【作者】 刘绍泽；

【导师】 杨兴易； 陈德昌； 林兆奋； 赵良； 单红卫；

【作者基本信息】 第二军医大学 ， 急诊医学， 2010， 博士

【摘要】 本课题提出如下假说：严重创伤打击下,肠道微生态环境及屏障遭受破坏,在此基础上发生外来菌定植及细菌易位,广谱抗生素的应用大量杀灭肠道敏感菌群,逐步选择出条件致病菌及耐药菌株,耐药基因可进一步传递给肠道原籍菌,使其在停用抗生素及肠道菌群恢复后成为肠道耐药基因播散潜在和持久的危害；大黄能保护危重病肠道微生态环境,减弱广谱抗生素对肠道菌群的选择作用,减少肠道细菌易位,为肠源性感染的防治探索新的途径；第一部分烫伤及内毒素“二次打击”对大鼠肠道菌群及细菌易位的影响目的：建立烫伤和内毒素“二次打击”的大鼠模型,观察不同创伤打击及大黄对肠道菌群及细菌易位的影响。方法：采用大鼠背部烫伤模型,烫伤后24h给予内毒素进行“二次打击”,大黄组于内毒素打击同时即予大黄灌胃。分别于建模后24h取大鼠结肠肠内容物行细菌、真菌定量培养、菌种鉴定及取大鼠心脏血、肝右叶、左下肺、回盲部肠系膜淋巴结行细菌定量培养、菌种鉴定。结果：1)烫伤导致大鼠肠道乳酸杆菌数量显著减少,真菌数量明显增加,肠杆菌、肠球菌数量无明显变化；内毒素“二次打击”后,肠杆菌及肠球菌数量较烫伤组分别增加103和102数量级,差异有显著性(P均<0.01)；真菌及乳酸杆菌数量与烫伤组比较未见明显变化。2)正常对照组大鼠结肠内容物肠杆菌选择培养均为大肠杆菌,肠球菌以粪肠球菌占绝对优势,头地霉为肠道内优势真菌；烫伤及内毒素“二次打击”后,肠道内兼性需氧杆菌、肠球菌、真菌的优势菌种未见变化,仍分别为大肠杆菌、粪肠球菌及头地霉；但打击后大鼠肠道兼性需氧杆菌种类发生了明显变化,分别出现铜绿假单胞菌、肺炎克雷伯杆菌、鲍曼不动杆菌及变形杆菌等条件致病菌。3)正常对照组肠道细菌的基础易位率为12.5%,易位仅发生在肠系膜淋巴结,烫伤大鼠肠道细菌易位率未见增加。内毒素“二次打击”后,肠道细菌易位率显著增加(同烫伤组比较,个体易位率P<0.05；脏器总体易位率P<0.01),肠道细菌广泛易位至血液及肝脏、肺等远隔脏器。4)烫伤和内毒素“二次打击”后粘膜菌群中乳酸杆菌数量较正常对照组明显减少(P<0.05),给予大黄治疗后乳酸杆菌的数量有所增加,与正常对照组比较差异无显著性,即大黄减弱了烫伤和内毒素打击对乳酸杆菌的作用；大黄治疗可以明显减少脓毒症大鼠肠外脏器总体细菌易位率,特别是其减少了肠道细菌引起的远隔脏器易位及条件致病菌易位。结论：烫伤及内毒素“二次打击”后,肠道微生态环境遭到破坏,益生菌数量减少,肠道内条件致病菌(如铜绿假单胞菌、肺炎克雷伯杆菌、鲍曼不动杆菌、变形杆菌及真菌)失去其定植抗力而大量增殖；脓毒症状态下,肠道屏障功能严重受抑,肠道杆菌(特别是铜绿假单胞菌、变形杆菌、肺炎克雷伯菌及阴沟肠杆菌等条件致病菌)广泛易位至血流、肝脏和肺,引发肠源性感染；大黄使脓毒症大鼠肠道益生菌的数量有所恢复,从而部分维持胃肠道微生态平衡,提高了对肠道内条件致病菌的定植抗力,通过保护粘膜屏障,减少了铜绿假单胞菌、变形杆菌、肺炎克雷伯菌及阴沟肠杆菌等院内感染常见致病菌所致的肠源性感染的发生。第二部分广谱抗生素对烫伤、脓毒症大鼠肠道微生态环境及细菌易位的影响目的：观察头孢曲松、亚胺培南对烫伤、脓毒症大鼠肠道菌群及细菌易位的影响方法：采用大鼠背部烫伤模型,烫伤后24h给予内毒素进行“二次打击”,大黄组于内毒素打击同时即予大黄灌胃。烫伤组、脓毒症组、大黄组分别细分为头孢曲松治疗组及亚胺培南治疗组,各组于建模24h后开始抗生素治疗,分别于抗生素治疗0d、3d、9d取大鼠结肠肠内容物行细菌、真菌定量培养、菌种鉴定及取大鼠心脏血、肝右叶、左下肺、回盲部肠系膜淋巴结行细菌定量培养、菌种鉴定。结果：1)广谱抗生素对肠道杆菌数量的影响：烫伤大鼠肠道杆菌数量约为105数量级,应用头孢曲松后肠杆菌数量锐减至103数量级(P<0.01)；而应用亚胺培南后,肠杆菌数量则在烫伤基础上进一步增加,治疗9天后明显高于烫伤未治疗组(P<0.05)。脓毒症大鼠肠道杆菌数量约为108—109数量级,应用头孢曲松后肠杆菌数量减少至104数量级(P<0.01)；亚胺培南治疗后,脓毒症大鼠肠道杆菌数量减少至107数量级(P<0.01)；亚胺培南治疗组肠道杆菌数量明显高于相同模型、相同治疗时限的头孢曲松治疗组(P<0.01)。2)广谱抗生素对肠道杆菌种类的影响：烫伤及脓毒症大鼠应用头孢曲松后其肠道内大肠杆菌被大量杀灭,数量剧减甚至消失,逐渐选择出铜绿假单胞菌和鲍曼不动杆菌,取代了大肠杆菌成为肠道优势菌群；亚胺培南治疗后大肠杆菌虽然丧失了其优势菌地位,但其数量仍维持在基本水平,同时表现出对肺炎克雷伯杆菌、变形杆菌的高选择性,伴有铜绿假单胞菌、鲍曼不动杆菌及其它杆菌的存在,并以这些条件致病菌占据数量上的绝对优势。3)广谱抗生素对肠道球菌数量的影响：烫伤及脓毒症大鼠应用头孢曲松和亚安培南治疗后,肠球菌数量均在原打击基础上进一步增加,达到109数量级(P<0.01),此现象与抗生素治疗时限无关。4)广谱抗生素对肠道球菌种类的影响：烫伤及脓毒症大鼠肠道中致病力较弱的粪肠球菌占绝对优势,头孢曲松和亚安培南治疗后,屎肠球菌所占比例明显增加,屎肠球菌在数量上超过粪肠球菌成为优势菌群。5)广谱抗生素对肠道真菌的影响：烫伤及脓毒症大鼠应用头孢曲松、亚胺培南后肠道真菌数量均在原有基础上进一步增加,此情况随着广谱抗生素应用天数的增加而有所加重；白杰尔孢子菌、白色念珠菌、光滑念珠菌、克柔氏念珠菌及毛霉菌等临床上常见的IFI病原菌的检出率明显增加。6)广谱抗生素对肠道乳酸杆菌的影响：烫伤及脓毒症大鼠应用头孢曲松、亚胺培南后肠道乳酸杆菌在原有基础上未见明显的数量变化。7)广谱抗生素对肠道细菌易位的影响：烫伤及脓毒症大鼠应用头孢曲松、亚胺培南后肠道细菌易位率均在原有基础上进一步增加,并持续存在血液及远隔脏器细菌易位,易位菌种也由治疗前的肠道杆菌转变为肠球菌,并且屎肠球菌易位比例高于粪肠球菌。8)大黄对广谱抗生素压力下烫伤、脓毒症大鼠肠道微生态环境及肠道细菌易位的影响：大黄治疗并未减轻广谱抗生素压力下脓毒症大鼠肠道菌群紊乱程度,在广谱抗生素治疗的中后期也不能有效抑制肠道细菌易位。结论：在广谱抗生素压力下烫伤及脓毒症大鼠肠道菌群紊乱程度进一步加重,广谱抗生素大量杀灭了肠道内敏感菌群,给肠道内对抗生素不敏感的常居菌(如念珠菌等)和耐药菌留下大量增殖的空间,成为优势菌群,导致肠道微生态环境破坏,严重者出现胃肠功能衰竭,加剧危重病病理生理过程；广谱抗生素使烫伤、脓毒症大鼠细菌易位显著增加,易位波及血液及肝脏、肺等远隔脏器；肠球菌特别是耐药性及致病性更高的屎肠球菌成为易位主要菌种,更进一步加重了肠源性感染；长期应用抗生素削弱了大黄对胃肠道微生态环境的保护作用及肠道细菌易位的抑制作用。第三部分广谱抗生素压力下脓毒症大鼠肠道内耐药基因的传播目的：建立肺炎克雷伯标准耐药菌株(ATCC700603)在烫伤及内毒素“二次打击”大鼠肠道定植模型,连续性观察头孢曲松对肠道定植菌及肠道原籍大肠杆菌数量的影响及其耐药性征的变化。方法：采用大鼠背部烫伤模型,烫伤后24h给予内毒素进行“二次打击”,各组均在在建模后2h给予肺炎克雷伯杆菌标准菌株菌液Iml(1×108cfu/ml)灌胃,菌液灌胃24h后给予头孢曲松或生理盐水治疗,疗程9天。分别于建模前、菌液灌胃24h后(头孢曲松或生理盐水使用前)、治疗1天、治疗3天、治疗6天、治疗9天、停药2天、停药1周、停药2周、停药3周、停药4周无菌条件下收集各组大鼠新鲜粪粒,选择性培养定植菌—肺炎克雷伯杆菌及肠道原籍大肠杆菌及计数,并对纯化菌落行菌株ESBLs表型确证、耐药基因检测。结果：1)正常对照组大鼠未见肺炎克雷伯标准菌株的定植,全部烫伤和脓毒症大鼠发生了外来菌的肠道定植,烫伤组大鼠定植菌量为105/g数量级,脓毒症组大鼠肠道定植菌量达到108/g数量级,与烫伤组相比差异显著(P<0.001)。2)烫伤、脓毒症大鼠肠道定植的肺炎克雷伯标准菌株在定植后1-3天其数量逐渐减少并最终被清除；头孢曲松治疗后,烫伤及脓毒症大鼠肠道内定植菌持续存在于肠道中,停药后清除缓慢；脓毒症组大鼠治疗过程中查见了肺炎克雷伯杆菌菌株对头孢曲松耐药性变化。3)未应用头孢曲松(生理盐水治疗组)的烫伤及脓毒症外源菌定植大鼠粪便中大肠杆菌均未见有耐药性改变；烫伤及脓毒症大鼠菌液灌胃24h后给予头孢曲松治疗1天其粪便中大肠杆菌数量即急剧减少,常规平板培养结果阴性(低于102/g数量级),直至治疗9天及停用头孢曲松1周后,烫伤及脓毒症组大鼠肠道原籍大肠杆菌基本恢复。脓毒症头孢曲松治疗组5只大鼠(共8只)分别于治疗9天(4只)和停药2周(1只)时粪便中检测到的大肠杆菌获得了定植菌的SHV-18耐药基因,转变为产ESBLs菌株,其对头孢曲松也由敏感转变为中敏；结论：在烫伤及脓毒症情况下,外来耐药菌株可发生肠道内定植及增殖；在广谱抗生素压力下,肠道微生态环境遭到破坏,定植耐药菌失去肠道原籍菌的定植抗力,同时发生了抗生素的诱导耐药而存在于肠道中,不易被机体清除,并进而将耐药基因传递给肠道原籍菌,耐药基因得以伴随肠道原籍菌而存在于肠道中,形成持久的潜在危害。因此,慎用抑酸剂,保护肠道化学屏障,严格广谱抗生素的应用及时限,保护肠道微生态环境是避免肠道耐药菌株定植及耐药基因传播的有效及必要手段。更多还原

【Abstract】 Sepsis is a common complication after a serious trauma or burn injuries, severe shock, and major surgery. Furthermore, it may lead to septic shock and multiple organ dysfunction syndromes (MODS). MODS have become one of the important causes of death in clinical critically ill patients. The gastrointestinal tract has been considered as a stress central organ, and gut barrier failure played an important role in sepsis and MODS. The theory was emphasized that gut bacteria and their products could be an important trigger for the development of sepsis and MODS in critically injured or burned patients. In the pathogenesis of traumatic stress that the gastrointestinal tract involved, the intestinal bacterial translocation is the main pathological link. The endogenous infection caused by opportunistic pathogen that stored in the gastrointestinal tract becomes a formidable problem. Under the pressure of the trauma and antibiotics, nosocomial pathogens could colonize in the intestine and give rise to resistance gene transfer among different kinds of bacteria, causing intestinal origin bacteria to acquire new resistance features, so that the intestine become the largest spreading platform of drug resistance genes. In addition to causing own endogenous infection, patients with diarrhea and fecal incontinence will lead to the spread of resistant bacteria in the hospitals. There is no clear research results and understanding of intestinal colonization of alien bacteria caused by the trauma and propagation of antibiotic resistance genes under the pressure of antibiotics, making the lack of prevention and treatment on this issue. The hypothesis of this study is that the trauma can damage intestinal micro-ecological environment or barrier, following the foreign bacteria colonization and bacterial translocation. Broad-spectrum antibiotics kill a great quantity of sensitive intestinal flora and pick out opportunistic pathogen and drug-resistant strains. Resistance genes can be further passed to the gastro-intestinal origin bacteria and continue to exist in the gut. Rhubarb can protect gut ecological environment of critical illness, diminish the choice of antibiotics on intestinal flora, reduce bacterial translocation and explore some new way to prevent the nosocomial infections caused by intestinal resistant. Avoiding colonization of alien drug-resistant strains and limiting on the use of broad-spectrum antibiotics are the important means to reduce acquired drug resistance in the gut.PARTⅠEffect of Burn and "Second Hit" by Endotoxin on Gut Microflora and Bacterial Translocation in RatsObjective:To approach the impact of different degrees of trauma on the intestinal flora and bacterial translocation and the effect of rhubarb on intestinal microecology in sepsis rats.Methods:32 SD rats with 30%Ⅲdegree burns on back were hit by endotoxin at a dose of 20 mg/kg 24 hours after the burn injury. The rhubarb was given at a dose of 100mg (50mg/kg, q12h) orally just after the second hit. At the end of the treatment, the bacteria and fungi in colon were cultured and the incidence of bacterial translocation of rats in each group was detected.Results:1) The impact of different degrees of trauma on the number of the intestinal flora in colon:The number of the enterobacteriaceae and enterococci did not change significantly after burn, but the number of fungi significantly increased (P<0.05) and the number of lactobacilli decreased from 108 cfu/g to 106-107 cfu/g (P<0.05). With the "second-hit" by endotoxin, the number of enterobacteriaceae and enterococci were increased 103 and 102 orders of magnitude, which were significantly different to those of burn group (P<0.01), and the number of fungi was slightly higher than that of the normal control group, but the difference was no statistical significance, the number of lactobacilli significantly less than that of the normal control group (P<0.05).2) The impact of different degrees of trauma on the species of the intestinal flora in colon:In normal control group, escherichia coli was the sole species of Enterobacter, Enterococcus faecalis and geotrichum capitatum accounted for the majority of enterococci and fungi respectively. After the burn and "second hit" by endotoxin, escherichia coli, enterococcus faecalis and geotrichum capitatum remained the dominant bacterial strains in gut. The significant change after trauma was that the opportunistic pathogen such as pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii or bacillus proteus appeared.3) Bacterial translocation ratio in normal control group was 12.5%, bacterial translocation only occurs in mesenteric lymph nodes, translocation to blood and distant organs was not found. There is no increase in the rate of intestinal bacterial translocation after burn. With the "second-hit" by endotoxin, the rate of intestinal bacterial translocation was significantly increased (compared with the burn group, the individual translocation rate P<0.05; overall organs translocation rate P<0.01) and intestinal bacteria widely translocated to the blood, liver, lungs and other distant organs.4) The effect of rhubarb on intestinal microecology in sepsis rats:Rhubarb weakened the harm of burn and endotoxin on lactobacilli. Rhubarb can significantly reduce overall organs translocation rate in septic rats, especially reduce the translocation of opportunistic pathogen and intestinal bacterial translocation to the distant organs.Conclusion:1) Trauma can destroy the intestinal microecology and the decrease of probiotics in gut lead to the lost of colonization resistance that allow proliferation of opportunistic pathogen (such as pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii or bacillus proteus).2) In septic state, intestinal barrier function was severely inhibited, intestinal bacilli (especially pseudomonas aeruginosa, bacillus proteus, klebsiella pneumoniae and enterobacter cloacae) widely translocated to the blood flow, liver and lungs, which lead to intestinal infections.3) The rhubarb can prevent the loss of the number of anaerobic bacteria. In some extent, the rhubarb can protect gut biogeocoenosis and reduce the incidence of intestinal infection.PARTⅡEffect of Broad-spectrum Antibiotics on Gut Microflora and Bacterial Translocation in Scalded Rats and Sepsis RatsObjective:To approach the selection of broad-spectrum antibiotics on the intestinal flora and the effect on bacterial translocation in scalded rats and sepsis rats.Methods:120 SD rats with 30%Ⅲdegree burns on back were hit by endotoxin at a dose of 20 mg/kg 24 hours after the burn injury. The rhubarb was given at a dose of 100mg (50mg/kg, q12h) orally just after the second hit. The animals were treated with intraperitoneal injection of ceftriaxone or imipenem 24h after the injury (60mg/kg, q12h). At the end of the treatment, the bacteria and fungi in colon were cultured and the incidence of bacterial translocation of rats in each group was detected. Results:1) The impact of broad-spectrum antibiotics on the intestinal flora in colon: Ceftriaxone obviously reduced the number of enteric bacilli in scalded rats (P<0.01). On the contrary, the number of enteric bacilli significantly increased after nine-day treatment of imipenem (P<0.05). In sepsis rats, ceftriaxone and imipenem both obviously reduced the number of enteric bacilli (P<0.01). After the rats received antibiotics treatment, opportunistic pathogen became the dominant enteric bacilli, ceftriaxone showed the high selectivity for pseudomonas aeruginosa and acinetobacter baumannii and imipenem showed the selectivity for klebsiella pneumoniae and proteus. The antibiotics significantly increased the number of Enterococcus and fungi, E.faecium become the predominant flora in the gut and Candida albicans, Candida glabrata, Candida krusei and etc have emerged in gut. There is no significant change in the number of lactobacilli after antibiotics treatment.2) The impact of broad-spectrum antibiotics on the intestinal bacterial translocation:The rate of intestinal bacterial translocation was significantly increased and persistence of bacterial translocation to blood and distant organs was been seen after antibiotics treatment. Species of translocated bacteria is also change from bacilli enterococcus, specifically enterococcus faecium.3) The effect of rhubarb on intestinal microecology and bacterial translocation in scalded and sepsis rats:Rhubarb did not relieve the intestinal flora disturbance and can not effectively inhibit bacterial translocation caused by broad-spectrum antibiotics.Conclusion:1) Broad-spectrum antibiotics further increase the degree of intestinal flora disorder in scald and sepsis rats. Broad-spectrum antibiotics kill a large number of the sensitive intestinal flora and left space to drug-resistant strains to become dominant microorganisms in gut.2) The rate of intestinal bacterial translocation was significantly increased and persistence of bacterial translocation to blood and distant organs was been seen after antibiotics treatment. Species of translocated bacteria is also changing from bacilli enterococcus, specifically enterococcus faecium, further aggratated the enterogenic infections.3) Long-term use of antibiotics weakens the effects of rhubarb on gastrointestinal protective action of micro-ecological environment and intestinal bacterial translocation in vivo.PARTⅢThe Transmission of Drug Resistance Gene in Gut of Sepsis Rats under the Pressure of Broad-spectrum AntibioticsObjective:To establish intestinal colonization model of rats by Klebsiella pneumoniae resistant strains and continuously observe the drug resistance change of Klebsiella pneumoniae resistant strains and original E. coli under the pressure of broad-spectrum antibiotics.Methods:40 SD rats with 30%Ⅲdegree burns on back were hit by endotoxin at a dose of 10 mg/kg 24 hours after the burn injury. Two hours after burn or injection of endotoxin, esophageal inoculation of 108 CFU of ESBL-producing K.pneumoniae suspended in 1ml of phosphate buffered saline was performed. The animals were treated with intraperitoneal injection of ceftriaxone 24h after the lavage. At 1,3,6,9 day of the treatment and 2, 7,14,21,28 day of drug discontinuance, stool samples were collected and detected.Results:1) Intestinal colonization by Klebsiella pneumoniae resistant strains happened in scald and sepsis rats but not rats in normal control group. The amount of colonized Klebsiella pneumoniae resistant strains is higher in sepsis rats than that in scald rats.2) Colonized Klebsiella pneumoniae resistant strains disappeared soon from intestine in rats without broad-spectrum antibiotics use. Under the pressure of broad-spectrum antibiotics, Colonized Klebsiella pneumoniae resistant strains repeatedly appeared in the intestinal tract and the change of resistance to ceftriaxone occurred with part of the strains.3) Original E.coli in sepsis rats was found acquired the resistance gene from Klebsiella pneumoniae resistant strains on day 9 of the treatment with ceftriaxone.Conclusion:Intestinal colonization by external resistant strains can occur after trauma. Due to the use of broad-spectrum antibiotics, the loss of colonization resistance permit external resistant strains continuing presence in the intestinal tract and cause simultaneous induction of antibiotic resistance. Most dangerous hazard is that intestinal original bacteria can acquire the resistance gene from external resistant strains and persist in the gut.更多还原