【作者】 崔冬清；

【导师】 孙铁英； 黎健； 方保民； 柯会星；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 背景:铜绿假单胞菌可以引起各种急慢性感染,并且由该菌引起的院内获得性感染的发病率远高于社区获得性感染,其原因目前并未完全明确。在众多学说中,目前认为产生生物被膜是铜绿假单胞菌耐药和躲避人体免疫系统攻击的重要手段之一。藻酸盐是生物被膜的重要组成成分,在生物被膜病的发病机制中有重要作用。氧疗是临床上常用的治疗方法,目前还没有关于高氧如何影响铜绿假单胞菌产生生物被膜的报道。密度感应系统是细菌协调群体生物学效应的一种有效手段,对于细菌毒力、耐药性等方面有重要影响,但是否在生成生物被膜这种群体行为中起调控作用目前也争议颇多。Ⅲ型分泌系统是铜绿假单胞菌主要的毒力因素之一,在急性感染中发挥重要作用,对于Ⅲ型分泌系统和生物被膜形成这两种生物学行为的关系学术界也有一些分歧。目的:观察氧环境变化对铜绿假单胞菌产生生物被膜及藻酸盐的影响,并探讨密度感应系统中Las系统和Rhl系统在生物被膜调控中的作用、以及Ⅲ型分泌系统表达和生物被膜形成的相互关系。方法:1.随机扩增DNA多态性基因分型法对23株铜绿假单胞菌临床株进行基因型鉴定,以区别是否有来源于同一克隆的菌株。2.23株基因型不同的铜绿假单胞菌临床株分别在无氧(0%氧浓度)、低氧(10%)、正常氧(20%)、高氧(30%、40%、50%、60%)七个不同的氧浓度下连续培养3天。用结晶紫染色法检测生物被膜产生量;对-羟基联苯法测定藻酸盐产生量。3.硝酸银染色法观察标准菌株ATCC27853在不同氧浓度环境下产胞外多糖的时间窗。4.观察ATCC27853在不同氧浓度环境下生长曲线的差异。5.采用实时定量PCR测定23株临床菌株在不同氧浓度环境下密度感应系统中信号分子合成酶调控基因LasⅠ和RhlⅠ的表达。6.Western blot方法观察23株临床菌株在不同氧浓度环境下分泌Ⅲ型分泌系统的代表毒素——胞外酶ExoS的情况。结果:1.基因分型显示23株菌株均能产生指纹图谱,分型率为100%。根据所得DNA片段数目及大小对铜绿假单胞菌菌株进行相似度比较,当使用随机引物A时,菌株1、3、6、20、22的DNA扩增条带相似,菌株4和23、8和21分别相似;其余菌株条带均不相同;再采用引物B对菌株1、3、4、6、8、20、21、22、23 DNA进行随机扩增,结果显示扩增条带均不相同。2.检测23株临床株分别在七种氧浓度环境下培养3天的生物被膜产生量,结果表明在氧浓度为50%时生物被膜产生量达到最高值(4.05 OD/mg/ml),趋势性分析表明随着氧浓度的升高生物被膜产生量逐渐上升,相关性分析显示生物被膜产生量与氧浓度成正相关,R为0.455,P为0.000。3.测定23株临床株分别在七种氧浓度环境下培养3天的藻酸盐生成量,结果表明藻酸盐产生量是在氧浓度为60%的条件下达到最高值(85.22 ug/mg蛋白),趋势性分析表明随着氧浓度的升高藻酸盐产生量逐渐上升,相关性分析显示藻酸盐产生量与氧浓度成正相关,R为0.367,P为0.000。4.银染法观察ATCC27853产生胞外多糖的时间窗,结果显示在氧浓度分别为40%、50%、60%时,菌株培养一小时即可出现胞外多糖;氧浓度分别为20%、30%时,菌株需培养2小时以上才出现胞外多糖;氧浓度为10%时,胞外多糖最早出现时间为4小时;无氧条件下,菌株培养8小时出现胞外多糖。5.ATCC27853生长曲线结果显示当氧浓度分别为0%、10%、20%时,菌株培养3小时开始由延迟期进入对数生长期;氧浓度为30%时,培养2小时进入对数生长期;氧浓度为40%、50%、60%时,菌株培养1小时即进入对数生长期。6.实时定量PCR结果显示相关性分析未见LasⅠ、RhlⅠ的表达和氧浓度(R分别为0.025和-0.044,P大于0.05)、对应菌株生物被膜产生量(R分别为0.001和0.11,P大于0.05)、藻酸盐产生量(R分别为0.029和0.193,P大于0.05)有统计学意义。7.Western blot结果表明菌株1、2、3、4、5、7、8、9、10、15、16、19、23共13株菌在氧浓度为20%条件下分泌胞外酶ExoS;当氧浓度为10%时,只有菌株15、16能够分泌ExoS;当氧浓度为30%时,菌株1、2、4、5、9、10、15能够分泌ExoS;氧浓度为0%、40%、50%、60%的条件下均未见菌株分泌ExoS。结论:1.铜绿假单胞菌在高氧环境下更容易产生生物被膜,且随着环境氧浓度的升高,生物被膜和藻酸盐产生量增加,表明高氧促进了铜绿假单胞菌产生生物被膜的能力,从而有利于铜绿假单胞菌在人体内的耐药和寄植,不利于肌体对病原菌的清除和抗生素杀菌作用的发挥,所以针对有铜绿假单胞菌感染高危因素的患者应该避免过高浓度的氧疗。2.在不同氧浓度环境下虽然铜绿假单胞菌产生生物被膜的能力有显著差异,但是生物被膜形成的早期密度感应系统中Las系统和Rhl系统关键基因表达无明显趋势性变化,因此Las系统和Rhl系统可能在生物被膜形成早期阶段因为菌群数量处于低水平而未参与生物被膜的调控过程。3.随着氧浓度的升高铜绿假单胞菌产生生物被膜增多,但是Ⅲ型分泌系统的代表毒素胞外酶ExoS分泌下调,表明铜绿假单胞菌生物被膜产生增多的同时Ⅲ型分泌系统的表达下调,侵袭力下降。更多还原

【Abstract】 Backgroud:The morbidity of hospital acquired infection of Pseudomonas aeruginosa is high.But the real cause is unknown.Producing biofilm barrier is an important way for P.aeruginosa to develop resistance and elude immune attack. Being the important component of biofilm,alginate play a significant role in bacterial biofilm related infection.Oxygen therapy is a usual medical management in hospital,which can offer hyperoxic situation in respiratory tract.Oxygen situation would influence the production of biofilm.Now there are not any reports about the relationship between hyperoxia and biofilm production of P.aeruginosa.On the other hand,Quorum Sensing System is a generic regulatory mechanism employed by P.aeruginosa,which consists of two separate but interrelated system,Las and Rhl.TypeⅢsecretion system is a kind of bacterial device for close combat with cells of their eukaryotic host,which allow bacteria to inject bacterial proteins across the eukaryotic cell membrane to destroy or subvert the target cell.But at present the scholars have not agreed with whether Quorum Sensing System and TypeⅢsecretion system play a part in the process of biofilm production.Objective:The aims of this study were to investigate the relationship between oxygen concentration and biofilm production of P.aeruginosa,and whether Las/Rhl system take effects on biofilm.Furthermore,the relationship between TypeⅢsecretion system and biofilm was explored.Methods:1.The genotypes of 23 clinical strains of P.aeruginosa were identified by random amplified polymorphic DNA assay.2.23 genetically different clinical strains of P.aeruginosa were cultured at different levels of environmental oxygen respectively.Then biofilm mass was quantified by crystal violet dye,and alginate by meta-hydroxydiphenyl reaction.3.Silver nitrate staining to determine the time window of extrapolysaccharide production under different levels of environmental oxygen.4.Growth curves of ATCC27853 under different levels of environmental oxygen were made.5.The expression level of LasI and RhlI was detected by real-time polymerase chain reaction.6.The secretion of exoenzyme S was examined by western blotting assay.Results:1.The genotype of 23 clinical strains of P.aeruginosa were assessed to be different by two random primers.2.For quantifying the amount of biofilm,the highest amount of biofilm (4.05 OD/mg/ml) was produced when the oxygen concentration was at 50%.There was positive correlation between the amount of biofim production and oxygen concentration(R=0.455,P=0.000).3.For quantifying the amount of alginate,the highest amount of alginate (85.22 ug/mg protein) was produced when the oxygen concentration was 60%. Linear correlation analysis indicated that a significant positive correlation existed between the oxygen concentration and alginate production(r=0.367,P=0.000).4.The production of extrapolysaccharide by ATCC27853 was not visualized until culture for 1 hour at＞40%environmental oxygen concentration,2 hours for 20%and 30%oxygen concentrations,4 hours for 10%oxygen concentration, and 8 hours for anaerobic conditions.5.The results of growth curve of ATCC27853 showed that when the concentration of oxygen was 0%,10%,or 20%,the bacteria culture required＞3 hours to shift from the lag period to the logarithmic growth phase,2 hours for 30%oxygen concentration,and 1 hour for＞40%oxygen concentration.6.The results of real-time polymerase chain reaction did not suggest any correlation between the expression level of LasI and oxygen concentration,or the production of biofilm and alginate.It’ s the same for RhlI.7.The detection of exoenzyme S showed the optimal oxygen concentration range for exoenzyme S secretion of P.aeruginosa was from 10%to 30%.Conclusion:1.Hyperoxia promoted the ability of producing biofilm and alginate of P.aeruginosa,which is in favor of resistance and colonization.To avoid and manage P.aeruginosa infection should obviate unnecessary hyperoxia therapy.2.Las/Rhl System may not take effects on biofilm production at earlier period,maybe for low level of bacteria amount.3.The results of exoenzyme S showed that although hyperoxia promoted the ability of producing biofilm of P.aeruginosa,TypeⅢsecretion system was down regulated,which may mean that the expression of TypeⅢsecretion system was repressed by the overproduction of biofilm.更多还原