【作者】 邢铭友；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2006， 博士

【摘要】 表皮葡萄球菌(Staphylococcus epidermidis,SE)是人体皮肤和粘膜上定居的正常菌群之一,通常情况下致病力很低。但随着留置静脉导管等侵袭性操作的增多以及大量广谱抗生素的使用,表皮葡萄球菌作为人类机会性致病菌成为院内感染的重要病原菌。表皮葡萄球菌感染人体后,容易在感染部位形成生物膜(Biofilm),特别是容易在高分子生物材料表面形成生物膜,如留置导管、植入材料等。生物被膜中的细菌不仅对抗生素耐药,而且抵抗机体的免疫吞噬作用,引起慢性感染甚至败血症,危害极其严重。所以,目前对于生物膜形成的机制研究非常活跃,这对于防治此类感染,寻找治疗此类感染的抗生素非常有意义。同时,研究生物膜形成的机制对于寻找抑制生物膜形成的措施从而最终消灭产膜菌株具有非常重要的实际意义。生物膜的形成是一个非常复杂的过程,有多种因子参与,一般将生物膜的形成过程分为两个阶段:粘附阶段和聚集阶段。粘附阶段是表皮葡萄球菌与生物材料(主要成分是聚乙烯)之间通过物理因素和各种粘附因子的作用,细菌与生物材料表面发生特异性结合。这种结合作用发生后就进入聚集阶段,开始只有一层细胞层,在细胞间多糖粘附素(Polysaccharide Intercellular Adhesion,PIA)等因子介导下,细菌之间相互粘附,继而分化、增殖,形成多层的细胞团块,并产生大量的粘液。最后,在生物材料表面形成厚实的粘液层,而表皮葡萄球菌多层细胞团块镶嵌在其中,这就形成了电镜下可观察到的生物膜。PIA是表皮葡萄球菌在生物膜形成过程中至关重要的物质,研究发现缺乏PIA的表皮葡萄球菌初期粘附到生物材料上的能力正常,而后期细菌间相互粘附能力却大大下降,无法形成生物膜,提示PIA在介导细菌间相互粘附中是不可缺少的。PIA合成的基础是ica基因,研究表明,ica基因位点包含一个操纵子结构icaADBC,是合成PIA所需的结构基因。其中icaA及icaD基因的联合表达是促进PIA合成最重要的环节,而icaR基因位于ica操纵子上游,对icaA/icaD的表达有抑制作用,可以减少PIA的合成,从而抑制生物膜的形成。鉴于目前国内对表皮葡萄球菌合成PIA的分子机制研究较少,本研究拟在研究表皮葡萄球菌耐药情况的基础上初步探讨PIA合成的分子机制,明确PIA合成与icaA、icaD、icaR表达之间的关系。目的:研究我院最近两年来各临床科室住院患者所送检的各类标本培养的61株表皮葡萄球菌的耐药情况,分析耐药趋势。检测各菌株PIA的合成情况,扩增icaA、icaD、icaR基因,探讨PIA合成与icaA、icaD、icaR基因之间的关系。方法:1.对61株细菌复苏、培养,进行常用抗生素的药物敏感性试验。2.对药敏结果进行耐药统计,分析耐药趋势。3.利用刚果红琼脂培养基检测各菌株的PIA合成情况。4.扩增icaA、icaD、icaR基因,探讨与PIA合成之间的关系。5.检测icaA、icaR基因的mRNA表达,探讨icaR与PIA合成的关系。结果:1.61株临床分离菌全部复苏、培养成功,在61株表皮葡萄球菌中,41株对苯唑西林耐药(占67.21%),20株对苯唑西林敏感(占32.79%)。61株表葡中对三种以上抗生素(含苯唑西林)耐药的菌株达90.16%(55/61),其中甲氧西林耐药的表皮葡萄球菌(MRSE)的多重耐药率达到100.00%(41/41),甲氧西林敏感的表皮葡萄球菌(MSSE)的多重耐药率为70.00%(14/20),两组的多重耐药率比较有极显著性的差异(x2=10.47,P<0.005)。61株表皮葡萄球菌对全部13种抗生素的总耐药率为40.86%(324/793),其中MRSE的总耐药率为47.28%(252/533),MSSE的总耐药率为27.69%(72/260),两组的总耐药率比较有极显著性的差异(x2=27.75,P<0.005)。2.表皮葡萄球菌在体外药敏试验中,对头孢唑啉、头孢呋新、庆大霉素及氨苄-舒巴坦保持较低的耐药率(<30%),而对SMZ-TMP、青霉素、红霉素、克林霉素及左氧沙星耐药率较高(>50%)。在本研究中,没有发现对万古霉素耐药的表葡菌株。3.对61株表皮葡萄球菌的细胞间多糖粘附素(PIA)检测结果显示,总共有22株表葡表现为PIA阳性,占全部菌株的36.07%(22/61),其中MRSE中有19株,占46.34%(19/41),MSSE中仅有3株,占15.00%(3/20),两组比较有显著差异(x2=4.448,P<0.05)。4.对61株表皮葡萄球菌DNA的提取和ica基因的扩增,共检测出icaA阳性菌株25株,其中76.00%(19/25)为产PIA株,而icaA阴性菌株中仅有8.33%(3/36)为产PIA株,两者比较差异极其显著(x2=26.44,P<0.005)。icaD的检测结果与icaA完全吻合。5.通过对61株表皮葡萄球菌的icaA及icaR基因mRNA表达的研究发现,20株产PIA菌株的icaR基因在mRNA水平上不再表达,而icaA基因在此水平上表达正常;同时,不产PIA而icaA基因阳性的菌株在mRNA水平上不再表达icaA基因,而icaR基因表达正常。结论:1.表皮葡萄球菌耐药严重,MRSE的多重耐药率、总耐药率较高;2.icaA/icaD基因的联合表达是PIA合成的关键环节;3.icaR基因的表达抑制PIA的合成。更多还原

【Abstract】 Staphylococcus epidermidis （SE） is part of the normal mucosa and skin microflora, usually showing low pathogenicity. In recent years, however, S. epidermidis, being as an opportunistic organism, become an important etiologic agent of infections associated with venous catheters, other indwelling medical devices and abuse of broad-spectrum antibiotics.After infection in the host, S. epidermidis is liable to form biofilm in the involved sites, especially on the surface of high-molecular biomaterials, such as indwelling catheters, implanted materials and etc. The bacteria in the biofilm can not only be resistant to antibiotics, but also resistant to immunologic phagocytosis of the host. Therefore, they can produce chronic infection and even septicemia, causing critical results. Just because of that, in nowadays, the mechanism of biofilm formation becomes an actively ongoing study. It is of great significance for preventing such infections as well as looking for sensitive antibiotics against such infections. At the same time, the study about mechanism of biofilm formation may actually help to inhibit biofilm fonnation and then ultimately eliminate slime-producing bacteria.Biofilm fonnation is a very complex and multistep process. Usually it is divided into two phages: adherence phage and collection phage. In adherence phage, S. epidermidis specifically combine to the surface of biomaterials owing to some physical factors and various adherence factors. After the adherence, it is collection phage. Firstly there is only one cellular layer, and then, mediated by polysaccharide intercellular adhesion （PIA） and other factors, bacteria adhere to each other, followed by differentiation and proliferation, and hence produce multilayer cellular mass as well as abundant slime. At last, on the surface of biomaterials, a thick slime layer comes into being, in which S. epidermidis studded, forming biofilm that can be observed by electron microscopy.PIA is an extremely important substance during the process of biofilm formation. It is investigated that S. epidermidis lack of PIA shows normal ability of adherence to the biomaterials in early stage, and remarked decrease of adherence ability among bacteria in late stage, causing inability to form biofilm. It suggests that PIA is indispensable in mediation of intercellular adherence.ica gene is the basis of PIA synthesis. Studies indicate that ica gene comprises operon icaA, icaD, icaB, icaC, which are necessary structural genes for PIA production. Among those, co-expression of icaA and icaD is the most important link to promote PIA production. icaR gene locates in the upper stream of ica operon, being able to inhibit icaA/icaD. Therefore it can decrease PIA production, and consequently inhibit biofilm formation. Since it is poorly studied about the molecular mechanism of PIA synthesis in S. epidermidis, the aim of this study was to primarily investigate the molecular mechanism of PIA synthesis on the basis of analyzing the status of drug resistance of S. epidermidis, and to elaborate the relationship between PIA synthesis and expression of icaA, icaD, and icaR.Objective: to study the situation of drug resistance of 61 S. epidermidis stains collected from various specimens of different patients in all clinical departments of our hospital in the recent two years, and to analyze the tendency of its drug resistance. To detect the status of PIA syntheses and Amplifying icaA, icaD, icaR genes in various strains and search the relationship between PIA production and icaA, icaD and icaR genes.Methods: 1. After resuscitation and culture of 61 SE strains, drug sensitivity test for common antibiotics was performed.2. In order to analyze the tendency of drug resistance, statistics of drug resistance was made on the basis of the results of drug sensitivity test.3. The situation of PIA synthesis of various strains was detected in Congo red agar medium.4. Amplifying icaA, icaD, icaR genes, and then investigating the relationship between them and PIA synthesis. 5. Detecting mRNA expression of icaA and icaR genes, and investigating the relationship between icaR and PIA synthesis.Results: 1. All the 61 SE strains were completely resuscitated and cultured. Among them, 41 strains were resistant to Oxacillin （67.21%）, 20 strains sensitive to Oxacillin. The strains presenting drug resistance to over 3 kinds of antibiotics accounted for 90.16% （including Oxacillin） in all the specimens （55/61 ）₅ with multi-drug-resistance rate up to 100% （41/41） in MRSE and 70.00% （14/20） in MSSE. There was significant difference between the two groups in multi-drug-resistance rate （x² —10.47, P<0.005）. The 61 SE strains showed total drug resistance to all the 13 antibiotics at 40.86% （324/793）, with total drug resistance at 47.28 （252/533） for MRSE and 27.69% （72/260） for MSSE. It showed extremely significant difference in total drug resistance between the two groups （x² —27.75, P<0.005）.2. The drug sensitive test in vitro showed that SE preserved a relatively lower drug resistance to cefazolin, cefuroxime, gentamicin and ampicillin-sulbactam （<30%）, and a higher drug resistance to SMZ-TMP, penicillin, erythromycin, clindamycin and levofloxacin （>50%）. In this study, we did not find any SE strain resistant to vancomycin.3. The results of detecting PIA in the 61 SE strains showed that 22 strains were PIA positive, accounting for 36.07% （22/61）. Among them, there were 19 strains of MRSE, accounting for 46.34% （19/41）, while only 3 strains of MSSE, accounting for 15.00% （3/20）. There was significant difference between the two groups （X² =4.448, P<0.05）.4. After extraction of DNA and amplification of ica genes, 25 SE strains turned out icaA positive, among which PIA-producing strains accounted for 76.00% （19/25）, while there were only 8.33% （3/36） of icaA-negative strains being able to produce PIA. The two groups had extremely significant difference （x² =26.44, P<0.005）. The results of icaD detection was completely consistent with that of icaA.5. According to the investigation of mRNA expression of icaA and icaR genes in 61 SE strains, we found that icaR gene no longer expressed mRNA in 20 PIA-positive strains, but icaA remained normal expression of mRNA; meanwhile, in PIA-negative strains, icaA gene did not express at the level of mRNA, while icaR gene remained normal.Conclusions: 1. S. epidermidis showed a serious situation about drug resistance, with a higher multi-drug-resistance rate and total drug resistance rate in MRSE,2. Co-expression of icaA/icaD genes is the key link of PIA synthesis.3. The expression of icaR gene may inhibit the synthesis of PIA.更多还原