临床分离铜绿假单胞菌密度感应缺陷株致病性分析

摘要

Objective Clinically isolated Pseudornonas aeruginosa (PA)were used to explore the function of quorum sensing (QS)in the pathogenesis of PA infection and provide novel evidence for controlling the PA infection clinically. Methods Congo red elastase test was applied to screen QS disability in 84 clinically isolated PA strains. The expression level of elastase,pyocyanine secretion,biofilm formation and mobility capabilities were also examined in the identified QS defective strains. Results In 84 clinically isolated PA strains, we have screened out 4 QS defective strains(4.76％). PCR tests suggested that the rhl I gene and rhl R gene of C84 were missing. The sequence of the rhl I gene of C39 had multiple mutations,for example Ser62Gly and Asp83Glu. The mutations in the rhl R gene of C39 not only resultcd in multiple point mutation, more importantly, a readingframe shift caused the complete coding sequence changed after the residue 48. In the las R gene of C84 mutations of Arg66Lys,Asp136Ser,Ala138Val and Ser172Asp were also identified. In the genes of C104 and C117 rhl I, mutations of Ser62Gly and Asp83Glu were identified. Further causative agent tests showed that the elastase expression levels of C39 , C84 , C104 , C117 were similar to the QS defective strains( PAOJP2 ) but significantly lower than the wild strain( PAO1 ). The pyocyanine secretion levels of C39 and C84 were significantly lower than PAO1 ,and similar to PAOJP2 ,whereas the pyocyanine secretion of C104 and C117 was similar to PAO1 . The biofilm initial capacity of C39 , C84 , C104 and C117 was found lower than that of the PAO1. The mobility capacity of C39 ,C84 ,C104 and C117 was lower than PAO1. Conclusion The QS system of PA plays an important role in the process of its infection. Interfering the PA QS may provide a very promising new therapy.%目的 以临床分离的铜绿假单胞菌(PA)为研究对象,从临床角度探讨密度感应(QS)在PA感染发病机制中的作用,为临床控制PA感染提供新的思路.方法 采用刚果红弹性蛋白酶解实验对临床分离的84例PA株进行QS缺陷筛选,检测QS缺陷株的弹性蛋白酶表达、绿脓菌素分泌、生物被膜形成及泳动能力.结果 在84例临床分离PA株中筛选出4例QS缺陷株(C39,C84,C104,C117),检出率为4.76%.PCR检测显示:C84的rhl I、rhl R基因缺失.序列检测发现:C39 rhl I基因序列多个位点碱基突变,其中第184、249位碱基突变导致编码蛋白62、83位氨基酸由丝氨酸和天冬氨酸变成甘氨酸和谷氨酸;C39 rhl R基因序列多个位点碱基突变,其中第146位移码突变,147位替换突变,导致编码蛋白第48位以后氨基酸改变;C84 las R基因序列多个位点碱基突变,导致编码蛋白66、136、138和172位氨基酸分别由精氨酸、天冬酰胺、丙氨酸和丝氨酸变成赖氨酸、丝氨酸、缬氨酸和天冬酰胺;C104和C117 rhl I基因序列多个位点碱基突变,其中第184、249位碱基突变导致编码蛋白第62、83位氨基酸由丝氨酸和天冬氨酸变成甘氨酸和谷氨酸.致病因子检测显示:C39、C84、C104、C117弹性蛋白酶表达水平低于野生株(PAO1),与QS缺陷株(PAOJP2)相近;C39、C84绿脓菌素分泌明显低于PAO1,与PAOJP2相近,C104、C117绿脓菌素分泌正常;C39、C84 、C104、C117的生物被膜起始能力及泳动能力均低于PAO1.结论 QS系统在PA感染过程中发挥了非常重要的作用,干扰QS是控制PA感染非常有希望的新方法.