志賀菌膜孔蛋白OmpF及調(diào)控基因micF介導(dǎo)多重耐藥機制的研究
發(fā)布時間：2018-08-20 15:43
【摘要】：細菌性痢疾又稱志賀菌病,是我國最常見的感染性腹瀉之一,目前居于我國法定傳染病發(fā)病率的第三位。志賀菌具有高度傳染性、人群普遍易感、危害性嚴(yán)重的特點,能夠通過消化道傳播的腸道傳染病。近年來,由于抗生素的廣泛使用,志賀菌耐藥現(xiàn)象越來越多,且出現(xiàn)多重耐藥趨勢。外膜膜孔蛋白介導(dǎo)的細胞通透性改變是引起細菌多重耐藥的重要機制之一。某些抗生素首先要經(jīng)膜孔蛋白進入細胞才能發(fā)揮殺菌作用,所以外膜蛋白的結(jié)構(gòu)及調(diào)控膜孔蛋白表達因素的改變都會引起耐藥性的變化。研究目的了解micF和ompF基因在轉(zhuǎn)錄水平的相對表達量,探討外膜孔蛋白OmpF與耐藥性的關(guān)系,micF對ompF表達的調(diào)控作用,以及micF是否調(diào)控志賀菌的細胞膜耐藥。材料與方法1.收集2015年5月-10月天津醫(yī)科大學(xué)第二醫(yī)院腸道門診腹瀉患者糞便標(biāo)本的志賀菌臨床株13株,及天津醫(yī)科大學(xué)第二醫(yī)院感染性疾病研究所保存的部分志賀菌8株,共計21株,采用常規(guī)生化鑒定和血清凝集法確定血清型。2.選取環(huán)丙沙星、頭孢曲松、氨芐西林等6類常用抗生素和K-B紙片擴散法進行藥敏試驗,篩選出多重耐藥菌株和非多重耐藥菌株。3.PCR擴增兩組外膜孔蛋白ompF基因,檢測有無ompF基因缺失株。4.提取志賀菌總RNA,采用實時熒光定量RT-PCR技術(shù)檢測兩組菌株micF和ompF基因的mRNA轉(zhuǎn)錄情況,并使用SPSS20.0軟件對結(jié)果進行統(tǒng)計分析。5.制定環(huán)丙沙星標(biāo)準(zhǔn)濃度曲線,采用多功能酶標(biāo)儀測定兩組菌株胞內(nèi)環(huán)丙沙星的濃度。實驗結(jié)果1.經(jīng)常規(guī)生化鑒定和血清凝集實驗證實,所收集的21株菌株均為志賀菌,其中福氏志賀菌8株,鮑氏志賀菌6株,宋內(nèi)志賀菌6株,痢疾志賀菌1株。2.藥敏結(jié)果顯示:13株志賀菌對3類或3類以上抗生素耐藥,將其歸為多重耐藥組,8株僅對1類、2類抗生素耐藥或?qū)θ靠股鼐舾?將其歸為非多重耐藥組。3.21株志賀菌ompF基因PCR擴增結(jié)果均為陽性,未發(fā)現(xiàn)ompF基因缺失株。4.多重耐藥組micF基因相對表達水平明顯高于非多重耐藥組,且差異有統(tǒng)計學(xué)意義(P0.05)。多重耐藥組ompF基因相對表達水平明顯低于非多重耐藥組,且差異有統(tǒng)計學(xué)意義(P0.05)。對兩者進行相關(guān)性分析,相關(guān)系數(shù)r=-0.244,提示micF和ompF之間呈負相關(guān),但關(guān)聯(lián)程度不高。5.多重耐藥組胞內(nèi)環(huán)丙沙星濃度低于非多重耐藥組,且差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1.多重耐藥志賀菌ompF基因相對表達水平明顯低于非多重耐藥志賀菌,膜孔蛋白OmpF減少可能是志賀菌引起多重耐藥的重要機制之一。2.micF基因可能對膜孔蛋白OmpF的表達起負調(diào)控作用。3.多重耐藥菌胞內(nèi)環(huán)丙沙星濃度降低,可能與外膜孔蛋白的表達減少有一定關(guān)系。
[Abstract]:Bacillary dysentery, also known as Shigella, is one of the most common infectious diarrhea in China. Shigella has the characteristics of high infectivity, general susceptibility to the population and serious harmfulness, which can be transmitted through the digestive tract. In recent years, due to the widespread use of antibiotics, Shigella drug resistance phenomenon more and more, and the trend of multidrug resistance. The change of cell permeability mediated by adventitia porin is one of the important mechanisms of multidrug resistance of bacteria. Some antibiotics must enter the cells through the membrane pore protein to play a bactericidal effect, so the change of the structure of outer membrane protein and the regulation of membrane pore protein expression factors will cause the change of drug resistance. Objective to investigate the relative expression of micF and ompF genes at the transcriptional level, and to investigate the relationship between the expression of ompF and the relationship between OmpF and drug resistance, and whether micF regulates the cell membrane resistance of Shigella. Materials and methods 1. From May to October 2015, 13 clinical strains of Shigella and 8 strains of Shigella from the Institute of Infectious Diseases, second Hospital of Tianjin Medical University, were collected from fecal specimens of diarrhea patients in the second Hospital of Tianjin Medical University. Routine biochemical identification and serum agglutination method were used to determine the serotype. 2. Six common antibiotics, such as ciprofloxacin, ceftriaxone, ampicillin, and K-B disk diffusion test were used to screen out the multidrug resistant strains and non-multidrug resistant strains. 3. PCR amplification of two groups of outer membrane pore protein ompF gene. Detection of ompF gene deletion strain. 4. The total RNAs of Shigella were extracted and the mRNA transcription of micF and ompF genes in two groups of strains were detected by real-time fluorescence quantitative RT-PCR. The results were statistically analyzed by SPSS20.0 software. The standard concentration curve of ciprofloxacin was established. Experimental results 1. The collected 21 strains were Shigella flexneri 8 strains, Shigella baumannii 6, Shigella sonnei 6 strains and Shigella dysenteriae 1 strain. The results of antimicrobial susceptibility showed that 13 strains of Shigella spp were resistant to 3 or more kinds of antibiotics, which were classified as multidrug resistant group. 8 strains were only resistant to class 1 or class 2 antibiotics or were sensitive to all antibiotics. The results of PCR amplification of ompF gene of Shigella spp. 3.21 strains were all positive, and no ompF gene deletion strain. 4 was found. The relative expression level of micF gene in multidrug resistance group was significantly higher than that in non multidrug resistance group (P0.05). The relative expression level of ompF gene in multidrug resistance group was significantly lower than that in non multidrug resistance group (P0.05). The correlation coefficient between micF and ompF was negative, but the correlation degree was not high (r = -0.244), which indicated that there was a negative correlation between micF and ompF, but the correlation degree was not high. The concentration of ciprofloxacin in multidrug resistant group was lower than that in non-multidrug resistant group, and the difference was statistically significant (P0.05). Conclusion 1. The relative expression level of ompF gene in multidrug resistant Shigella was significantly lower than that in non-multidrug resistant Shigella. The decrease of membrane porin OmpF may be one of the important mechanisms of multidrug resistance induced by Shigella. 2.MicF gene may play a negative role in regulating the expression of OmpF. The decrease of intracellular ciprofloxacin concentration in multidrug resistant bacteria may be related to the decreased expression of adventitia pore protein.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R516.4
本文編號：2194156
[Abstract]:Bacillary dysentery, also known as Shigella, is one of the most common infectious diarrhea in China. Shigella has the characteristics of high infectivity, general susceptibility to the population and serious harmfulness, which can be transmitted through the digestive tract. In recent years, due to the widespread use of antibiotics, Shigella drug resistance phenomenon more and more, and the trend of multidrug resistance. The change of cell permeability mediated by adventitia porin is one of the important mechanisms of multidrug resistance of bacteria. Some antibiotics must enter the cells through the membrane pore protein to play a bactericidal effect, so the change of the structure of outer membrane protein and the regulation of membrane pore protein expression factors will cause the change of drug resistance. Objective to investigate the relative expression of micF and ompF genes at the transcriptional level, and to investigate the relationship between the expression of ompF and the relationship between OmpF and drug resistance, and whether micF regulates the cell membrane resistance of Shigella. Materials and methods 1. From May to October 2015, 13 clinical strains of Shigella and 8 strains of Shigella from the Institute of Infectious Diseases, second Hospital of Tianjin Medical University, were collected from fecal specimens of diarrhea patients in the second Hospital of Tianjin Medical University. Routine biochemical identification and serum agglutination method were used to determine the serotype. 2. Six common antibiotics, such as ciprofloxacin, ceftriaxone, ampicillin, and K-B disk diffusion test were used to screen out the multidrug resistant strains and non-multidrug resistant strains. 3. PCR amplification of two groups of outer membrane pore protein ompF gene. Detection of ompF gene deletion strain. 4. The total RNAs of Shigella were extracted and the mRNA transcription of micF and ompF genes in two groups of strains were detected by real-time fluorescence quantitative RT-PCR. The results were statistically analyzed by SPSS20.0 software. The standard concentration curve of ciprofloxacin was established. Experimental results 1. The collected 21 strains were Shigella flexneri 8 strains, Shigella baumannii 6, Shigella sonnei 6 strains and Shigella dysenteriae 1 strain. The results of antimicrobial susceptibility showed that 13 strains of Shigella spp were resistant to 3 or more kinds of antibiotics, which were classified as multidrug resistant group. 8 strains were only resistant to class 1 or class 2 antibiotics or were sensitive to all antibiotics. The results of PCR amplification of ompF gene of Shigella spp. 3.21 strains were all positive, and no ompF gene deletion strain. 4 was found. The relative expression level of micF gene in multidrug resistance group was significantly higher than that in non multidrug resistance group (P0.05). The relative expression level of ompF gene in multidrug resistance group was significantly lower than that in non multidrug resistance group (P0.05). The correlation coefficient between micF and ompF was negative, but the correlation degree was not high (r = -0.244), which indicated that there was a negative correlation between micF and ompF, but the correlation degree was not high. The concentration of ciprofloxacin in multidrug resistant group was lower than that in non-multidrug resistant group, and the difference was statistically significant (P0.05). Conclusion 1. The relative expression level of ompF gene in multidrug resistant Shigella was significantly lower than that in non-multidrug resistant Shigella. The decrease of membrane porin OmpF may be one of the important mechanisms of multidrug resistance induced by Shigella. 2.MicF gene may play a negative role in regulating the expression of OmpF. The decrease of intracellular ciprofloxacin concentration in multidrug resistant bacteria may be related to the decreased expression of adventitia pore protein.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R516.4
【參考文獻】
相關(guān)期刊論文 前4條
1 茹維平;黃麗莉;趙嘉詠;夏勝利;;河南省2005～2009年細菌性痢疾流行概況及病原特性分析[J];現(xiàn)代預(yù)防醫(yī)學(xué);2010年21期
2 王睿,柴棟;細菌耐藥機制與臨床治療對策[J];國外醫(yī)藥(抗生素分冊);2003年03期
3 張文偉,李曉輝,楊梅;乳酸環(huán)丙沙星的熒光特性及其應(yīng)用[J];光譜實驗室;1999年02期
4 丁建強,馬亦林,龔正,陳亞崗;志賀菌流行菌株耐藥性及R質(zhì)粒的研究[J];中華傳染病雜志;1998年03期
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