败毒梭菌免疫原性的研究——Ⅰ.败毒梭菌的血清学分型及交互免疫

本文对27株败毒梭菌进行了抗原的血清学分型。结果表明,按“H”抗原可将所试菌株分成四个型和不定型;按“O”抗原可分成三个型。选取具有不同型“O”抗原的三株菌株制成菌苗所作的交叉保护试验证明,不同型“O”抗原菌株间缺乏有效的共同性保护抗原。     

艰难梭菌毒素致病基因调控机制和抗毒素治疗

艰难梭菌(C.difficile)是一种重要的院内感染病原体,是抗菌药物相关性腹泻的主要致病菌。C.difficile产毒菌株主要通过释放肠毒素A(Tcd A)和细胞毒素B(Tcd B)引起结肠损伤和炎症发生。研究发现C.difficile相关性疾病(CDAD)的严重程度与宿主体内细菌毒素水平相关。然而,不同菌株产毒能力差异较大,与毒素产生过程中涉及的基因调控密切相关。本文就C.difficile毒素致病基因调控机制和抗毒素治疗作一综述。     

表达LTB乳酸杆菌提高艰难梭菌类毒素疫苗黏膜免疫效应的研究

目的 通过构建大肠埃希菌热敏性肠毒素B亚单位 (heat labile enterotoxin B subunit, LTB) 乳酸杆菌表达载体,研究其对艰难梭菌类毒素疫苗的黏膜免疫保护效应。方法 克隆LTB基因,制备pSIP411-LTB重组质粒,电转化到乳酸杆菌中,制备乳酸杆菌表达LTB载体;通过透析和层析等技术纯化艰难梭菌A毒素和B毒素,经甲醛处理获得的艰难梭菌A毒素和B毒素的类毒素作为疫苗候选抗原;在第0 d、第14 d和第28 d皮下接种小鼠艰难梭菌类毒素和口服乳酸杆菌表达LTB载体;在初次接种后第0 d、10 d、24 d和38 d检测抗A毒素和B毒素的血清IgG和粪便IgA;第34 d,小鼠经口感染艰难梭菌,观察疫苗保护效果。结果 成功构建了LTB乳酸杆菌佐剂表达载体和制备了艰难梭菌A毒素和B毒素的内毒素;各疫苗组和疫苗+LTB佐剂组小鼠在感染艰难梭菌后,均无腹泻、体重减轻和死亡现象,而对照组老鼠腹泻发病率为100%,死亡率42.86%,5 d体重下降0.83 g;在初次接种后的10 d、24 d和38 d,与空白组比较,各疫苗组抗A毒素或B毒素的血清IgG及粪便IgA显著升高(F=284.93,1 037.05,696.82,74,184.07,166.95,P<0.01);与疫苗组比较,接种疫苗+LTB佐剂组的血清IgG及粪便IgA显著升高(F=21.88,27.22,8.31,56.49,63.76,101.78,P<0.05)。结论 艰难梭菌A毒素和B毒素的类毒素作为疫苗的候选抗原对艰难梭菌的感染具有很好的保护效应,LTB乳酸杆菌载体表达佐剂能够提高免疫动物的抗体效价,增强黏膜免疫效应。     

致病性嗜水气单胞菌保护性抗原的研究

本试验采用SDS－PAGE对10株嗜水气单胞菌（Ah）的外膜蛋白（OMP）和脂多糖（LPS）进行分析，结果显示大多数菌株具有相似的OMP图谱，而LPS则有较大的差异，用鲫鱼抗AhJ-1株全菌血清进行免疫转印，发现S蛋白（S－layerProtein）是70％Ah菌株的共同抗原，而LPS则有明显的抗原多样性，AhJ－1株抗血清仅能与同源菌株起反应。用AhJ－1株HEC毒素（Hemolvticactivity，Enterotoxicity，Cytotoxicity，HEC）的单克隆抗体检测此10株Ah，8株细菌可产生抗原性相同的HEC毒素。用纯化的AhJ－1株的S蛋白、LPS及HEC毒素（预先天活）分别免疫小鼠，强化免疫后，对同源菌株的攻击均能完全保护；S蛋白免疫组和HEC毒素免疫组对异源菌株TPS－30的攻击均能保护，而LPS免疫组则不能完全保护。上述结果表明，S蛋白和HEC毒素是Ah的主要共同保护抗原。AhJ-1株有望成为预防嗜水气单胞菌败血症的疫苗生产菌株。     

土拉杆菌无血培养基的研究

本文报告了土拉杆菌无血培养基。通过该培养基质量的初步试验证明,对土拉杆菌的变异、抗原性、凝集原性均无影响。培养基中葡萄糖含量在1％时产菌浓度最高。细菌活存率与卵黄培养基无区别。该培养基适用于土拉杆菌的培养和菌苗生产。     

艰难梭菌感染:临床表型与细菌毒素相关吗?

艰难梭菌(C.difficile)是院内获得性腹泻和伪膜性肠炎的首要病原体,产毒菌株主要通过产生毒素A和毒素B引起腹泻、肠道炎症甚至肠坏死。有研究表明,C.difficile在体内的产毒水平是影响C.difficile相关性疾病(CDAD)临床表型的重要因素,而细菌毒素与临床表型的具体联系尚不明确。本文主要针对CDAD临床表型以及毒素蛋白的结构、功能及其调控等方面进行论述,探讨细菌毒素与临床表型的关系,为进一步深入研究C.difficile的致病机制并探索新的药物治疗靶点奠定理论基础。     

耻垢分枝杆菌新型毒素-抗毒素系统MSMEG_3435-3436基因功能的初步研究

目的 初步探讨耻垢分枝杆菌(Mycobacterium smegmatis)毒素-抗毒素(toxin-antitoxin,TA)系统基因的功能及其在细菌药物耐受中的作用。方法 利用无水四环素(ATc)诱导穿梭质粒构建毒素基因(MSMEG_3436和MSMEG_6760)表达系统,检测毒素基因表达的抑菌作用。应用CRISPR-Cas12a基因编辑技术构建ΔMSMEG_3435-3436和ΔMSMEG_6762-6760敲除菌株,探究毒素-抗毒素系统对菌株生长的影响。通过计算菌株存活率检测MSMEG_3435-3436基因对异烟肼(96μg/ml)和利福平(40μg/ml)的耐受相关性。在耻垢分枝杆菌中用LacZ报告基因分别替换毒素-抗毒素基因(MSMEG_1277-1278、 MSMEG_1283-1284、 MSMEG_3435-3436、 MSMEG_4447-4448和MSMEG_5635-5634),构建5个启动子活性检测突变菌株(SY3328、SY3309、SY6407、SY3310和SY3311),并将pMV261空载体和pMV261-抗毒素系列质粒分别电转至5个突变菌株中,通过测定吸光度值(A₆₀₀、A₅₅₀和A₄₂₀)计算β-半乳糖苷酶活性[酶活性单位为“Miller单位(MU)”],以检测毒素抗毒素系统的启动子活性。结果 在耻垢分枝杆菌中,ATc诱导表达毒素基因MSMEG_3436可抑制细菌生长,而同时表达对应的抗毒素基因MSMEG_3435可消除抑制作用;ATc诱导表达毒素基因MSMEG_6760未发现明显的抑菌作用。与野生株相比,ΔMSMEG_3435-3436和ΔMSMEG_6762-6760敲除菌株在7H9液体培养基中生长表型无明显差异。野生株和ΔMSMEG_3435-3436敲除菌株经异烟肼和利福平处理后的存活率[分别为(4.38±1.48)%和(3.49±0.66)%,(0.15±0.04)%和(0.03±0.02)%]显示毒素-抗毒素基因MSMEG_3435-3436与药物耐受性无关(t=0.548,P=0.613;t=2.663,P=0.056)。启动子(SY3328、SY3309、SY6407、SY3310和SY3311)在携带pMV261-空载体和pMV261抗毒素表达质粒的报告菌株中的β-半乳糖苷酶活性分别为(376.50±17.13)和(315.50±20.71)、(189.00±12.24)和(160.70±9.89)、(225.20±9.95)和(211.70±2.57)、(221.40±12.07)和(186.60±13.17)、(179.10±5.87)和(127.70±19.21)MU,差异均无统计学意义(t=2.272,P=0.086;t=1.795,P=0.147;t=1.319,P=0.258;t=1.949,P=0.123;t=2.562,P=0.063)。结论 成功构建了MSMEG_3435-3436和MSMEG_6762-6760在耻垢分枝杆菌中的诱导表达体系及敲除菌株,并发现MSMEG_3435-3436是一个新的有功能的毒素-抗毒素系统,以及这两个毒素-抗毒素系统与菌株生长表型及异烟肼和利福平耐受性无关,最后发现耻垢分枝杆菌中5对毒素-抗毒素系统的抗毒素基因可能在自身启动子调控中不发挥关键作用,可为进一步研究结核分枝杆菌毒素-抗毒素系统的功能提供线索。     

艾希氏大肠杆菌83549菌株菌毛抗原化学分析

<正> 大肠杆菌菌毛(K88ac)抗原与致病性和免疫性能有密切的关系。我所分离的83549菌株具有K88ac抗原,免疫性能良好。为了明确83549菌株菌毛抗原的产量、化学组成和抗原性是否与15012菌株(法国引进)相同,我们作了比较试验,结果如下。 材料与方法 一、菌株:大肠杆菌15012株从法国引     

B型肉毒梭菌的分离与鉴定

目的建立用于B型肉毒梭菌的分离鉴定方法,为今后快速检测B型肉毒梭菌奠定基础。方法通过革兰氏染色、动物试验、胶体金免疫层析试验对待测食品标本进行检测,确定为肉毒梭菌,用特异性引物进行PCR扩增分型。结果根据菌株特点分离出肉毒梭菌,菌株和标本均能在253 bp位置扩增出目的片段,测序结果与Gen Bank的已知序列对照检索分析,其与已知B型肉毒毒素序列的一致性达到99%以上。结论此方法可用于B型肉毒梭菌的快速分离和鉴定。     

人源大肠杆菌不耐热肠毒素单克隆抗体的研究

本文应用杂交瘤细胞技术,获得5株对人源产毒性大肠杆菌不耐热肠毒素(H-LT)单克隆抗体(McAb)细胞株,它们分泌的抗体属IgG_1亚类。应用平板免疫溶血试验检测抗体的效价和特异性,其用H-LT抗毒素作对照,证明检测人源产毒性大肠杆菌二者结果一致,而对猪源产毒性大肠杆菌单克隆抗体无交叉反应,可用H-LT单克隆抗体作临床诊断试剂。     

Case report: Clostridium septicum infection presenting as liver abscess in a case of choriocarcinoma with liver metastasis

Clostridium septicum is an anaerobic, gram-positive bacillus. Infection with this organism has a known association with malignant diseases, especially colon and haematological cancers. Clostridium septicum is rarely found to be a pathogen of liver abscess. Herein, we report the case of a female choriocarcinoma patient with liver metastasis in which C. septicum infection presented as a gas-forming liver abscess. This case and previous reports indicate that once C. septicum is identified as a pathogen in liver abscess, metastatic liver tumours should be highly suspected.     

Clostridium septicum infection and associated malignancy. Report of 2 cases and review of the literature

We report 2 patients with myonecrosis due to Clostridium septicum and associated colon carcinoma and have reviewed the English language literature for all reported cases of atraumatic C. septicum infection. A total of 162 cases of C. septicum infection have been reported. Eighty-one percent of these patients had an associated malignancy. Thirty-four percent of all patients had associated colon carcinoma, while 40% had a hematologic malignancy. Thirty-seven percent of reported patients had an occult malignancy at the time of their infection with C. septicum. In many patients, the portal of entry was found in the large intestine. In a particularly lethal form (79% mortality) of C. septicum infection, known as "distant myonecrosis," infection metastatic from the initial site of infection causes severe myonecrosis, gangrene, and often death within hours of clinical detection. Overall, survival of patients with C. septicum infection is only 35%. Review of all cases of C. septicum infection suggests several conclusions. 1) Patients with malignancy, particularly colonic or hematologic, and patients with cyclic neutropenia who develop signs and symptoms of sepsis, especially with associated findings of abdominal pain or pain in an extremity, should be treated for possible clostridial infection. 2) C. septicum infection does not appear to be a result of a single specific defect in either humoral or cell-mediated immunity. Rather, it may occur in patients who are granulocytopenic and therefore prone to an enterocolitis. 3) Patients in whom an infection with C. septicum is found must undergo a vigorous search for malignancy following acute therapy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycotic aortic aneurysm infected by Clostridium septicum--a case history

The authors describe a sixty-seven-year-old hypertensive, diabetic man with a mycotic abdominal aortic aneurysm infected with Clostridium septicum. The patient had colonic polyps but no malignant disease. They could find only one other report of a mycotic aneurysm infected with C. septicum. In that case, as in most other cases of C. septicum bacteremia, the patient had gastrointestinal cancer. Their case suggests that treatment for a clostridial infection should be considered in patients with known gastrointestinal disease, signs and symptoms of sepsis, and abdominal pain. Conversely, patients known to have a C. septicum infection should be evaluated for gastrointestinal lesions.     

Clostridium septicum infection associated with perforation of colon diverticulum.

Non-traumatic or spontaneous gas gangrene by Clostridium septicum is a rare infection in humans, characterised by extensive destruction of muscle tissue, a mortality rate of 100% if left untreated (1), and often associated with haematological or colorectal diseases. An associated malignancy was found in 80% of patients with Clostridium septicum infection: 34% had a colorectal carcinoma and 40% had a haematological malignancy (2); the infection usually lies in an often-ulcerated intestinal lesion (3). We report here a case of spontaneous gas gangrene by Clostridium septicum in the right thigh due to perforation of a diverticulum in the sigmoid colon.     

Clostridium septicum septicemia with identical metastatic myonecroses in a granulocytopenic patient. Infectious disease emergency

Clostridium septicum is a gram-positive, sporulating spindle-shaped rod. Gas gangrene secondary to trauma is not uncommon. However, nontraumatic clostridial infection causing myonecrosis is quite unusual. This is a unique case report of Clostridium septicum bacteremia with two simultaneously evolving metastatic foci of myonecrosis of the left arm and right thigh that developed in a patient with lymphoma when he became granulocytopenic during his hospital course.     

Vaccination with parenteral toxoid B protects hamsters against lethal challenge with toxin A-negative, toxin B-positive clostridium difficile but does not prevent colonization

Toxin A has historically been regarded as the primary virulence determinant in Clostridium difficile infection, but naturally occurring toxin A-negative, toxin B-positive (A-/B+) C. difficile strains are known to be virulent. To determine the role of toxin B in these strains, we immunized hamsters with a toxoid prepared from purified toxin B to determine whether they would be protected from lethal challenge with an A-/B+ strain of C. difficile.     

Spontaneous, nontraumatic gangrene due to Clostridium septicum

Clostridium septicum is a major cause of spontaneous, nontraumatic gas gangrene. Unlike Clostridium perfringens, C. septicum is relatively aerotolerant and thus appears to be more capable of initiating infection in the absence of obvious damage to tissues. Six cases illustrate the clinical setting and fulminant nature of spontaneous gangrene caused by C. septicum. A lesion in the colon such as carcinoma is often present and is presumed to serve as a portal of entry to the bloodstream. Diabetes and leukopenia are also common predisposing conditions; compromise of vital host responses may facilitate proliferation of those organisms that settle out in the tissues. Acute lymphoma or leukemia during a course of chemotherapy is accompanied by damage to bowel mucosa and granulocytopenia, thus predisposing to spontaneous clostridial gangrene. Infection progresses in a fulminating manner; the majority of patients die within 24 hours of onset. Characteristic symptoms and signs include excruciating pain (although a sense of heaviness may be the only early symptom), swelling of tissues, crepitance, and bulla formation. A hallmark of C. septicum infection is the absence of acute inflammatory cells in involved tissues or in bulla fluid. A series of laboratory investigations demonstrated that fluid obtained from a bulla adversely affected the viability, morphology, and function of polymorphonuclear leukocytes (PMNs), which may explain the paucity of PMNs in involved tissues and may in part contribute to the fulminant progression observed in infection due to this organism.     

CNS infection with clostridium septicum

We present an unusual case of Clostridium septicum brain infection in a 72-yr-old woman who had no underlying malignant disease. The infection spread from a localized sit to the CNS causing gas formation. The patient died rapidly.     

Clostridium difficile

Clostridium difficile is a major cause of antibiotic-associated diarrhea and colitis. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Pathogenic strains of C. difficile produce two protein exotoxins, toxin A and toxin B, that cause colonic mucosal injury and inflammation. Many patients who are colonized are asymptomatic, and recent evidence indicates that diarrhea and colitis occur in those individuals who lack a protective antitoxin immune response. In patients who do develop symptoms, the spectrum of C. difficile disease ranges from mild diarrhea to fulminant pseudomembranous colitis. Prevention of nosocomial C. difficile infection involves judicious use of antibiotics and multidisciplinary infection control measures to reduce environmental contamination and patient cross-infection. Ultimately, active or passive immunization against C. difficile may be an effective means of controlling the growing problem of nosocomial C. difficile diarrhea and colitis.     

Mycotic aortic aneurysm infected by Clostridium septicum: a case report and review of the literature

We report on a case of Clostridium septicum aortic mycotic aneurysm in an 83-year-old patient. This is a rare infectious disease. To our knowledge, only 31 cases have been reported in the literature. The clinical presentation is dominated by abdominal pain with fever. Although only the bacteriological samples can definitely identify the germ, the presence of gas bubbles around an aneurysm on abdominal computed tomography scan suggests C. septicum aortic infection. This infectious disease is frequently associated with neoplasia, mainly colonic. Treatment is primarily surgical resection with bypass or graft, associated with prolonged antibiotic therapy. The prognosis is poor due to vascular complications and pathogenesis of C. septicum. Patients cumulate the death risk from mycotic aneurysm and C. septicum sepsis.