产气荚膜梭菌双圈溶血现象之培养及其毒素分析

通过实验介绍了产气荚膜梭菌在血琼脂平板上经数次传代培养后恢复毒力产生双圈溶血现象的培养方法,并阐明了导致该现象产生的两种毒素(θ毒素和α毒素)的作用机理。     

A型产气荚膜梭菌α毒素全基因的分子克隆与核苷酸序列分析

应用PCR技术,从A型产气荚膜梭菌菌株NCTC64609中扩增出A型产气荚膜梭菌α毒素全基因(cpa 1229基因)并将其克隆至pMDl8-T载体中。经转化、IPTG／X—gal选择培养,提取质粒,PCR和EcoRI／Pstl双酶切鉴定,筛选阳性重组克隆。经核苷酸序列分析证实,cpa1229基因阅读开放框架由1194bp组成。经GenBank检索对照分析,cap1229基因序列与国外献报道同源性达98．3％,表明本实验所克隆的cpa1229基因即为A型产气荚膜梭菌α毒素基因。     

产气荚膜梭菌α毒素基因的克隆及结构分析

应用PCR技术,从产气荚膜梭菌菌株NCTC64609中,扩增出A型产气荚膜梭菌α毒素基因C端片段(cpa408),并将其克隆至pMDl8-T载体中．经转化,α互补蓝白菌落选择培养,提取质粒,进行PCR和Eco RⅠ、PstⅠ双酶切鉴定,筛选出阳性重组克隆．经核苷酸序列分析证实,cpa408基因阅读开放框架由372个核苷酸组成,编码124个氨基酸．经计算机分析,cpa408基因序列与国外文献报道的A型产气荚膜梭菌α毒素基因C端片段同源性达99％以上,表明所克隆的基因即为α毒素基因C端片段．     

产气荚膜梭菌ε毒素导致多器官组织损伤

【背景】产气荚膜梭菌ε毒素(ε toxin,ETX)是由B型和D型产气荚膜梭菌产生的一种肠毒素,具有迅速的致命性和经济上的破坏性。【目的】研究ETX对小鼠多个脏器的损伤情况及其在不同小肠肠段的结合情况。【方法】构建纯化偶联红色荧光蛋白的mScarlet-ETX,使用小动物活体成像观察其在小鼠体内的累积情况;使用病理学技术观察ε毒素对小鼠各个脏器和不同肠段的结合情况,以及对脏器的损伤情况。【结果】产气荚膜梭菌感染后,产生的ETX能累积于脑、肾、肺、肝、脾和心脏组织中,并能导致这些脏器的损伤。ETX在小鼠的小肠中主要累积于结肠中。【结论】产气荚膜梭菌ε毒素导致多器官组织损伤,并主要在结肠上被吸收,为治疗和预防携带ε毒素的产气荚膜梭菌感染提供了理论依据。     

重组A型产气荚膜梭菌α毒素C端片段的分离纯化

押在构建出高表达、高活性的A型产气荚膜梭菌α毒素保护性抗原工程菌株pBV/cpa408的基础上,对表达产物进行了分离纯化研究。诱导表达菌液离心后,超声破碎沉淀细菌,上清用80%饱和硫酸铵沉淀,沉淀蛋白经透析,上凝胶过滤层析柱分离纯化,得到纯度达95%以上的表达目的蛋白,经SDS-PAGE测定,相对分子质量为15.5×103,序列与文献报道的相符。     

产气荚膜梭菌前噬菌体的分布特点及遗传进化分析

【目的】分析和研究产气荚膜梭菌中前噬菌体的分布情况、基因组特点及遗传进化关系。【方法】利用PHASTER (phage search tool enhanced release)软件预测产气荚膜梭菌携带的前噬菌体,基于ANI (average nucleotide identity)值对前噬菌体进行分群,利用CARD (comprehensive antibiotic research database)、Res Finder 4.1、VFDB (virulence factors database)和Bac Met(antibacterial biocide&metal resistance genes database)分析前噬菌体携带的耐药基因、毒力基因、抗菌剂/金属离子抗性基因,利用CRISPRCas Finder分析产气荚膜梭菌的CRISPR-Cas系统,利用MEGA 7.0进行前噬菌体的遗传进化关系分析。【结果】产气荚膜梭菌平均携带前噬菌体2.67条,其长度呈双峰分布,平均占基因组2.23%;前噬菌体不携带耐药基因,但携带了α毒素、唾液酶和溶血素等毒力基因以及重金属...     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌a, b, e, t 毒素基因cpa、cpb、etx 及iA序列合成了针对4种毒素基因的4对特异引物, 建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法。结果本所保存的A, B, C, D, E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带, 而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性; 将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段。并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定, 并与毒素中和试验鉴定结果进行了比较, 结果表明两种方法具有较高的符合率。本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义。     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌α,β,ε,τ毒素基因cpa、cpb,etx及iA序列合成了针对4种毒素基因的4对特异引物,建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法.结果本所保存的A,B,c,D,E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带,而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性;将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段.并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定,并与毒素中和试验鉴定结果进行了比较,结果表明两种方法具有较高的符合率.本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义.     

兼性厌氧复合菌群H纤维素降解和产乙醇能力及生态组成初探

通过限制性培养条件和连续继代培养,筛选获得了一组具有高效稳定降解纤维素能力的复合菌群H。该菌群在传代30代以上仍能保持各项性状稳定,其工作pH为6～9,3 d可以完全降解置于100 mL PCS缓冲液培养基中的滤纸,发酵液中能够检出1.54 g/L乙醇。通过16S rDNA扩增和DGGE的方法,对菌群在不同阶段的微生物组成进行了研究,确定了琥珀酸嗜热梭菌Clostridium thermo succinogene、产气荚膜梭菌Clostridium straminisolvens和紫色板蓝根梭菌Clostridium isatidis等多种可直接实现纤维素到乙醇转化的菌株。菌群通过菌种之间的协同作用,共同维持了体系的稳定及降解能力的稳定。明确菌系的组成,对于进一步研究菌群降解机理、优化菌群和提高乙醇产率意义重大。     

厌氧菌的译名问题

本世纪70年代厌氧细菌学的崛起,是近代微生物学的重要进展。近十年来由于国内有关厌氧菌的工作日益发展壮大,有关厌氧菌书籍也日益增多。但由于未经统一,厌氧菌译名多种多样,同一种细菌可有几种名称,使人莫衷一是,这些问题表现在下列几个方面。 1.一种细菌有许多译名例如Clostridium perfringens,过去译成产气荚膜梭状芽胞杆菌或产气荚膜杆菌,现在译成产气荚膜梭菌,但也有译成突破芽胞梭菌或镶边梭菌。从人名上更有称之为魏氏(威氏、韦氏)梭菌的。Clostridium现在称为梭菌属,但也有称为小梭菌的。除非在每个译名之后都附有原文,否则读者见此如堕五里雾中,无法识别了。 2.菌属名称翻译有错误的如Eubacterium,根据Bergey氏鉴定细菌学手册第8版(1974)和Borgey氏系统细菌学手册第一版第一卷(1984)和第二卷(1986)的记载,Eu是good,well,beneficial(not as opposed to pseudo),应译成良好,有益的,不是假的反面。Eubacterium是beneficial bacterium,即有益的,良好的细菌,并且特别注明不是假的反面,即不是“真”。这个错误名称     

Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.     

Growth of Clostridium perfringens in Food Proteins Previously Exposed to Proteolytic Bacilli

Proteolytic sporeforming bacteria capable of surviving processing heat treatments in synthetic or fabricated protein foods exhibited no antagonistic effects on growth of Clostridium perfringens, but instead shortened the lag of subsequent growth of C. perfringens in sodium caseinate and isolated soy protein. Bacillus subtilis A cells were cultured in 3% sodium caseinate or isolated soy protein solutions. The subsequent effect on the lag time and growth of C. perfringens type A (strain S40) at 45 C was measured by colony count or absorbance at 650 nm, or both. B. subtilis incubation for 12 h or more in sodium caseinate reduced the C. perfringens lag by 3 h. Incubation of 8 h or more in isolated soy protein reduced the lag time by 1.5 h. Molecular sieving of the B. subtilis-treated sodium caseinate revealed that all molecular sizes yielded a similar reduced lag time. Diethylaminoethyl-Sephadex ion exchange fractionation and subsequent amino acid analysis indicated that the lag time reduction caused by B. subtilis incubation was not related to charge of the peptides nor to their amino acid composition. Apparently the shortened C. perfringens lag in these B. subtilis-hydrolyzed food proteins was a result of the protein being more readily available for utilization by C. perfringens.     

Correlations Between Virulence and Other Characters of Clostridium perfringens Type A

The correlation analysis which has already proved its value in ecology has not yet been applied to the determination of virulence indicators. Its application to a group of Clostridium perfringens type A strains has brought out some characters that may be considered as virulence indicators. This study suggests that the toxicity of the culture supernatant fluids for mice is significantly correlated with the virulence for mice and guinea pigs. A significant correlation was found between the virulence of the fluid cultures for mice or guinea pigs and the coagulation of milk, production of gas (in deep agar), hydrogen sulfide production, and fermentation of glucose, sucrose, maltose, and levulose.     

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.     

Construction of a fusion protein carrying antigenic determinants of enteric clostridial toxins

Clostridium difficile and Clostridium perfringens type A are infectious agents of enteric diseases. The main virulence factors of these microorganisms include toxins A and B of C. difficile (ToxA and ToxB) and enterotoxin of C. perfringens (Cpe). In this study genetic constructions have been created for the expression of ToxA, ToxB and Cpe fragments either as individual components or as a hybrid multidomain (ToxA-ToxB-Cpe) protein. Rabbit monospecific sera raised against individual peptides reacted with the chimeric product indicating that the corresponding antigenic determinants were correctly expressed on the hybrid molecule. Furthermore, mice immunized with the fusion protein produced antibodies specific to each of the three separate components. These data suggest that the constructed three-domain molecule could be used in future studies for development of a vaccine against enteric clostridial diseases.     

Response of White Mice to Cells and Culture Constituents of Clostridium perfringens

Broth cultures of Clostridium perfringens (ATCC 10543) were fractionated by ammonium sulfate precipitation and Sephadex G-150 chromatography. Components isolated, as well as some enzymes present in the culture, were assayed for toxicity by feeding to white mice. Early work indicated that when a meat-fat-starch slurry, infected with C. perfringens, was fed to mice, the intestinal passage time was reduced. By using large numbers of mice as test animals and analyzing the data statistically, we found that C. perfringens and several fractions from the culture supernatant significantly affected the mice. A toxic material present in the supernatant was not identifiable as phospholipase C. Phospholipase C and physphorylcholine affected the intestinal passage time of the mice only when large amounts were given. The enzyme, neuraminidase, and another unidentified compound present in the supernatant affected the passage time when very small amounts were fed to mice.     

Cloning and expression of the Mycobacterium fortuitum Superoxide dismutase gene

Abstract The beta-toxin gene from Clostridium perfringens type C was cloned and expressed as a glutathione S-transferase fusion protein in Escherichia coll . The DNA sequence was determined and compared to the type B sequence. Two nucleotide differences were found in the protein coding sequence, resulting in one amino acid difference between the two proteins. The purified beta-toxin fusion protein is not toxic in mice, but rabbit antiserum raised against it neutralises the toxic effect of C. perfringens type C culture filtrate in mice.     

Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D.

To obtain high yields of extracellular enzymes and toxins for immunological analysis, type culture collection strains of Clostridium perfringens types A to D and 28 fresh isolates of C. perfringens type A from humans were grown in fermenters under controlled conditions in a pre-reduced proteose peptone medium. The type culture collection strains all showed different characteristics with respect to growth rates and pH optima for growth. Production of phospholipase C (alpha-toxin), haemolysin and lethal activity varied considerably between the different types. Growth and extracellular protein production in fermenters with pH control and static or stirred cultures were compared. Production of all extracellular proteins measured was markedly improved by cultivation in fermenters with pH control. Strain ATCC13124 produced five times more phospholipase C than any of 28 freshly isolated strains of C. perfringens type A, grown under identical conditions. Haemolytic and lethal activities of the ATCC strain were equal or superior to the activities of any of the freshly isolated strains. There were no differences in the bacterial yields and in the production of extracellular toxins between type A strains isolated from clinical cases of gas gangrene and abdominal wounds, and those isolated from faecal samples from healthy persons.     

TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks

Clostridium perfringens type A and type C are causative agents of necrotic enteritis (NE) in poultry. TpeL, a recently-described novel member of the family of large clostridial cytotoxins, was found in C. perfringens type C. Others have since reported TpeL in type A isolates from NE outbreaks, suggesting that it may contribute to the pathogenesis of NE. The virulence of TpeL-positive and -negative C. perfringens strains from cases of NE was examined by challenge of broiler chicks. Gross lesions typical of NE were observed in all challenged birds, and those inoculated with TpeL(pos) strains had higher average macroscopic lesion scores than those inoculated with a TpeL(neg) strain. Infection with TpeL(pos) strains may yield disease with a more rapid course and higher case fatality rate. Thus, TpeL may potentiate the effect of other virulence attributes of NE strains of C. perfringens. However, TpeL(pos) and Tpel(neg) strains compared here were not isogenic, and definitive results await the production and testing of specific TpeL mutants.     

Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.