抗生素所致肠道菌群失衡及乳杆菌对其调节作用的研究

发布时间：2018-04-05 18:14

本文选题：肠道菌群　切入点：多样性　出处：《第三军医大学》2007年博士论文

【摘要】： 抗生素的不当使用将直接危害人体健康:服用治疗剂量抗生素会增加临床病人二重感染的发病几率,被动摄入残留剂量的抗生素则有可能导致肠道菌群失调症的发生。这两大类疾病在本质上都与抗生素导致的肠道菌群变化密切相关,因此,基于微生态学基本观点开展抗生素对肠道菌群作用的深入研究,对于预防和治疗相关疾病具有重要意义。能够充分模拟抗生素与肠道菌群作用的标准化动物模型是阐述相关疾病发生机理和探索防治策略的重要基础,而菌株选择是微生态疗法发挥有效性的关键。因此,本研究建立了两大类标准化动物模型以充分模拟人暴露于抗生素的两种主要途径,使用体外筛选获得的乳杆菌菌株对抗生素诱导的菌群失衡进行调节,结合经典技术与现代分子微生态技术评价乳杆菌调节菌群的有效性,以期阐明肠道微生态区系改变与感染发生的关系,同时为防治菌群失调和感染提供实验数据。课题分为三部分: 第一部分目的:构建不同剂量头孢曲松诱导的小鼠肠道菌群失衡模型,分别为研究微生态区系与感染之间的关系和残留剂量抗生素对菌群的影响提供标准化的实验工具和整体评价体系。方法:(1)通过灌胃方式,连续5d给予SPF小鼠头孢曲松,人为灌胃白假丝酵母菌造成小鼠肠道真菌过增殖模型;(2)通过随机饮水方式,连续45d分别给予SPF小鼠三个浓度的低剂量头孢曲松,模拟残留剂量抗生素的持续作用;(3)革兰染色光镜和扫描电境直接观察小鼠菌群变化、活菌计数研究菌群数量变化及对白假丝酵母的清除作用、PCR-DGGE技术研究菌群结构多样性变化、模拟肠道离体模型研究菌群定植抗力的变化。结果:(1)50mg/d的头孢曲松会导致小鼠肠道菌群重度紊乱,厌氧总菌、乳杆菌和肠球菌在处理期间数量出现极显著下降(P0.01),双歧杆菌和肠杆菌缺如(P0.001);DGGE图谱显示,总菌和乳杆菌丰富度和多样性极显著低于正常水平(P0.01),肠球菌多样性指数也出现显著下降(P0.05);通过对DGGE条带进行回收、测序,发现Bacillus sp.部分菌株失去优势菌群的地位,一株Lactobacillus reuteri演替为优势菌;肠黏膜表面膜菌群脱落,肠道菌群定植抗力严重受损。(2)300μg/ml及30μg/ml CRO水溶液持续处理45d会导致小鼠肠道菌群紊乱,厌氧总菌、乳杆菌和肠杆菌数量均出现先降后升的趋势,而肠杆菌数量异常增殖,双歧杆菌数量显著减少(P0.01)且无法恢复,肠球菌无显著变化(P0.05);3μg/ml头孢曲松处理对小鼠肠道菌群数量无显著影响(P0.05);DGGE分析可见总菌和乳杆菌菌群多样性显著下降,肠球菌多样性指数无显著变化;通过对DGGE条带进行回收、测序,发现处理中期出现两株Ruminococcus torques,但其数量未能增殖,而对头孢曲松耐药的一株Enterococcus faecium优势效应贯穿处理始终;小鼠肠道乳杆菌的优势菌群为两株Lactobacillus reuteri和两株Lactobacillus salivarius subsp. Salivarius,数量优势不受头孢曲松作用的影响;定植抗力受损,但仍可显著抑制白假丝酵母的增殖(P0.05)。第二部分目的:通过体外模型筛选出拮抗白假丝酵母能力最强的乳杆菌用于体内实验,研究乳杆菌对小鼠肠道失衡菌群的调节效果。方法:(1)结晶紫芽管染色法比较8株乳杆菌及其代谢产物体外拮抗白假丝酵母芽管形成的能力,共孵育法和琼脂打孔扩散法比较8株乳杆菌活菌与培养乏液对白假丝酵母的生长抑制能力;(2)对治疗剂量抗生素作用后的小鼠,于不同时间点,采用预防、同步、治疗三种策略,通过灌胃方式给予肠道白假丝酵母过增殖小鼠模型108CFU的LGG;对低剂量抗生素作用的小鼠,向饮用头孢曲松水溶液中直接混合109CFU乳杆菌LGG;(3)革兰染色光镜直接观察小鼠菌群变化、活菌计数研究菌群数量变化及对白假丝酵母的清除作用、PCR-DGGE技术研究菌群结构多样性变化、模拟肠道离体模型研究菌群定植抗力的变化。结果:(1)鼠李糖乳杆菌LGG对白假丝酵母的芽管抑制及生长抑制能力均显著高于其他乳杆菌;短链脂肪酸中只有丁酸能够显著抑制白假丝酵母芽管的形成;(2)LGG对小鼠重度失衡的菌群调节作用明显,可显著促进厌氧总菌、双歧杆菌和乳杆菌数量的恢复;LGG干预后,受损菌群的丰富度和乳杆菌的多样性指数恢复至正常(P0.05),且预防处理恢复菌群的效果最显著;定植抗力显著恢复,LGG干预后的小鼠可以极显著清除肠道内的白假丝酵母,仍以预防处理组效果最好(3)LGG调节残留剂量头孢曲松诱导的菌群失调具有一定效果,促进了双歧杆菌数量的恢复,但无法抑制肠杆菌的过度增殖;可以恢复乳杆菌的丰富度和多样性,但总菌多样性指数无显著变化(P0.05),对肠球菌的数量和多样性均无显著影响(P0.05);能够显著增强菌群定植抗力,LGG干预组对于白假丝酵母的清除能力极显著的高于抗生素单独处理组(P0.01)。第三部分目的:对暴露在抗生素压力下的乳杆菌介导耐药性传递的风险进行评估。方法:(1)K-B法检测8株乳杆菌对于14种抗生素的敏感性;(2)双纸片扩散法检测体内实验菌株LGG产超广谱β-内酰胺酶的情况;(3)提取体内实验菌株LGG的质粒,进行菌体内可传递耐药因子的检测。结果:(1)8株乳杆菌对抗生素表现出多重耐药,LGG可以耐受5种受试药物,而耐受万古霉素的乳杆菌菌株为7株;(2)LGG不产超广谱β-内酰胺酶,不含质粒。主要结论: 1、临床剂量头孢曲松的作用可严重破坏小鼠肠道菌群的平衡,该模型可以成功模拟临床病人服用头孢曲松进而造成肠道真菌过增殖的现象。头孢曲松作用后,小鼠肠道菌数量显著减少、多样性降低、菌群固有定植抗力严重受损,白假丝酵母菌可大量增殖。因此,大剂量头孢曲松诱导的小鼠肠道菌群失衡模型证明了肠道菌群紊乱与肠源性感染具备因果关系。 2、食品残留剂量头孢曲松的长期作用可导致小鼠肠道菌群出现轻度失衡,该模型成功模拟了人体经由食物链被动摄入抗生素造成的菌群失调。 3、通过体外筛选获得的乳杆菌菌株LGG对两类模型进行干预,可有效调节头孢曲松导致的肠道菌群失衡,恢复菌群数量、多样性和定植抗力;使用乳杆菌调节肠道菌群,同时存在耐药性转移的风险,但乳杆菌LGG株在体内介导耐药性传递的风险性较小。创新性: 1、以肠道微生态学为着眼点,全面模拟人体暴露于抗生素的两种不同途径,首次使用同种抗生素的不同剂量构建了两类肠道菌群失衡模型,并且分别研究了乳杆菌对于不同程度菌群失衡的调节作用,具有微生态学特色的整体、系统化实验设计具有一定创新性; 2、使用临床剂量的头孢曲松处理小鼠,成功建立了肠道白假丝酵母局部感染的小鼠模型,以微生态学观点阐述了抗生素诱导的肠道菌群失衡与感染发生发展的关系; 3、利用乳杆菌LGG株稳定和恢复肠道菌群的方法来防治感染,为抗感染治疗提供了新的参考策略,为微生态疗法运用于抗感染治疗提供了理论基础,同时为本菌株开发为微生态制剂提供了新的实验依据。 4、结合传统活菌计数与现代分子微生态学PCR-DGGE技术,为研究抗生素对于肠道菌群的影响及其防治提供了新的评价方法; 5、本实验在研究乳杆菌调节菌群有效性的同时,对其介导肠道菌群之间耐药性传递的风险进行了初步评估,对乳杆菌耐药性的关注具有前瞻性。
[Abstract]:The improper use of antibiotics will directly harm human health : the administration of therapeutic doses of antibiotics will increase the incidence of secondary infection in clinical patients , and the passive intake of residual doses of antibiotics may lead to the occurrence of intestinal dysbacteriosis . These two major diseases are intrinsically related to the changes in intestinal flora caused by antibiotics . Therefore , it is important to study the effect of antibiotics on intestinal flora based on the basic viewpoint of microecology .

A standardized animal model which can fully simulate the effects of antibiotics and intestinal flora is an important basis for the pathogenesis of related diseases and the exploration and prevention strategies , and the selection of strains is the key to the effectiveness of micro - ecological therapy . Therefore , this study establishes two major types of standardized animal models to fully simulate the two main routes of human exposure to antibiotics .

the first portion

Objective : To establish a model of intestinal flora imbalance induced by different doses of cephalosporin , and to provide a standardized experimental tool and an overall evaluation system for the study of the relationship between microecological region and infection and the influence of residual dose antibiotics on the flora .

Methods : ( 1 ) SPF mice were given SPF mice by gavage for 5 days .

Results : ( 1 ) There was a significant decrease in the number of intestinal flora in mice ( P0.01 ) . Salivarius , the superiority of quantity was not influenced by the action of cephalosporin ; the colonization resistance was damaged , but the proliferation of Candida albicans could be significantly inhibited ( P0.05 ) .

the second part

Objective : To study the effect of lactobacillus on the intestinal imbalance flora of mice .

Methods : ( 1 ) The growth inhibition ability of 8 strains of Lactobacillus reuteri and its metabolites in vitro was compared with 8 strains of lactobacillus and its metabolites .

Results : ( 1 ) The inhibition and growth inhibition ability of Lactobacillus rhamnosus LGG was significantly higher than that of other lactobacillus .

PART III

Objective : To evaluate the risk of drug resistance transfer mediated by Lacan exposed to antibiotic pressure .

Methods : ( 1 ) The sensitivity of 8 strains of Lactobacillus to 14 antibiotics was detected by K - B method . ( 2 ) In vivo experiment strain LGG was detected by double - paper diffusion method .

Results : ( 1 ) Eight strains of lactobacillus showed multiple resistance to antibiotics , and LGG could tolerate 5 kinds of tested drugs , and the strains resistant to vancomycin were 7 strains ; ( 2 ) LGG did not produce the super - broad - spectrum 尾 - lactamase and did not contain plasmids .

Main conclusions :

1 . The effect of the clinical dose of Cefomycin can seriously destroy the balance of intestinal flora in mice . The model can successfully simulate the phenomenon of excessive intestinal flora in clinical patients . After the action of cephalosporin , the number of intestinal bacteria in mice is obviously reduced , the diversity is reduced , the inherent colonization resistance of the flora is severely damaged , and the Candida albicans can proliferate . Therefore , the model of intestinal flora imbalance induced by the large - dose cephalosporin has proved the causal relationship between intestinal flora disturbance and enterogenous infection .

2 . The long - term effects of the food residual dose of cephalosporin could lead to mild imbalance in the intestinal flora of mice . The model successfully simulated the dysbacteriosis caused by the passive ingestion of antibiotics through the food chain .

3 . The lactobacillus strain LGG obtained by in vitro screening can effectively regulate the imbalance of intestinal flora , restore the quantity , the diversity and the resistance of the intestinal flora caused by the cephalosporin , and use lactobacillus to adjust the intestinal flora , and meanwhile , the risk of drug resistance transfer exists , but the risk of the LGG strain mediated drug resistance transmission in vivo is small .

Innovative :

1 . Based on the microecology of intestinal tract , two different routes of human exposure to antibiotics were fully simulated . Two kinds of intestinal flora imbalance models were constructed for the first time using different doses of the same antibiotics .

2 . A mouse model of local infection of Candida albicans was successfully established by using the clinical dose of cefsuspine , and the relationship between the imbalance of intestinal flora and the development of infection was described by microecology .

3 . The method for stabilizing and recovering the intestinal flora by using the lactobacillus LGG strain provides a new reference strategy for the anti - infection treatment , and provides a theoretical basis for the application of the micro - ecological therapy to the anti - infection treatment , and provides a new experimental basis for the development of the strain as a micro - ecological preparation .

4 . Combining the traditional living bacteria counting and modern molecular microecology PCR - DGGE technology , this paper provides a new evaluation method for the study of the effect of antibiotics on intestinal flora and the prevention and control of antibiotics .

5 . In this experiment , the risk of drug - resistance transfer between intestinal flora and intestinal flora was preliminarily assessed at the same time of studying the efficacy of Lactobacillus plantarum , which was forward - looking .

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363;R96

【引证文献】