细菌黏附分子介导的定植抗力抑制肠道耐药基因产生与传播的机制研究

Gut is the platform of production and transmission of antibiotic-resistant strain. Antibiotic can kill a lot of gut flora under pathological condition. Our previous research demonstrated that gut mucosa flora was closely related to the production and transmission of antibiotic resistance strain. In this research, the alteration of gut mucosa flora, the expression of bacterial adhesion molecule on gut epithelial cell and its relationship with microbial colonization resistance were detected by confocal laser scanning, immunoblotting and quantitative PCR from in vitro ,animal and clinical experiments. The selective action of antibiotics on intestinal cavity flora, the alteration of antibiotic-resistance and pathogenicity of intestinal cavity flora were investigated. The gut colonization of exogenous antibiotic-resistant bacteria, the expression and exposure of epithelial adhesion molecule were studied. Meanwhile, the effect of gut mucosa flora and its adhesion molecule on expression of gut conditional pathogenic bacteria adhesion molecule,its exposure on epithelialcell and transmission of antibiotic-resistant gene among gut flora were studied. The expression and mutual effect of mucosa flora, cavity flora and exogenous conditional pathogenic bacteria colonization factor under pathological condition was explored. Furthermore, the gut mucus distribution under the pressure of antibiotics and the effect of exogenous probiotics as well as original bacteria on expression and affinity of bacterial adhesion molecule on gut epithelial cell ,intestinal microbial colonization resistance and transmission of antibiotic-resistant gene were investigated. Therefore, as new mechanism of gut bacterial resistance was studied, a new target is to be provided for preventing the production of bacterial antibiotic-resistant strain .

肠道是耐药菌株产生与传播平台，病理情况下抗生素的应用大量杀灭原籍菌。作者研究提示肠道膜菌群与肠道耐药菌株的产生与传播关系密切。本项目通过体外、动物实验和临床研究三个层面，采用激光共聚焦、免疫印迹和定量PCR等技术研究病理情况下肠道膜菌群变化、肠黏膜上皮细胞膜菌群黏附分子表达，及其与肠道定植抗力关系；抗生素对肠道腔菌群选择作用，腔菌群耐药性及其致病性改变；外源性条件致病菌在肠道内定植，及其上皮细胞黏附分子表达与暴露程度；膜菌群及其黏附分子对肠道条件致病菌黏附分子表达及其在上皮细胞表面暴露的影响、对耐药基因在肠道菌群间传播的影响；病理情况下膜菌群、腔菌群和外源性条件致病菌定植因子表达及其相互间的作用；抗生素压力下外源性和内源性益生菌在肠黏膜分布规律，对上皮细胞细菌黏附分子表达和亲和力影响，对外源性和内源性耐药菌株在肠道定植和耐药基因传播的影响。研究肠道细菌耐药新机制，为阻断细菌耐药获得新靶点。

本项目首先研究脓毒症对肠黏膜上皮细胞膜菌群和条件致病菌黏附分子和定植因子表达的影响。SD大鼠随机分为正常组、假手术组、脓毒症组、脓毒症治疗组。采用盲肠结扎穿孔脓毒症模型，脓毒症治疗组术后给予环丙沙星治疗。各组相应处理后1、2、3、4周采集大鼠结肠黏膜和结肠内容物检测后发现：脓毒症导致肠粘膜上皮细胞膜菌群黏附分子和致病菌、益生菌定植因子的表达上升，而伴随着感染趋于稳定，上皮细胞膜菌群黏附因子表达有所降低；随着广谱抗生素的应用，部分膜菌群黏附因子，如asialo-GM2、硫肝脂粘膜受体的表达也会提高，而细菌定植因子的表达能显著降低。. 基于上述研究，作者通过封闭肠黏膜上皮细胞膜菌群和条件致病菌黏附分子探索外源性条件致病菌在肠道内定植和耐药基因的传播。SD大鼠随机分为假手术（Sham）、脓毒症（Sepsis）、环丙沙星（CPFX）、CPFX +双歧杆菌组（CPFX+Bif）、CPFX +定植因子抗原-1组（CPFX+CFA/I）。Sham、Sepsis组术后2h给予pGFPuv-H10407灌胃检测外源性致病菌的定植；肺炎克雷伯杆菌（ATCC700603）灌胃检测SHV-18耐药基因的传播；CPFX、CPFX+Bif和CPFX+CFA/I组在术后予CPFX治疗，后2组在术后2h给予Bif或CFA/I灌胃，3个组在CPFX后第2d予pGFPuv-H10407和ATCC700603灌胃。在相应处理后的1、3、7、14、21、30、60d取粪便检测后发现：脓毒症肠道膜菌群明显增多，外源性致病菌大量定植，并出现耐药基因在肠道内的传播；抗生素治疗后E.coli数量锐减，外源性条件致病菌也出现大量定植，原籍E.coli出现对氟喹诺酮类抗生素的少量耐药及其MIC的轻度升高，未发现耐药基因的传播；封闭肠黏膜上皮细胞膜菌群或条件致病菌黏附分子可以维持肠道膜菌群的数量稳定，同时抵御外源性条件致病菌的定植和耐药基因的传播，但是在抗生素压力下产生大量对氟喹诺酮类抗生素耐药的E.coli。. 结论：肠粘膜上皮细胞膜菌群黏附分子和致病菌、益生菌定植因子在脓毒症状态下表达上升，广谱抗生素主要降低了细菌定植因子的表达。封闭肠黏膜上皮细胞膜菌群和条件致病菌黏附分子可以维持脓毒症状态下肠道膜菌群的稳定，抵御外源性致病菌的定植和耐药基因的传播，但是会诱发氟喹诺酮类抗生素耐药的产生。.