發(fā)布時間：2018-04-18 10:20

  本文選題：表皮葡萄球菌 + 耐藥研究�。� 參考：《華中科技大學》2006年博士論文

【摘要】： 表皮葡萄球菌(Staphylococcus epidermidis,SE)是人體皮膚和粘膜上定居的正常菌群之一,通常情況下致病力很低。但隨著留置靜脈導管等侵襲性操作的增多以及大量廣譜抗生素的使用,表皮葡萄球菌作為人類機會性致病菌成為院內感染的重要病原菌。 表皮葡萄球菌感染人體后,容易在感染部位形成生物膜(Biofilm),特別是容易在高分子生物材料表面形成生物膜,如留置導管、植入材料等。生物被膜中的細菌不僅對抗生素耐藥,而且抵抗機體的免疫吞噬作用,引起慢性感染甚至敗血癥,危害極其嚴重。所以,目前對于生物膜形成的機制研究非�；钴S,這對于防治此類感染,尋找治療此類感染的抗生素非常有意義。同時,研究生物膜形成的機制對于尋找抑制生物膜形成的措施從而最終消滅產膜菌株具有非常重要的實際意義。 生物膜的形成是一個非常復雜的過程,有多種因子參與,一般將生物膜的形成過程分為兩個階段:粘附階段和聚集階段。粘附階段是表皮葡萄球菌與生物材料(主要成分是聚乙烯)之間通過物理因素和各種粘附因子的作用,細菌與生物材料表面發(fā)生特異性結合。這種結合作用發(fā)生后就進入聚集階段,開始只有一層細胞層,在細胞間多糖粘附素(Polysaccharide Intercellular Adhesion,PIA)等因子介導下,細菌之間相互粘附,繼而分化、增殖,形成多層的細胞團塊,并產生大量的粘液。最后,在生物材料表面形成厚實的粘液層,而表皮葡萄球菌多層細胞團塊鑲嵌在其中,這就形成了電鏡下可觀察到的生物膜。 PIA是表皮葡萄球菌在生物膜形成過程中至關重要的物質,研究發(fā)現(xiàn)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,而后期細菌間相互粘附能力卻大大下降,無法形成生物膜,提示PIA在介導細菌間相互粘附中是不可缺少的。 PIA合成的基礎是ica基因,研究表明,ica基因位點包含一個操縱子結構icaADBC,是合成PIA所需的結構基因。其中icaA及icaD基因的聯(lián)合表達是促進PIA合成最重要的環(huán)節(jié),而icaR基因位于ica操縱子上游,對icaA/icaD的表達有抑制作用,可以減少PIA的合成,從而抑制生物膜的形成。鑒于目前國內對表皮葡萄球菌合成PIA的分子機制研究較少,本研究擬在研究表皮葡萄球菌耐藥情況的基礎上初步探討PIA合成的分子機制,明確PIA合成與icaA、icaD、icaR表達之間的關系。 目的:研究我院最近兩年來各臨床科室住院患者所送檢的各類標本培養(yǎng)的61株表皮葡萄球菌的耐藥情況,分析耐藥趨勢。檢測各菌株PIA的合成情況,擴增icaA、icaD、icaR基因,探討PIA合成與icaA、icaD、icaR基因之間的關系。 方法:1.對61株細菌復蘇、培養(yǎng),進行常用抗生素的藥物敏感性試驗。 2.對藥敏結果進行耐藥統(tǒng)計,分析耐藥趨勢。 3.利用剛果紅瓊脂培養(yǎng)基檢測各菌株的PIA合成情況。 4.擴增icaA、icaD、icaR基因,探討與PIA合成之間的關系。 5.檢測icaA、icaR基因的mRNA表達,探討icaR與PIA合成的關系。 結果:1.61株臨床分離菌全部復蘇、培養(yǎng)成功,在61株表皮葡萄球菌中,41株對苯唑西林耐藥(占67.21%),20株對苯唑西林敏感(占32.79%)。61株表葡中對三種以上抗生素(含苯唑西林)耐藥的菌株達90.16%(55/61),其中甲氧西林耐藥的表皮葡萄球菌(MRSE)的多重耐藥率達到100.00%(41/41),甲氧西林敏感的表皮葡萄球菌(MSSE)的多重耐藥率為70.00%(14/20),兩組的多重耐藥率比較有極顯著性的差異(x2=10.47,P0.005)。61株表皮葡萄球菌對全部13種抗生素的總耐藥率為40.86%(324/793),其中MRSE的總耐藥率為47.28%(252/533),MSSE的總耐藥率為27.69%(72/260),兩組的總耐藥率比較有極顯著性的差異(x2=27.75,P0.005)。 2.表皮葡萄球菌在體外藥敏試驗中,對頭孢唑啉、頭孢呋新、慶大霉素及氨芐-舒巴坦保持較低的耐藥率(30%),而對SMZ-TMP、青霉素、紅霉素、克林霉素及左氧沙星耐藥率較高(50%)。在本研究中,沒有發(fā)現(xiàn)對萬古霉素耐藥的表葡菌株。 3.對61株表皮葡萄球菌的細胞間多糖粘附素(PIA)檢測結果顯示,總共有22株表葡表現(xiàn)為PIA陽性,占全部菌株的36.07%(22/61),其中MRSE中有19株,占46.34%(19/41),MSSE中僅有3株,占15.00%(3/20),兩組比較有顯著差異(x2=4.448,P0.05)。 4.對61株表皮葡萄球菌DNA的提取和ica基因的擴增,共檢測出icaA陽性菌株25株,其中76.00%(19/25)為產PIA株,而icaA陰性菌株中僅有8.33%(3/36)為產PIA株,兩者比較差異極其顯著(x2=26.44,P0.005)。icaD的檢測結果與icaA完全吻合。 5.通過對61株表皮葡萄球菌的icaA及icaR基因mRNA表達的研究發(fā)現(xiàn),20株產PIA菌株的icaR基因在mRNA水平上不再表達,而icaA基因在此水平上表達正常;同時,不產PIA而icaA基因陽性的菌株在mRNA水平上不再表達icaA基因,而icaR基因表達正常。 結論:1.表皮葡萄球菌耐藥嚴重,MRSE的多重耐藥率、總耐藥率較高; 2.icaA/icaD基因的聯(lián)合表達是PIA合成的關鍵環(huán)節(jié); 3.icaR基因的表達抑制PIA的合成。
[Abstract]:Staphylococcus aureus ( SE ) is one of the normal flora settled on human skin and mucosa . Generally , the pathogenic force is very low . However , with the increase of invasive operation such as indwelling venous catheter and the use of a large number of broad - spectrum antibiotics , Staphylococcus epidermis is an important pathogenic bacterium in nosocomial infection as human opportunistic pathogen .









The mechanism of biofilm formation is very active not only to antibiotics , but also to the immune phagocytosis of organisms . Therefore , the mechanism of biofilm formation is very active . At the same time , it is very important to study the mechanism of biofilm formation to find the measures to inhibit biofilm formation and finally eliminate the membrane - producing strain .









The formation of biofilm is a very complex process . There are many factors involved . Generally , the formation process of biofilm is divided into two stages : adhesion stage and aggregation stage .









As a matter of vital importance in the process of biofilm formation , it was found that the ability to adhere to the biological material was normal at the initial stage of biofilm formation . However , the ability to adhere to each other in the late stage was greatly decreased , and the biofilm could not be formed . It was suggested that it was indispensable to mediate inter - bacteria adhesion .









On the basis of ica gene , it is shown that ica gene locus contains one operon structure ica ADBC , which is the structural gene necessary for the synthesis .









Objective : To study the drug resistance of 61 S . S . S . S . S . S . S . S . in all kinds of specimens collected from various clinical departments in our hospital for the last two years , and to analyze the drug resistance .









Methods : 1 . 61 strains of bacteria were recovered and cultured , and the susceptibility test of antibiotics was carried out .









2 . The drug - sensitive results were analyzed and the drug - resistant trends were analyzed .









3 . Using the Congo red agar culture medium to test the synthesis of each strain .









4 . To amplify the gene of ica A , ica D , ica R , and to explore the relationship between the synthesis and the synthesis .









5 . To detect the mRNA expression of ica A and ica R genes .









Results : 1 . 61 strains of clinical isolates recovered successfully . Among 61 strains of S . S . aureus , 41 strains were resistant to oxacillin ( 67.21 % ) , 20 strains were sensitive to benazosin ( 32.79 % ) . The multiple drug - resistant rates of staphylococcus aureus ( MRSE ) in 61 strains reached 100 . 00 % ( 41 / 41 ) . The total drug resistance rate was 47.28 % ( 252 / 533 ) . The total drug resistance of MSSE was 27.69 % ( 72 / 260 ) .









2 . In vitro susceptibility test , the drug resistance rate ( 30 % ) was lower for Cefazolin , ceftiofur , gentamicin and ampicillin , while the resistance to SMZ - TMP , penicillin , erythromycin , klincomycin and levofloxacin was high ( 50 % ) . In this study , it was not found that vancomycin - resistant strains were resistant to vancomycin .









3 . The results showed that there were 22 strains in total 22 strains , which accounted for 36.07 % of all strains ( 22 / 61 ) , among them 19 in MRSE , 46.34 % ( 19 / 41 ) , only 3 in MSSE and 15 . 00 % ( 3 / 20 ) , there was significant difference between the two groups ( x2 = 4.448 , P0.05 ) .









4 . The extraction of 61 strains of S . S . S . DNA and the amplification of ica gene were detected . Among them , 25 strains were detected , of which 76.00 % ( 19 / 25 ) was the yield - producing strain , and only 8.33 % ( 3 / 36 ) in the negative strains of ica A was the yield - producing strain , the difference was extremely significant ( x2 = 26.44 , P0.05 ) .









5 . Through the study of the mRNA expression of ica A and ica R genes of 61 strains of S . S . S . , it was found that the expression of ica R gene of 20 strains was no longer expressed at mRNA level , and the expression of the gene was normal at this level . At the same time , it was not produced , and the positive strain of ica A gene was no longer expressed at mRNA level , and the expression of ica R gene was normal .









Conclusion : 1 . The resistance to staphylococcus aureus is serious , the multiple drug resistance rate of MRSE is high , and the total drug resistance rate is high ;









2 . The joint expression of the two genes is a key link in the synthesis of the two genes .









3 . The expression of the R gene inhibited the synthesis of PIAs .

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R378

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