具有临床代表性的艰难梭菌感染小鼠稳定模型的构建

目的构建临床代表性好、稳定性佳的艰难梭菌感染小鼠模型,为艰难梭菌感染相关疾病提供研究工具。方法选择C57BL/6、BALB/c、昆明小鼠,经抗生素诱导后,分别予以不同浓度(108 CFU/mL~10~(10) CFU/mL)的临床分离菌株混悬液灌胃,观察不同品系小鼠不同时间点腹泻、全身情况及结肠组织病理学变化。结果灌菌后,BALB/c小鼠10~(10) CFU/mL浓度组全部出现腹泻,死亡率为16.7%;其他实验组小鼠腹泻程度差异较大或仅部分出现轻度腹泻。腹泻小鼠表现为稀烂便甚至水样便和湿尾现象,体重减轻,肠道病理显示结肠黏膜充血水肿伴炎性细胞浸润。结论经5种抗生素灌胃9d+克林霉素腹腔注射诱导,10~(10) CFU/mL艰难梭菌活菌混悬液灌胃后构建的BALB/c小鼠艰难梭菌感染模型最稳定。     

对引入仓鼠菌群的C_3H无菌小鼠拮抗艰难梭菌定居实验模型的研究

正常成年仓鼠肠道菌群可拮抗艰难梭菌在该动物肠道中定居。将该菌群转移到无菌小鼠并经过抗生素和热(70℃,10min)简化处理后,获得一组由严格厌氧菌组成的菌群,仍有拮抗艰难梭菌的能力,由此建立了一研究拮抗艰难梭菌在肠道中定居的实验动物模型。作者研究了屏障菌群与艰难梭菌相互作用的可能机制,并对与艰难梭菌有关的伪膜性结肠炎的病原学进行了讨论。     

重组艰难梭菌毒素B对小鼠结肠癌CT26细胞的诱导凋亡作用

本文探讨了重组艰难梭菌毒素B(rTcd B)对小鼠结肠癌CT26细胞的诱导凋亡作用。采用不同浓度rTcd B处理CT26细胞, 通过MTT法检测细胞增殖抑制率; 比色法测定Caspase 3活性; 细胞形态学和流式细胞技术检测细胞凋亡。结果表明, rTcd B显著抑制了CT26细胞的增殖, 并呈时间?剂量依赖性; Caspase 3活性在处理6 h后显著升高, 至18 h达到最大值, 与对照组相比差异显著, 具有统计学意义(P<0.05); 荧光显微镜观察到典型细胞凋亡形态学变化, 细胞膜内侧的磷脂酰丝氨酸(PS)异位到了膜外侧, 细胞膜呈明亮的绿色荧光; 通过流式细胞仪检测结果表明, 细胞凋亡率呈时间?剂量依赖性增加。实验结果表明, 重组艰难梭菌毒素B能够诱导小鼠结肠癌CT26细胞凋亡。     

艰难梭菌研究现状

艰难梭菌(Clostridium difficile,CD)是产芽胞革兰阳性厌氧菌,产毒素艰难梭菌可导致艰难梭菌感染(Clostridium difficile infection,CDI)。产毒素艰难梭菌已被公认是抗菌药物相关性腹泻最常见的病原体,也是发达国家最常见的导致住院患者腹泻感染的原因之一。高毒力菌株导致全世界范围内CDI的发病率和死亡率增加。如何快速而准确地检测CD一直是备受关注的问题,国内外多推荐两步法和三步法进行检测。艰难梭菌治疗的一线用药是甲硝唑、万古霉素以及非达霉素,应用粪便微生物移植(faecal microbiota transplantation,FMT)也可作为治疗轻度或严重复发CDI(recurrence Clostridium difficile infection,rCDI)的可选方法。艰难梭菌的芽胞能抵抗不良环境,但抗菌药物的使用,医院环境的复杂性和医务工作者的手等因素会导致艰难梭菌易于传播,因此医疗机构应加强艰难梭菌的预防。本文主要对艰难梭菌的流行病学、致病机制、检测及治疗进行综述。     

酪酸梭菌对艰难梭菌感染的防治研究

目的:观察酪酸梭菌对艰难梭菌感染的防治效果.方法:用艰难梭菌产毒株人工感染BALB/C小鼠,感染前后分别用酪酸梭菌进行预防与治疗,并检测盲肠内容物细胞毒性和进行肠黏膜病理观察.结果:酪酸梭菌不能预防艰难梭菌的感染,但在艰难梭菌感染后则能明显降低艰难梭菌的产毒力和盲肠黏膜的病理损伤.结论:酪酸梭菌对小鼠艰难梭菌感染有明显的治疗作用.     

粪菌移植治疗艰难梭菌感染的研究进展

艰难梭菌感染(Clostridium difficile infection,CDI)是院内抗生素相关性腹泻的最重要因素之一,其主要临床表现包括血便、腹泻、中毒性巨结肠、伪膜性肠炎等。近年来,CDI发病率、复发率、死亡率和治疗费用均明显增加,但其各种治疗方法均有局限性,尤其是抗生素治疗复发性艰难梭菌感染已面临许多棘手问题。目前证实肠道菌群失调和CDI感染关系密切,肠道菌群紊乱后导致艰难梭菌过度繁殖并释放毒素,可导致艰难梭菌感染。粪菌移植作为重建肠道菌群的重要方式,已成为复发性、难治性艰难梭菌感染最有效的治疗方式之一,不良反应极少。本文就国内外粪菌移植治疗艰难梭菌感染的研究进展作一综述。     

上海部分地区儿童艰难梭菌相关性腹泻的临床分析

本文旨在监测儿童腹泻中艰难梭菌相关性腹泻(CDAD)的发病情况,并对其进行回顾性临床分析。对复旦大学附属儿科医院2007年2月～9月111例腹泻患儿的粪便标本进行艰难梭菌毒素A检测及粪便厌氧菌培养,对所有患儿进行回顾性病史采集及分析,并对粪便培养所得的4株艰难梭菌菌株用多位点可变数目串联重复序列分析(MLVA)技术进行同源性分析。111例患者中,艰难梭菌毒素A检测及艰难梭菌培养均为阳性者无,艰难梭菌毒素A阳性而艰难梭菌培养阴性者16例,艰难梭菌毒素A阴性而艰难梭菌培养阳性者4例,艰难梭菌毒素A及艰难梭菌培养均阴性者91例。比较院内、院外组3种不同病程腹泻CDAD的发病率,无显著差异。MLVA分析发现粪便培养得到的4株艰难梭菌菌株有部分同源性。结果提示,目前上海部分地区儿童CDAD发病情况为散发,但彼此之间有部分同源性;院内、外获得性腹泻的CDAD发病率无显著差异;艰难梭菌毒素A阳性或艰难梭菌培养阳性的病例在临床表现上与艰难梭菌毒素A阴性且艰难梭菌培养阴性的腹泻病例无显著差异。     

无菌大白鼠肠道中破伤风梭菌的生长和毒素的产生

<正>梭菌是在全世界范围内广泛分布的厌气性芽胞菌。肉毒梭菌和破伤风梭菌能产生较强的神经毒素,分别引起典型的肉毒中毒和破伤风疾病。肉毒中毒大都和吃污染肉毒毒素的食物有关。过去一直认为肉毒梭菌在肠道中不能生长和产生毒素;但是,近来的研究表明,肉毒梭菌能在新生小鼠和新近给了广谱抗菌素治疗的成鼠肠道中生长并产生毒素。最近认识到,新生儿肉毒中毒乃是婴儿猝死症的原因之一。因为肉毒梭菌芽胞能在     

艰难拟梭菌毒素致病机制及毒素基因调控的研究进展

艰难拟梭菌(Clostridioides difficile)是一种产芽孢的革兰氏阳性专性厌氧杆菌,是引起抗生素相关性腹泻的主要致病菌。艰难拟梭菌产生的毒素A和毒素B在其致病过程中发挥关键作用。毒素发挥毒性作用依赖其4个功能结构域：葡萄糖基转移酶结合域、半胱氨酸蛋白酶结合域、易位域和受体结合域。毒素的受体结合域识别并结合细胞表面受体,介导毒素内吞并形成内体。经过自体催化切割,毒素将真正的毒性片段——葡萄糖基转移酶结合域释放到胞浆中,葡萄糖基转移酶能够失活宿主肠上皮细胞内的GTP酶导致细胞骨架解聚和坏死,进而引起腹泻和伪膜性结肠炎等临床症状。艰难拟梭菌毒素产生受致病基因座内及基因座外许多调控因子的调节。tcdR和tcdC基因位于致病基因座内,对毒素基因的表达分别起促进和抑制作用,而基因座外如spo0A、codY等基因则通过抑制tcdR的表达从而间接影响毒素蛋白产生。本文将重点介绍艰难拟梭菌毒素的致病过程和影响毒素基因表达的分子调控机制,以期为开发针对毒素的治疗手段提供新思路。     

艰难拟梭菌毒素致病机制及毒素基因调控的研究进展

艰难拟梭菌(Clostridioides difficile)是一种产芽孢的革兰氏阳性专性厌氧杆菌,是引起抗生素相关性腹泻的主要致病菌。艰难拟梭菌产生的毒素A和毒素B在其致病过程中发挥关键作用。毒素发挥毒性作用依赖其4个功能结构域:葡萄糖基转移酶结合域、半胱氨酸蛋白酶结合域、易位域和受体结合域。毒素的受体结合域识别并结合细胞表面受体,介导毒素内吞并形成内体。经过自体催化切割,毒素将真正的毒性片段——葡萄糖基转移酶结合域释放到胞浆中,葡萄糖基转移酶能够失活宿主肠上皮细胞内的GTP酶导致细胞骨架解聚和坏死,进而引起腹泻和伪膜性结肠炎等临床症状。艰难拟梭菌毒素产生受致病基因座内及基因座外许多调控因子的调节。tcdR和tcdC基因位于致病基因座内,对毒素基因的表达分别起促进和抑制作用,而基因座外如spo0A、codY等基因则通过抑制tcdR的表达从而间接影响毒素蛋白产生。本文将重点介绍艰难拟梭菌毒素的致病过程和影响毒素基因表达的分子调控机制,以期为开发针对毒素的治疗手段提供新思路。     

Intestinal bacteria antagonistic to Clostridium difficile in mice

Overgrowth by Clostridium difficile has been reported in conventional mice injected intraperitoneally with ampicillin. In this study, we aimed to determine which types of indigenous intestinal bacteria were eliminated by ampicillin to allow overgrowth by C. difficile. C. difficile overgrowth was associated with a decrease in the numbers of lactobacilli, an increase in bacteroidaceae and a slight decrease in the frequency of isolation of fusiform-shaped bacteria (clostridia). C. difficile cytotoxin was detected in caeca from mice in which the numbers of C. difficile were greater than 10(5) per gram of faeces. Gnotobiotic mice were inoculated with various groups of intestinal anaerobes to determine which members of the indigenous flora would antagonize C. difficile. Gnotobiotic mice inoculated with three strains of lactobacilli, 37 strains of bacteroides or 46 strains of clostridia isolated from limited-flora mice were unable to eliminate C. difficile. C. difficile was eliminated, however, from the gastrointestinal tracts of gnotobiotic mice inoculated with whole faeces or chloroform-treated faeces from conventional mice or whole faeces from limited-flora mice containing only clostridia.     

Prevention of Clostridium difficile induced mortality in gnotobiotic mice by Saccharomyces boulardii

Oral preventive treatment of gnotobiotic mice by Saccharomyces boulardii significantly decreased mortality following Clostridium difficile infection. A single S. boulardii ingestion protected 16% of mice, whereas 56% were protected when S. boulardii was given continuously in the drinking water. No direct antagonistic effect of the yeast on C. difficile numbers was detected, whereas a modulation of fecal cytotoxin production was demonstrated.     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。     

Survey of susceptibility of clinical Clostridium diffiicile strains isolated from patients hospitalised in different departments of paediatric hospital to antimicrobial agents

This study was performed to determine the susceptibility of 50 C. difficile strains isolated from faecal samples of children suspected to antibiotic associated diarrhea (AAD) to antimicrobial agents: metronidazole, vancomycin, erythromycin, clindamycin, ciprofloxacin, moxifloksacin, gatifloksacin and imipenem. The all C. difficile strains were sensitived to metronidazole and vancomycin. Twenty six per cent of strains were resistant to erythromycin and clindamycin (MLS(B) type resistance). Resitance to ciprofloxacin, moxifloxacin, gatifloxacin and imipenem was detected in 98%, 8%, 8% and 30% of C. difficile strains, respectively.     

Effect of Streptococcus parvulus and Peptostreptococcus magnus on cytotoxin levels of Clostridium difficile in anaerobic continuous flow culture

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.     

气相色谱—质谱技术对艰难梭菌细胞的挥发性脂肪酸成份的鉴定

应用气相色谱技术测定艰难梭菌培养物和悉生小鼠盲肠内含物中的挥发性脂肪酸含量,并用气相色谱—质谱技术加以鉴定。结果表明2-甲基丁酸是艰难梭菌的特征性产物,以前曾被其他作者错误地鉴定为异戊酸。     

Modulation of cytotoxin production by Clostridium difficile in the intestinal tracts of gnotobiotic mice inoculated with various human intestinal bacteria

Gnotobiotic mice died 2 days after inoculation of a cytotoxigenic Clostridium difficile strain. Protection occurred when mice were previously inoculated with a strain of Escherichia coli or Bifidobacterium bifidum. Intestinal cytotoxin production was highly reduced in the surviving mice, whereas the C. difficile population level did not decrease to a great extent.     

Modulation of cytotoxin production by Clostridium difficile in the intestinal tracts of gnotobiotic mice inoculated with various human intestinal bacteria.

Gnotobiotic mice died 2 days after inoculation of a cytotoxigenic Clostridium difficile strain. Protection occurred when mice were previously inoculated with a strain of Escherichia coli or Bifidobacterium bifidum. Intestinal cytotoxin production was highly reduced in the surviving mice, whereas the C. difficile population level did not decrease to a great extent.     

Effect of toxins produced by various Clostridium difficile strains on cecum size reduction in gnotobiotic mice

Inoculation of axenic mice with Clostridium difficile strains induced a significant reduction in ceca weight (dry or wet), whereas a nontoxinogenic strain led to a partial reduction. A strain, which produces cytotoxin and no enterotoxin in vivo, caused a reduction similar to that observed with a nontoxinogenic strain. Simultaneous cytotoxin and enterotoxin production by various C. difficile strains caused the cecum size to diminish to that observed for conventional control mice.