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.     

气相色谱法检出和鉴定厌氧菌的实用研究

根据目前气相色谱法检出和鉴定厌氧菌工作中存在的实际问题,本文提出了一种去除蛋白胨、酵母浸膏、葡萄糖培养基(PYG)本底干扰物的简便方法,提高了对色谱图定性和半定量解析的准确性。同时又推荐了一套简易价廉的实验仪器和实验试剂。通过对若干厌氧菌株的检出和鉴定,得到了比较满意的结果。上述工作将有利于气相色谱检测厌氧菌技术的推广和应用。     

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。     

抗生素在体内外对厌氧菌和艰难梭菌细胞毒素生成的作用

氯林霉素、灭滴灵和甲砜霉素对大多数肠道厌氧菌的生长具抑制作用。氯林霉素还会破坏肠道菌群平衡,使原来受抑制的艰难梭菌得以定植,并在艰难梭菌浓度达10~8/g盲肠内含物时,检测到艰难梭菌细胞毒素。培养基中亚抑菌浓度的氯林霉素和灭滴灵会推迟艰难梭菌细胞霉素的生成。灭滴灵还可保护无菌小鼠及受氯林霉素处理的悉生小鼠免遭艰难梭菌细胞毒素的致死作用,从而证实了灭滴灵在伪膜性结肠炎临床治疗中的可用性。     

一株D型肉毒梭菌的分离和鉴定

从我国东海地区的海泥培养物中,分离出一株厌氧性芽孢杆菌。经细菌学、毒素血清学．细菌代谢产物的气相色谱分析及DNA中G+ct001％测定,鉴定为D型肉毒梭菌,编号为D85501。与国际参考菌株D359对照,D85 501菌株具有D型肉毒梭菌的总特性,还有自身的特点,是D型肉毒梭菌中的一新株,是我国分离的首株D型肉毒梭菌。     

艰难梭菌的研究进展

艰难梭菌为革兰阳性厌氧芽胞杆菌,可引起艰难梭菌相关性腹泻,导致一系列肠道感染症状和相关临床表现。近年来由于高致病株的出现、菌株耐药性的增加,艰难梭菌感染在全球呈蔓延趋势。本文就艰难梭菌的耐药机制、检测技术、防治及国内感染现状等作一简要综述。     

艰难梭菌感染的临床诊断与治疗研究进展

艰难梭菌感染可引起相关腹泻,且在住院患者中的发病率不断升高.根据腹泻的程度不同,艰难梭菌相关腹泻可分为轻、中、重和暴发型4型,临床诊断除了血常规外,毒素鉴定与内镜诊断也是重要的辅助检查手段.目前在临床治疗中仍以甲硝唑或万古霉素等抗生素进行药物治疗,可通过益生菌来调节肠道菌群实现艰难梭菌相关腹泻的预防.     

ICU患者艰难梭菌分离培养与 流行病学特征研究

目的了解ICU患者艰难梭菌的定植和感染情况,为预防艰难梭菌的流行提供参考。方法收集2016年9月至2017年6月福建医科大学附属第一医院ICU中139例住院时间7d的患者的粪便样本,对其进行选择性厌氧培养和质谱鉴定。对艰难梭菌培养阳性标本进行毒素基因(tcdA、tcdB、cdt A、cdtB)的PCR检测以及毒素A、B表型检测。收集所有患者的临床资料,并对艰难梭菌培养阳性患者的临床特征和实验室检查结果进行单因素分析和多因素回归分析。结果艰难梭菌检出率为17.27%(24/139)。其中,14株艰难梭菌的tcdA和tcdB基因检测阳性,占58.3%(14/24);10株为tcdA和tcdB基因检测阴性,占41.7%(10/24)。所有菌株二元毒素基因(ctdA/ctdB)均未检出。单因素分析提示,高龄、长时间住院、高淋巴细胞数、使用β-内酰胺类抗生素是艰难梭菌定植的高危因素;多因素回归分析提示,使用β-内酰胺类抗生素是艰难梭菌定植的独立危险因素(OR=3.881,P=0.039)。结论我院ICU可能存在艰难梭菌感染和传播的风险,对具有高龄、长期住院以及使用抗生素等高危因素的患者应进行艰难梭菌的监测,以防艰难梭菌的传播、感染和艰难梭菌相关性腹泻的发生。     

艰难梭菌基因编辑技术研究进展

艰难梭菌Clostridioesdifficile是一种革兰氏阳性、产芽孢、专性厌氧细菌,是医院相关性腹泻的主要病原体。近年来,随着强毒株的出现(如核糖体027型),其流行性与致死率逐年上升,因此对艰难梭菌生理、生化特征及致病机制的研究受到广泛重视。艰难梭菌生理、生化特征及致病机制研究又以建立其稳定、高效的基因编辑方法为必要前提。借助基因编辑工具,研究者可以扰动艰难梭菌核心生物学过程,在分子水平研究其分子致病机制。如Clos Tron技术在艰难梭菌毒素A (Toxin A)和毒素B (Toxin B)与其致病力关系的研究中起到了关键作用。文中以时间为主线综述了艰难梭菌基因编辑技术的发展历程和最新进展,并对艰难梭菌基因编辑技术未来的研究方向进行展望。     

艰难梭菌的快速鉴定试验

<正>现已充分证明,假膜性结肠炎几乎都是由艰难梭菌引起的,而且,抗生素相关性腹泻(约占25％)也与之有关。其实验诊断主要是取病人的粪便分离培养艰难梭菌,然后再行鉴定。然而现行的鉴定方法不是费时、需要贵重的仪器设备,就是重复性差或需要作补充鉴定试验。本文报道的是一种快速(30分钟内)、准确、重复性良好的确证方法。     

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

Abstract Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile . A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector λEMBL3. Out of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated λCd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone λCd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against λCd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.     

Secretome analysis of Clostridium difficile strains

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.     

酪酸梭菌与婴双歧杆菌对艰难梭菌体外生物拮抗作用的作用

用酪酸梭菌（Clostridium butyricum)和婴双歧杆菌（Bifidobacterium infantis)单独或联合艰难菌（Clostridium difficile)进行试管内的生物拮抗作用,将酪酸梭菌、婴儿双歧杆菌单独或酪酸梭菌和婴儿双歧杆菌联合分别与艰难梭菌以一定的比例等量混合后,接种于GAM液体培养基中进行厌氧培养。证明酪酸梭菌和婴儿双歧杆菌能明显抑制艰难梭菌的生长,并且两菌联合比各自单独培养时显示出更强的生物拮抗作用。     

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile. A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.     

The end products of the metabolism of aromatic amino acids by clostridia

The end products of the metabolism of phenylalanine, tyrosine and tryptophan by growing cultures of clostridia have been identified. The species used were Clostridium aminovalericum; C. bifermentans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. cochlearium; C. difficile; C. ghoni; C. histolyticum; C. lentoputrescens; C. limosum; C. lituseburense; C. malenomenatum; C. mangenoti; C. propionicum; C. putrefaciens; C. sordellii; C. sporogenes; C. sporosphaeroides; C. sticklandii; C. subterminale; C. tetani; C. tetanomorphum. The mixture of aromatic compounds formed, which depended upon the species, included phenyl acetic acid, phenyl propionic acid, phenyl lactic acid, phenol, p-cresol, p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, indole, indole acetic acid and indole propionic acid.Abbreviation GLC gas liquid chromatography     

In vivo lysogenization of a Clostridium difficile bacteriophage ФCD119

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic-associated diarrhea and colitis. In this study, we have documented the lysogeny of a C.?difficile bacteriophage in hamsters during C.?difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in?vivo isolated lysogens was affected due to ФCD119 lysogenization as in the case of in?vitro isolated ФCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C.?difficile host even during the infection process and highlights the importance of lysogeny of C.?difficile phages as an evolutionary adaptation for survival.     

Identification and susceptibility to antimicrobial agents of strictly anaerobic bacteria isolated from hospitalized patients

The aim of this study was to identify anaerobic strains isolated in 2001 from clinical specimens obtained from patients of Warsaw hospital and to evaluate a susceptibility of these strains to antimicrobial agents. In 2001 two hundred and twenty five clinical strains of obligate anaerobes were cultured, which were identified in the automatic ATB system (bioMérieux, France) using biochemical tests API 20 A. Drug-susceptibility of strains was determined also in ATB system with the use of ATB ANA strips. C. difficile strains were isolated on selective CCCA medium. Toxins A/B of C. difficile directly in stool specimens were detected by means of ELISA test (TechLab, USA). Fifty four strains of Gram-negative anaerobes (B. fragilis strains dominated) and 171 strains of Gram-positive anaerobes (the greatest number of strains belonged to genus Peptostreptococcus) were cultured from clinical specimens. In the cases of antibiotic-associated diarrhea 28 C. difficile strains were isolated and C. difficile toxins A/B were detected in 39 stool samples. The most active in vitro antimicrobials against Gram-negative anaerobes were metronidazole, imipenem, ticarcillin combined with clavulanic acid and piperacillin with tazobactam. Gram-positive, clinical strains of anaerobes were the most susceptible in vitro to beta-lactam antibiotics combined with beta-lactamase inhibitors (amoxicillin/clavulanate, piperacillin/tazobactam, ticarcillin/clavulanate) and imipenem.     

Production of p-cresol by Clostridium difficile on different basal media

The production of p -cresol by Clostridium difficile on a variety of agar basal media was investigated using gas-liquid chromatography. None of the basal media studied supported the production of large amounts of p -cresol. The addition of 0–1% p -hydroxyphenylacetic acid to a basal medium stimulated the production of p -cresol. If detection of p -cresol is to be used for the presumptive identification of C1. difficile then the basal medium should be supplemented with p -hydroxyphenylacetic acid.     

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.     

Mapping interactions between germinants and Clostridium difficile spores

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.