【作者】 陈一强；

【导师】 施焕中；

【作者基本信息】 广西医科大学 ， 内科呼吸病学， 2010， 博士

【摘要】 铜绿假单胞菌(Pseudomonas aeruginosa,P.a)是一种常见的条件致病菌,是引起免疫力低下患者的慢性感染的常见病原体,该菌对多种常见抗生素耐药,感染易反复发作,难以清除。与其慢性感染密切相关的是P.a可以形成生物膜(biofilm,BF),并能分泌多种毒力因子(virulence factors)。BF的形成和维持、毒力因子的合成释放主要由密度感应系统(quorum sensing, QS)调控。我课题组经体外实验研究证明金银花水煎液可抑制P.a的BF形成,并对已形成的P.a的BF有明显的破坏作用[58];金银花的主要活性成分为绿原酸(Chlorogenic acid, CA)。因此,本研究采用体外BF模型的方法,探讨亚抑菌浓度的CA对P.a的BF形成的抑制作用;然后研究亚抑菌浓度的CA对P.a毒力因子(藻酸盐、弹性蛋白酶、蛋白水解酶、过氧化物酶、绿脓菌素、鼠李糖脂)释放的抑制作用;进而采用质谱方法检测亚抑菌浓度的CA对P.a的QS系统信号分子的影响,探讨其干预P.a生物膜和毒力因子的分子机制;再以大鼠腹腔感染模型为基础,研究QS系统在体内BF感染中的作用,以及CA对体内P.a的BF的抑制作用。第一部分绿原酸对铜绿假单胞菌生物膜的抑制作用及其机制的体外研究第一节绿原酸对铜绿假单胞菌体外生物膜形成的抑制作用目的:观察CA对P.a体外BF形成的抑制作用。方法:(1)检测CA、红霉素(erythromycin,EM)对PAO1的最低抑菌浓度(minimal inhibitory concentration, MIC)。(2)采用体外静置BF模型,分为空白对照组、EM组和CA组。开始建模时就加入EM(1/8MIC)和CA(1/4MIC)。建模第3天和第7天,采用SEM观察载体表面BF形态、结晶紫染色法半定量载体表面BF。结果:(1)CA对P.a的MIC是3000ug/ml,EM对P.a的MIC是128ug/ml。(2)建模3天,SEM观察,空白对照组载体表面可形成早期BF,CA组和EM组载体表面形成的BF规模明显小于空白对照组;建模7天,空白对照组载体表面可见浓厚黏液样物质, BF呈立体结构,而CA组和EM组仅见薄层BF。不论是建模3天,还是建模7天,结晶紫染色法BF半定量,CA组不仅少于空白对照组(P<0.01),而且少于EM组(P<0.05)。结论:CA可抑制P.a体外BF的形成,且其抑制作用强于EM。第二节绿原酸对铜绿假单胞菌毒力因子的抑制作用目的:观察CA对P.a毒力因子的抑制作用。方法:(1)建立体外静置BF模型,分为空白对照组、EM组和CA组,加入EM(1/8MIC)和CA(1/4MIC)。(2)检测培养液藻酸盐含量;弹性蛋白-刚果红法测定菌液中弹性蛋白酶活性;偶氮酪蛋白法测定建模3、7天的菌液中蛋白水解酶活性;氯仿萃取法测定建模1、3、5、7天菌液的绿脓菌素的相对浓度;测定CA作用后游离菌及BF细菌对H2O2的敏感性;苔黑酚—浓硫酸法测定EM、CA作用后,菌液的鼠李糖脂含量。结果:(1)培养液中藻酸盐含量检测,CA组不仅少于空白对照组(P<0.01),而且少于EM组(P<0.05)。(2)与空白对照组相比,EM组、CA组的弹性蛋白酶活性明显较低(P<0.01),而CA组弹性蛋白酶活性与EM组相当(P>0.05)。(3)无论是3天,还是7天,与空白对照组相比,EM组、CA组的蛋白水解酶活性明显较低(P<0.01),但是CA组高于EM组(P<0.05)。(4)在BF形成的各个时期,菌液绿脓菌素相对浓度测定,EM组和CA组绿脓菌素相对浓度均低于空白对照组(P<0.01),CA组绿脓菌素相对浓度高于EM组(P<0.05);(5)在进入对数生长期(4-6h)前, CA组游离P.a与空白组对H2O2敏感性相当(P>0.05),进入对数生长期后,CA组铜绿假单胞菌对H2O2敏感性强于PAO1空白组(P<0.05);3天和7天的H2O2敏感性测定, EM组和CA组均高于空白对照组(P<0.05)。早期生物膜细菌CA组对H2O2敏感性高于EM组(P<0.05),而成熟期,CA组对H2O2敏感性则低于EM组(P<0.05)。(6)在作用后48h或72h, EM组及CA组的鼠李糖脂均少于空白对照组(P<0.01),作用48h时CA组鼠李糖脂含量高于EM组(P<0.05),作用72h时CA组鼠李糖脂含量与EM组相当(P>0.05)。结论:CA可以抑制P.a的多种毒力因子的释放,对蛋白水解酶活性、各时期绿脓菌素,48h鼠李糖脂及成熟期的氧化应激的抑制作用弱于EM;对弹性蛋白酶活性及72h鼠李糖脂抑制作用与EM相当;但对藻酸盐和早期生物膜细菌的氧化应激的抑制作用高于EM组。第三节绿原酸对铜绿假单胞菌QS系统信号分子的抑制作用目的:探讨CA对P.a QS系统信号分子的抑制作用。方法:(1)乙酸乙酯萃取法提取EM,CA作用后菌液的信号分子。(2)HPLC–MS鉴定PAO1产生的AHLs信号分子的种类,并检测其信号分子3-oxo-C12-HSL,C4-HSL的含量。结果:(1)PAO1产生的AHLs信号分子中3-oxo-C12-HSL和C4-HSL含量最高,且在胞外中,C4-HSL的含量高于3-oxo-C12-HSL(P<0.05)。(2)EM组和CA组的C4-HSL, 3-oxo-C12-HSL含量都明显少于空白对照组(P<0.05),CA对3-oxo-C12-HSL的抑制作用弱于EM(P<0.05), EM组和CA组中,C4-HSL含量无差别(P>0.05)。结论:PAO1产生的AHLs信号分子主要是3-oxo-C12-HSL和C4-HSL;胞外AHLs信号分子中,C4-HSL含量高于3-oxo-C12-HSL;CA可以抑制QS系统AHLs信号分子的产生,其对3-oxo-C12-HSL的抑制作用比EM弱,而对于C4-HSL,二者效应一致。第二部分绿原酸对铜绿假单胞菌生物膜的抑制作用的体内研究第一节QS系统在铜绿假单胞菌体内生物膜形成中的作用目的:构建大鼠腹腔P.a BF感染模型,初步探讨QS系统在BF形成中的作用。方法:(1)体外准备:以PVC导管为载体,铜绿假单胞菌PAO1野生株和PAO1 ?lasR rhlR缺陷株(PA-JP3)在体外培养20h,使其在黏附在载体表面。(2)构建大鼠腹腔P.a BF感染模型: 48只Wistar大鼠,随机分为PAO1组和PA-JP3组,将上述载体植入Wistar大鼠腹腔,建立P.a BF感染模型。建模后分别在第3天,7天和第14天,病理学观察腹腔局部组织炎症变化,SEM观察载体表面BF变化,连续稀释法载体表面菌落计数。结果:PAO1组腹腔局部组织炎症反应明显较PA-JP3组严重。第3天、第7天和第14天SEM观察,载体表面细菌粘附量均较PA-JP3组多,形成的BF多且稠厚;载体表面菌落计数明显高于PA-JP3组(P<0.05);与PA-JP3组相比,机体对PAO1组载体细菌清除缓慢,在第7天后有增多趋势(P<0.05)。结论:PVC材料可以在大鼠腹膜腔成功构建PAO1体内BF模型; QS系统在体内BF形成中起重要作用。第二节绿原酸对铜绿假单胞菌体内生物膜形成的抑制作用目的:探讨CA对P.a体内BF形成的抑制作用。方法:以铜绿假单胞菌PAO1野生株为实验菌株,32只大鼠随机分为对照组和CA组,建立腹腔感染模型后,腹腔注射CA溶液40mg/kg·d,每12小时一次,移植物置入后就开始腹腔注射,连续用3d、7d。对照组用等量的无菌生理盐水腹腔注射。建模后分别在第3天和第7天,病理学观察腹腔局部组织炎症变化,SEM观察载体表面BF变化,连续稀释法载体表面菌落计数。结果:CA干预后,PAO1野生株组SEM观察载体表面细菌粘附数量较对照组明显减少,BF少而且稀薄,局部组织炎症反应明显较生理盐水对照组明显减轻,第3天和第7天载体表面菌落计数均明显少于对照组(P<0.05)。结论:CA可以抑制P.a体内BF的形成。更多还原

【Abstract】 Pseudomonas aeruginosa (P.a) is a common opportunistic pathogen, often causes chronic infection in immunocompromised patients. Resistant to many kinds of common antibiotics, so P.a infections can repeat easily, and it is difficult to be removed. P.a can form and maintain biofilms (BF), synthesize and release a variety of virulence factors. The BF formation and virulence synthesis are under the regulation of P.a quorum sensing (QS) system. There is possible relationship between chronic P.a infections and its QS system. Then, inhibition of QS system may be one of the key points to control BF infections. The preliminary studies of my group show that honeysuckle aqueous extracts can inhibit BF formation by P.a in vitro and destruct the BF of P.a in vitro effectively[58]. Chlorogenic acid (CA) is the main active ingredient of honeysuckle. Therefore, in this study, the inhibitory effect of sub-inhibitory concentration of CA on P.a BF in vitro was detected firstly; then, the inhibitory effect of sub-inhibitory concentration CA on the P.a virulence factors (including alginate, elastase, protease, pyoverdine, peroxidase and rhamnolipids) was studied. To understand the mechanism of CA on P.a BF and virulence factors, the level of the AHLs signaling molecule synthesized by P.a were detected after the intervention of sub-inhibitory concentration CA. Lastly, the BF model of intraperitoneal infection in rats was constructed to explore the role of QS system and the inhibitory effect of CA on the P.a BF in vivo.PART I THE INHIBITORY EFFECT AND MECHANISM OF CHLOROGENIC ACID ON BIOFILM OF PSEUDOMONAS AERUGINOSA IN VITROSection I THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON BIOFILM FORMATION OF PSEUDOMONAS AERUGINOSA IN VITROOBJECTIVES: To observe the inhibitory effect of CA on the BF formation of P.a.METHODS: (1) Detect the minimal inhibitory concentration (MIC) of CA and erythromycin (EM) against PAO1. (2) Constructed the static BF model in vitro, and divided them into control group, EM group and CA group. EM (1/8MIC) or CA (1/4MIC) was added at the beginning of making BF models. After cultivated for 3 days and 7 days, SEM was used to observe the form of BF and crystal violet staining was used for semi-quantitative analysis of the P.a BF.RESULTS: (1) The MIC of CA to P.a is 3000ug/ml, EM is 128ug/ml.(2)After cultivated for 3 days, yong BF can be seen in the control group under SEM. The scale of the BF both in the CA group and EM group formed are smaller than the control group. After cultivated for 7 days, there is a large number of mature BF in the control group, but a few of thin BF can be seen both in the CA group and EM group. Whether it is cultivated for 3 days or 7 days, the semi-quantitative BF by crystal violet staining in CA group is less than the control group (P<0.01) and EM group (P<0.05).CONCLUSIONS: CA can inhibit P.a to form BF in vitro,and is more effective than EM.Section II THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA VIRULENCE FACTORSOBJECTIVES: To observe the inhibitory effect of CA on P.a virulence factors.METHODS: (1) Set up the static BF formation model of P.a in vitro. There are three groups: control group, EM group with 1/8 MIC EM and CA group with 1/4 MIC CA in final concentration. (2)Detect the alginate concentration in culture medium after treatment with EM or CA. The LasB elastolytic activity in the EM or CA treated bacterium was determined by using Elastin-Congo red. The protease activity in the bacterium on 3d and 7d of the static BF formation model were determined by using Azo-casein. Chloroform extraction method was used for assaying pyoverdine level in the bacterium of BF formation model in different periods. The H2O2 sensitivity of planktonic bacteria and BF bacteria were determined after EM or CA intervention. The orcinol-sulfuric acid method was used to directly assess the amount of rhamnolipids.RESULTS: The alginate contents in culture medium in the CA group is not only less than the control group (P<0.01), but also less than the EM group (P<0.05). Compared with the control group, the elastase activity of EM group and CA group were significantly lower (P <0.01), CA group were even reduced to the same as with the EM group (P>0.05). In different periods, the protease activity of EM group and CA group, while comparing with the control group, were significantly lower (P<0.01), on 3d and 7d, the protease activity of CA group was higher than that of the EM group (P <0.05). In various periods of BF formation, the pyoverdine level in culture medium were detected, the EM group and CA group were lower than that of the control group (P<0.01), compared with the EM group, the CA group is higher (P<0.05). Before the logarithmic growth phase (4-6h), the planktonic bacteria H2O2 sensitivity was not different between CA group and control group (P>0.05), but after entering the logarithmic growth phase, the bacteria of the CA group was more sensitive to H2O2 than the control group (P<0.05 ); Both young and mature BF bacteria in EM group and CA group were more sensitive to H2O2 than the control group (P<0.05), the young BF bacteria in CA group were more sensitive to H2O2 than EM group (P<0.05), while the maturity, CA group was lower than that of the EM group (P<0.05). After the intervention of EM or CA for 48h and 72, the rhamnolipids in this two group were less than the control group (P<0.01), in 48h the inhibitory effect of EM was better than CA, while in 72h, they had the same effect.CONCLUSIONS: Sub-inhibitory concentration of CA can inhibit the release of a variety of virulence factors of P.a, weak the functions of the QS system. On the inhibition of protease activity, 48h rhamnolipids, pyocyanin and the mature BF bacteria H2O2 sensitivity, CA was weaker than EM, about the elastase, 72h rhamnolipids, CA and EM had the same effect, while to alginate and the young BF bacteria H2O2 sensitivity, CA was more effective than EM. Section III THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA QS SIGNALING MOLECULESOBJECTIVES: To explore the inhibitory effect of CA on p.a QS signaling molecules.METHODS: (1)The QS signaling molecules in the bacteria intervented by EM or CA were extracted by acidified ethyl acetate. (2) AHLs produced by PAO1 were detected by High performance liquid chromatography - mass spectrometry (HPLC-MS), and the signal molecule 3-oxo-C12-HSL, C4-HSL levels were further tested.RESULTS:(1)PAO1 produced two main AHLs signaling molecules: 3-oxo-C12-HSL and C4-HSL, In addition, the content of C4-HSL is higher than in 3-oxo-C12-HSL (P<0.05) in extracellular AHLs. (2) For C4-HSL and 3-oxo-C12-HSL detected, both EM group and CA group were significantly less than the control group (P <0.05), about the 3-oxo-C12-HSL, the inhibitory effect of EM was stronger than CA (P<0.05), while the concentration of C4-HSL, EM group and the CA group had no difference (P> 0.05).CONCLUSIONS: PAO1 can produced two main AHLs signaling molecules: 3-oxo-C12-HSL and C4-HSL; about the extracellular AHLs, the content of C4-HSL is higher than 3-oxo-C12-HSL, the C4-HSL is easier to diffusion through the cell membrane to the extracellular; CA can inhibit the QS system signaling molecule AHLs production, its inhibitory effect of 3-oxo-C12-HSL was weaker than EM, but for the C4-HSL, EM and CA has the same effect. PART II THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA BIOFILM FORMATION IN VIVOSection I THE EFFECT OF QS SYSTEM OF PSEUDOMONAS AERUGINOSA ON BIOFILM FORMATION IN VIVOOBJECTIVE: Construction of peritoneal P.a infection model to explore the role of QS system played on BF formation.METHODS: (1) Preparation: The PAO1 wild strains andΔlasR rhlR deficient strains (PA-JP3) were cultivated in vitro for 20h to make them adhere to the PVC tube carriers’surface. (2) Construction of peritoneal P.a BF infection model: divide 48 wistar female rats randomly into two groups: the PAO1 group and the PA-JP3 group, then implant the carriers into the wistar rats’intra-abdominal to establish the P.a BF infection model. Histopathological observation of local peritoneal tissues inflamation, SEM observation of the BF on the carriers’surface and the viable bacteria counts on the carriers’surface were carried out on the 3rd, 7th and 14th day respectively.RESULTS: The inflammation response of the local peritoneal tissue in PAO1 wild strains group was more serious than the PA-JP3 group. On the day 3, 7 and 14, the BF formation of PAO1 on the carriers’surface were thicker than the PA-JP3 on the SEM. The viable colony count of PAO1 on the carriers’surface was significantly higher than the PA-JP3 (P<0.05), the colonies of PAO1 on the carriers’surface were cleared more slowly than the PA-JP3 group, and even rebounded on day 7(P<0.05).CONCLUSION: With the PVC tube carrier, the peritoneal PAO1 BF infection model was constructed successfully; the QS system plays an important role on the formation of BF in vivo.Section II THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA BIOFILM FORMATION IN VIVOOBJECTIVE: Explore the role of CA played on P.a BF in rat peritoneal infection.METHODS: Choosing the PAO1 wild strains as the experimental strains, 32 female rats were randomly divided into 2 groups: the control group and the CA group. The rats were given CA 40mg/kg ? d (intraperitoneal injections) for 3 or 7 days, starting just after implantation of the PVC tube carrier. The control group was injected with the same amount of sterile saline. Histopathological observation of local peritoneal tissues inflamation, SEM observation of the BF on the carriers’surface and the viable bacteria counts on the carriers’surface were carried out on the 3rd and 7th day respectively.RESULTS: After CA administration, the adhesion of PAO1 on the carriers’surface reduced more significantly than the control group, and got fewer and thinner BF by SEM. Compared with the control group, the local inflammatory reactions of the CA treatment group were significantly reduced. On day 3 and 7, the viable colony counts on the carriers’surface were significantly less than the control group (P <0.05).CONCLUSION: CA can inhibit the P.a BF formation in vivo.更多还原