应用多重PCR鉴定对人致病的产气荚膜梭菌毒素

目的研究用多重PCR的方法鉴定产气荚膜梭菌及分型毒素,为开展基因诊断做好准备。方法根据GenBank已经发表的产气荚膜梭菌毒素基因序列,设计出针对CPα、CPβ、CPE毒素基因的3对特异引物,运用多重PCR的方法鉴定出产气荚膜梭菌并对其毒素基因进行分型。结果经电泳鉴定,多重PCR成功的扩增出预先设计的3条特异的目的条带。结论所设计的多重PCR反应体系能够得到产气荚膜梭菌的特异序列,同时能够鉴定分型3种对人致病的毒素序列,为进一步开展基因诊断打下基础。     

一起由C型产气荚膜梭菌引起的食源性疾病致病因子检测

目的检测食物中毒样本中的产气荚膜梭菌,为同类事件检测和判定提供依据。方法利用快速显微镜便检锁定检测方向;利用TSC培养基厌氧培养产气荚膜梭菌;利用产气荚膜梭菌α、β、ε、ι毒素和CPE、β2肠毒素PCR扩增检测产气荚膜梭菌所含肠毒素类型。结果在食品留样、环境涂抹拭子和患者粪便标本中共检出产气荚膜梭菌阳性样本8例。1份食品样本和5份患者粪便样本中分离株毒素携带情况完全一致,为含有α、β和肠毒素β2的C型产气荚膜梭菌;从环境试子中分离的菌株6种毒素均为阴性。结论此次事件是一起由C型产气荚膜梭菌毒素导致的细菌性食物中毒,食品与环境之间存在产气荚膜梭菌的交叉污染。作为重要食物中毒致病因的产气荚膜梭菌的监测与检测工作有待开展与标准化。     

运用压电石英晶体生物传感器检测产气荚膜梭菌α毒素的实验研究

目的研究运用压电石英晶体生物传感器对产气荚膜梭菌α毒素（CPα）进行检测的方法和反应条件。方法按照碱基配对的原则,设计特异的产气荚膜梭菌α毒素基因寡核苷酸探针并利用巯基自组装技术固定在石英晶体上,运用压电石英晶体生物传感器的“质量效应”对聚合酶链反应（PCR）扩增得到的产气荚膜梭菌α毒素靶基因进行杂交检测,并探讨不同pH值和不同离子强度的缓冲液对检测的影响。结果压电石英晶体生物传感器成功检测出产气荚膜梭菌α毒素;pH7.6的缓冲液和0.64mol/L的NaCl溶液最适合于检测。结论压电石英晶体生物传感器能够有效地检测产气荚膜梭菌α毒素,有望对临床上开展毒素诊断提供一种新的手段。     

利用产气荚膜梭菌作为泳滩水质污染指标菌的检测方法

目的评估产气荚膜梭菌作为泳滩水质污染指标菌,建立更快速及可靠的方法。方法抽取澳门黑沙海滩及竹湾海滩的泳滩水作为样本,比较双套管法定量检测产气荚膜梭菌,利用滤膜法定量检测大肠埃希菌数。结果产气荚膜梭菌及大肠埃希菌结果数据无相关性(P0.05)。粪大肠菌群与大肠埃希菌呈正相关(P0.05)。厌氧亚硫酸盐还原梭菌与产气荚膜梭菌呈正相关(P0.05)。产气荚膜梭菌采样点平均值不合格数远多于大肠埃希菌不合格数(P0.05)。结论利用双套管法定量检测产气荚膜梭菌方便快捷,符合公共卫生检测上的效益,更全面反映水质污染情况,故此建议加入产气荚膜梭菌作为澳门泳滩水的指标微生物。     

食品中产气荚膜梭菌α毒素基因快速检测方法的研究

目的建立分子信标荧光PCR体系检测产气荚膜梭菌的快速检测方法,应用于食物中毒快速诊断和食品微生物检验。方法根据GenBank公布的保守序列,针对α毒素基因设计一对引物和分子信标探针,用FAM荧光剂标记探针的5’端,并进行特异性和灵敏度分析,同时以10种细菌作对照。结果分子信标荧光PCR反应体系检测10种细菌,只有产气荚膜梭菌出现特异荧光信号,其他均无荧光信号,而且与其他细菌无交叉反应。对40份食品样品进行检测,3份产气荚膜梭菌PCR阳性,其余样品为阴性,检测仅需2 h。3份阳性样本经传统方法培养,2份有检出产气荚膜梭菌。结论分子信标荧光PCR检测体系快速、灵敏度高,特异性强,可用于产气荚膜梭菌食物中毒的快速诊断和食品污染物及感染性腹泻等监测工作中,为食源性疾病的分子流行病学调查提供新的检测手段。     

南平市肉与肉制品产气荚膜梭菌污染调查

目的了解南平市生畜肉及含肉馅速冻米面制品产气荚膜梭菌污染状况,发现安全隐患,为食品安全风险评估提供依据。方法按2017年福建省卫生计生系统食品中微生物及致病因子监测方案,采集南平市农贸市场、超市、肉制品小摊的生畜肉和含肉馅速冻米面制品共136份,按GB 4789.13-2012检测产气荚膜梭菌。结果样品中产气荚膜梭菌总检出率19.1%(26/136),其中生猪肉检出率35.0%(21/60)、生羊肉(3/10)、生牛肉(2/10);含肉馅速冻米面制品56份未检出。结论南平市生畜肉中存在不同程度产气荚膜梭菌污染,应加强食品卫生监管和食物中毒样品中产气荚膜梭菌检测。     

应用DNA探针技术快速检测食品中产气荚膜梭菌产肠毒素基因的研究

产气荚膜梭菌是引起食物中毒的病原菌,其为G-厌氧菌。有芽胞,常存在于自然界、健康人和动物肠道内。产气荚膜梭菌食物中毒的起因物质是该菌在形成芽胞时产生的A型肠毒素。因此,检测产气荚膜梭菌的产肠毒素株是非常必要的。本实验建立一种基因探针方法,能快速准确地检出产气荚膜梭菌的产毒株。我们于1999年5月～10月间,共检测样品180份,结果报告如下。     

用聚合酶链反应法检测水中产气荚膜梭菌

1980年ＢｉｓｓｏｎＪＷ和ＣａｂｅｌｌｉＶＪ指出[1],产气荚膜梭菌繁殖体的出现表示水受到新近粪便污染,而其芽胞的出现则表明污染为陈旧性的,并提出产气荚膜梭菌作为水污染的指示菌有一定的卫生学意义。但它存在的一个主要缺陷是没有简易、快速、经济的检测方法。为此,我们对检测水中产气荚膜梭菌的聚合酶链反应(ＰＣＲ)方法进行了研究。材料与方法1.菌株来源:产气荚膜梭菌20株(肠毒性产气荚膜梭菌14株;非肠毒性产气荚膜梭菌6株),其中产气荚膜梭菌肠毒株ＮＣＴＣ8239、Ｃ581购于卫生部生物药品鉴定所,其余由西京医院检验科提供。2.主要试…     

一起耐热型A型产气荚膜梭菌食物中毒的病原菌分离

一起耐热型Ａ型产气荚膜梭菌食物中毒的病原菌分离南京市卫生防疫站张彬彬陈晓蔚Ａ型产气荚膜梭菌与人类关系密切，其不耐热型主要产生α毒素，引起创伤感染。而耐热型主要产生肠毒素，引起食物中毒。禽、肉等食品易被该菌污染。在加热烹调后氧化还原电位降低，在冷却过程...     

三例产气荚膜梭菌感染病例医院感染防控并文献回顾

目的 了解不同部位产气荚膜梭菌感染患者的传播方式以及医院感染防控措施。方法 回顾性收集某院3例不同部位产气荚膜梭菌感染患者的临床资料、医院感染防控措施和环境采样结果,并查阅相关文献进行分析总结。结果 3例产气荚膜梭菌感染分别为盆腔气性坏疽、肝脓肿、血流感染,1例在在负压层流手术间和负压层流病房的回风口（栅栏+过滤网）培养出产气荚膜梭菌生长,余2例环境卫生学结果正常。结论 针对产气荚膜梭菌感染患者需严格执行环境清洁消毒与隔离,气性坏疽患者感染部位产气荚膜梭菌可远距离播散、污染环境,推荐采用独立非层流的感染手术间,其他部位感染者可实施单间、同病种隔离。     

Potential protective immunogenicity of recombinant Clostridium perfringens α-β2-β1 fusion toxin in mice, sows and cows

Clostridial toxins are main pathogenic virulence of Clostridium perfringens that have been associated with a wide range of diseases in both humans and domestic animals. Genetically engineered toxoids have been shown to function as potential vaccine candidates in the prevention of Clostridium derived infectious diseases. In this study, we have developed recombinant α-toxin (CPA), β2/β1-fusion toxin (CPB2B1) and α/β2/β1 trivalent fusion-toxin (CPAB2B1) as vaccine candidates that may be used to vaccinate against C. perfringens α, β1 and β2-toxins. Mice immunized with these recombinant toxoids demonstrated a strong protective immunological response when administered a lethal dose of C. perfringens type C culture filtrate with high titers of neutralizing antibodies to the toxins in the sera, as well as the intestinal mucosal s-IgA level. Specific neutralizing antibodies to the toxins were also detected in the sera and colostrum of sows and cows vaccinated with the toxoids. Furthermore, the CPA and CPB2B1 recombinant toxoid cocktail was capable of stimulating relatively higher levels of immune responses compared to that of CPA, CPB2B1 and CPAB2B1 alone. The CPAB2B1 trivalent fusion toxoid also displayed increased immunogenicity relative to CPA and CPB2B1 alone. These results suggest that recombinant toxoids are potential vaccine candidates against Clostridial toxins; the use of mixed cocktails and/or multivalent recombinant toxoids against different types of toxins may be an effective approach in the prevention of diseases caused by toxins produced by C. perfringens.     

三例产气荚膜梭菌感染病例医院感染防控并文献回顾

目的 了解不同部位产气荚膜梭菌感染患者的传播方式以及医院感染防控措施。方法 回顾性收集某院3例不同部位产气荚膜梭菌感染患者的临床资料、医院感染防控措施和环境采样结果，并查阅相关文献进行分析总结。结果 3例产气荚膜梭菌感染分别为盆腔气性坏疽、肝脓肿、血流感染，1例在在负压层流手术间和负压层流病房的回风口（栅栏+过滤网）培养出产气荚膜梭菌生长，余2例环境卫生学结果正常。结论 针对产气荚膜梭菌感染患者需严格执行环境清洁消毒与隔离，气性坏疽患者感染部位产气荚膜梭菌可远距离播散、污染环境，推荐采用独立非层流的感染手术间，其他部位感染者可实施单间、同病种隔离。     

Expression of Clostridium perfringens epsilon-beta fusion toxin gene in E. coli and its immunologic studies in mouse

Clostridium perfringens is an anaerobic spore-forming, pathogenic bacterium that is responsible for severe diseases in humans and livestock. In the present study, an epsilon-beta fusion toxin was expressed as a soluble protein in E. coli and the recombinant cell lysate was used for immunization studies in mouse. Potency of the toxin (as an antigen) induced 6 and 10 IU/ml of epsilon and beta anti-toxin in rabbit, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia for epsilon and beta toxins. Experimental challenge with the recombinant fusion toxoid revealed that it could protect mice against C. perfringens epsilon and beta toxins. Toxicity of the fusion toxin was studied by histopathological findings, which were the same as the native toxins. In conclusion, E. coli is a suitable expression host for immunogenic epsilon-beta fusion toxin of C. perfringens.     

Heterologous protection against alpha toxins of Clostridium perfringens and Staphylococcus aureus induced by binding domain recombinant chimeric protein

Clostridium perfringens and Staphylococcus aureus are the two important bacteria frequently associated with majority of the soft tissue infections. The severity and progression of the diseases caused by these pathogens are attributed primarily to the alpha toxins they produce. Previously, we synthesized a non-toxic chimeric molecule r-αCS encompassing the binding domains of C. perfringens and S. aureus alpha toxins and demonstrated that the r-αCS hyperimmune polysera reacts with both the native wild type toxins. In the present report, we evaluated efficacy of r-αCS in conferring protection against C. perfringens and S. aureus alpha toxin infections in murine model. Immunization of BALB/c with r-αCS was effective in inducing both high titers of serum anti-r-αCS antibodies after three administrations. Sub-typing the antibody pool revealed high proportions of IgG1 indicating a Th2-polarized immune response. The r-αCS stimulated the proliferation of splenocytes from the immunized mice upon re-induction by the antigen, in vitro. The levels of interleukin-10 increased while TNF-α was found to be downregulated in the r-αCS induced splenocytes. Mice immunized with r-αCS were protected against intramuscular challenge with 5 × LD₁₀₀ doses of C. perfringens and S. aureus alpha toxins with >80% survival, which killed control animals within 48–72 h. Passive immunization of mice with anti-r-αCS serum resulted in 50–80% survival. Our results indicate that r-αCS is a remarkable antigen with protective efficacy against alpha toxin mediated C. perfringens and S. aureus soft tissue co-infections.     

Indicator Microorganisms and Pathogens Removal Function Performed by Copepods in Constructed Wetlands

Removal efficiency of indicator and pathogenic microorganisms in constructed wetlands were analyzed, and microorganisms removal function performed by copepods was determined. The results showed that the constructed wetlands effectively reduced Escherichia coli, fecal streptococci, total coliforms, and fecal coliforms, the Salmonella spp. removal efficiency was relatively low and the Clostridium perfringens removal was the least. At copepods concentrations of 3.0 × 10²/L, and 6.0 × 10²/L, high die-off rates were observed for indicator and pathogenic microorganisms compared to the control group, and indicator and pathogenic microorganisms in samples with higher concentration of copepods decreased much more rapidly than those in samples with lower concentration. These results suggest that predation by copepods is an important mechanism for the removal of bacteria in constructed wetlands.     

A survey of urease-positive Vibrio parahaemolyticus strains isolated from traveller's diarrhea,sea water and imported frozen sea foods

The frequency of urease-positive Vibrio parahaemolyticus among isolates from patients, imported frozen sea foods and the environment (sea water) was studied. The highest isolation frequency of urease-positive V. parahaemolyticus was found in clinical isolates (11.2% out of 204 strains examined).Urease-positive V. parahaemolyticus was found in 5.7% of 88 frozen sea food-isolates examined, but no strains isolated from sea water were urease-positive. The isolates were further examined for the production of thermostable direct hemolysin (Vp-TDH) and its related hemolysin (Vp-TRH). Both are possible pathogenic toxins produced by mostly clinical isolates of V. parahaemolyticus. Urease-positive strains have a tendency to associate with clinical isolates producing both or neither Vp-TDH and Vp-TRH. Rabbit ligated ileal loops test was performed with several strains of ureasepositive and -negative clinical isolates, and we found that some strains producing urease, even those which do not produce Vp-TDH or Vp-TRH, caused intestinal fluid accumulation.Corresponding author.     

Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri

Naegleria fowleri, a worldwide distributed pathogen, is the causative agent of primary amoebic meningoencephalitis. Because it is such a fulminant disease, most patients do not survive the infection. This pathogen is a free-living amoeboflagellate present in warm water. To date, it is well established that there are several types of N. fowleri, which can be distinguished based on the length of the internal transcribed spacer 1 and a one bp transition in the 5.8S rDNA. Seven of the eight known types have been detected in Europe. Three types are present in the USA, of which one is unique to this country. Only one of the eight types occurs in Oceania (Australia and New Zealand) and Japan. In mainland Asia (India, China and Thailand) the two most common types are found, which are also present in Europe and the USA. There is strong indication that the pathogenic N. fowleri evolved from the nonpathogenic Naegleria lovaniensis on the American continent. There is no evidence of virulence differences between the types of N. fowleri. Two other Naegleria spp. are pathogenic for mice, but human infections due to these two other Naegleria spp. are not known.     

Morphological alterations and changes in cellular cations induced by Clostridium perfringens type a enterotoxin in tissue culture cells

The morphological alterations (bleb-balloon formation) induced by Clostridium perfringens type A enterotoxin in HeLa and Vero cells were studied under defined extracellular conditions. The action of enterotoxin was found to depend on the temperature but not on energy metabolism. The morphological alterations by the enterotoxin occurred in phosphate buffered saline containing Ca²⁺ and Mg²⁺. Of the constituents of the buffered saline, Ca²⁺ was essential for the morphological alterations and other ions were interchangeable. The morphological alterations by the enterotoxin occurred also in 10 mM Hepes-Na buffer, pH 7.2 containing NaCl, KCl or choline chloride at a concentration of over ca. 50 mM and in 10 mM Hepes-Ca buffer, pH 7.2 containing CaCl₂ at a concentration of over ca. 50 mM. Addition of sucrose to the medium prevented induction of the morphological alterations. The amount of sucrose necessary to protect the cells increased with increase in NaCl, KCl or CaCl₂ concentration in the medium. A calcium ionophore A23187 mimicked the action of enterotoxin. Examination of the cation contents of the cells by atomic absorption spectrophotometry showed early and rapid increase of Ca ²⁺ during intoxication with concomitant changes in Na⁺, K⁺ and Mg²⁺ that reduced the ion concentration gradients between inside and outside of the cell present before toxin treatment. The mechanism of action of C. perfringens type A enterotoxin is discussed on the basis of these findings.Corresponding Author.     

Detection of Clostridium perfringens in yearling lamb meat (barbacoa), head,and gut tacos from public markets in Mexico City

No reports on the incidence of Clostridium perfringens in popularly-consumed food from Mexico City have been published; neither are there any reports that have analyzed food consumed in popular markets and less established restaurants. Therefore, this study is aimed at providing data to evaluate the relevance of C. perfringens as an etiologic agent of food-borne diseases. Of the 650 analyzed samples, 106 (16.3%) were positive for C. perfringens; 6.4% (16/250) isolates were from barbacoa, 19% (38/200) from head, and 13% (52/200) from gut tacos. The presence of C. perfringens in these popular-consumed foods demonstrates its relevance as an etiologic agent of food-borne diseases, and confirms the great sanitary risk involved in their consumption. These results may serve as a basis for the Mexican sanitary authorities to control the microbiological quality of street-made foods.     

Preliminary phylogenetic identification of virulent Chlamydophila pecorum strains

Chlamydophila pecorum is an obligate intracellular bacterium associated with different pathological conditions in ruminants, swine and koala, which is also found in the intestine of asymptomatic animals. A multi-virulence locus sequence typing (MVLST) system was developed using 19 C. pecorum strains (8 pathogenic and 11 non-pathogenic intestinal strains) isolated from ruminants of different geographical origins. To evaluate the ability of MVLST to distinguish the pathogenic from the non-pathogenic strains of C. pecorum, the sequences of 12 genes were analysed: 6 potential virulence genes (ompA, incA, incB, incC, mip and copN), 5 housekeeping genes (recA, hemD, aroC, efp, gap), and the ORF663 gene encoding a hypothetical protein (HP) that includes a variant 15-nucleotides coding tandem repeat (CTR). MVLST provided high discriminatory power (100%) in allowing to distinguish 6 of 8 pathogenic strains in a single group, and overall more discriminatory than MLST targeting housekeeping genes. ompA was the most polymorphic gene and the phylogenetic tree based only on its sequence differentiated 4 groups with high bootstrap values. The number of CTRs (rich in serine, proline and lysine) in ORF663 detected in the pathogenic strains was generally lower than that found in the intestinal strains. MVLST appears to be a promising method for the differential identification of virulent C. pecorum strains, and the ompA, incA and ORF663 genes appear to be good molecular markers for further epidemiological investigation of C. pecorum.