选噬菌体C1进行形态学研究,发现噬菌体C1头部具有二十面体结构,尾部较长且无收缩性尾鞘,属于长尾噬菌体科.生物被膜控制实验结果显示,混合噬菌体能够较好地抑制TJC729生物被膜的形成.进一步实验结果显示,噬菌体C1、C10和C12分别与牛物被膜混合培养24 h后,牛物被膜的量分别下降到初始量的32.7%、57.6%、32.8%.结论 分离了13株铜绿假单胞菌烈性噬菌体,它们能够显著抑制宿主菌生物被膜的形成,并对生物被膜造成一定程度的破坏,为控制铜绿假单胞菌引起的感染提供了一个新方法.

Abstract：

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.

Isolation and classification of the bacteriophages of Pseudomonas aeruginosa and their application on biofilm control

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.