志贺菌膜孔蛋白OmpF及调控基因micF介导多重耐药机制的研究
发布时间：2018-08-20 15:43
【摘要】：细菌性痢疾又称志贺菌病,是我国最常见的感染性腹泻之一,目前居于我国法定传染病发病率的第三位。志贺菌具有高度传染性、人群普遍易感、危害性严重的特点,能够通过消化道传播的肠道传染病。近年来,由于抗生素的广泛使用,志贺菌耐药现象越来越多,且出现多重耐药趋势。外膜膜孔蛋白介导的细胞通透性改变是引起细菌多重耐药的重要机制之一。某些抗生素首先要经膜孔蛋白进入细胞才能发挥杀菌作用,所以外膜蛋白的结构及调控膜孔蛋白表达因素的改变都会引起耐药性的变化。研究目的了解micF和ompF基因在转录水平的相对表达量,探讨外膜孔蛋白OmpF与耐药性的关系,micF对ompF表达的调控作用,以及micF是否调控志贺菌的细胞膜耐药。材料与方法1.收集2015年5月-10月天津医科大学第二医院肠道门诊腹泻患者粪便标本的志贺菌临床株13株,及天津医科大学第二医院感染性疾病研究所保存的部分志贺菌8株,共计21株,采用常规生化鉴定和血清凝集法确定血清型。2.选取环丙沙星、头孢曲松、氨苄西林等6类常用抗生素和K-B纸片扩散法进行药敏试验,筛选出多重耐药菌株和非多重耐药菌株。3.PCR扩增两组外膜孔蛋白ompF基因,检测有无ompF基因缺失株。4.提取志贺菌总RNA,采用实时荧光定量RT-PCR技术检测两组菌株micF和ompF基因的mRNA转录情况,并使用SPSS20.0软件对结果进行统计分析。5.制定环丙沙星标准浓度曲线,采用多功能酶标仪测定两组菌株胞内环丙沙星的浓度。实验结果1.经常规生化鉴定和血清凝集实验证实,所收集的21株菌株均为志贺菌,其中福氏志贺菌8株,鲍氏志贺菌6株,宋内志贺菌6株,痢疾志贺菌1株。2.药敏结果显示:13株志贺菌对3类或3类以上抗生素耐药,将其归为多重耐药组,8株仅对1类、2类抗生素耐药或对全部抗生素均敏感,将其归为非多重耐药组。3.21株志贺菌ompF基因PCR扩增结果均为阳性,未发现ompF基因缺失株。4.多重耐药组micF基因相对表达水平明显高于非多重耐药组,且差异有统计学意义(P0.05)。多重耐药组ompF基因相对表达水平明显低于非多重耐药组,且差异有统计学意义(P0.05)。对两者进行相关性分析,相关系数r=-0.244,提示micF和ompF之间呈负相关,但关联程度不高。5.多重耐药组胞内环丙沙星浓度低于非多重耐药组,且差异有统计学意义(P0.05)。结论1.多重耐药志贺菌ompF基因相对表达水平明显低于非多重耐药志贺菌,膜孔蛋白OmpF减少可能是志贺菌引起多重耐药的重要机制之一。2.micF基因可能对膜孔蛋白OmpF的表达起负调控作用。3.多重耐药菌胞内环丙沙星浓度降低,可能与外膜孔蛋白的表达减少有一定关系。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.4
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.4
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