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.     

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＞7 d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、cdtA、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。     

婴幼儿艰难梭菌的无症状定植与防治措施研究进展

艰难梭菌是一种肠道条件致病菌,能形成芽胞有效抵抗抗生素的杀灭.由于临床上抗生素的不规范使用,导致人体肠道有益菌被杀死.艰难梭菌因抵抗力强而大量繁殖,破坏人体肠道菌群平衡,引起抗生素相关性腹泻.艰难梭菌同时也是院内感染的主要病原菌.艰难梭菌可在人体肠道无症状定植,不同年龄阶段艰难梭菌定植率有较大差异,相比于成人,婴幼儿时...     

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.     

Enhanced fermentation of mannitol and release of cytotoxin by Clostridium difficile in alkaline culture media.

Clostridium difficile ATCC 43255 fermented less than 10% of the mannitol in a medium at pH 7; however, when the initial pH of the medium was adjusted to 8.5 or 9, about 80% of the mannitol was fermented. Cell extracts of C. difficile phosphorylated mannitol with phosphoenolpyruvate, not ATP, indicating a phosphoenolpyruvate phosphotransferase system transport phosphorylation of mannitol. The phosphorylation product was dehydrogenated by D-mannitol-1-phosphate:NAD oxidoreductase. Growth at an initial pH of 8.5 yielded cytotoxin titers of 10(7) to 10(8) in Trypticase-yeast extract-mannitol medium, wit a titer of 10(8) as early as 13 h.     

粪便标本艰难梭菌三种培养方法的比较

目的 比较粪便标本中艰难梭菌三种培养方法的差异性,寻找适合临床实验室使用的艰难梭菌培养方法。方法 将健康儿童的300份粪便标本分别用快速芽孢孵育法、乙醇直接处理法、富集芽孢培养法进行培养,并对三种培养方法的差异性进行统计学比较。结果 300份粪便标本快速芽孢孵育法培养出艰难梭菌51例,检出率为17.0%;乙醇直接处理法培养出48例,检出率为16.0%;富集芽孢培养法培养出44例,检出率为14.7%。三种方法的检出率差异无统计学意义（P=0.7352,P>0.05）。结论 乙醇直接处理法、快速芽孢孵育法简单快速,适合临床实验室进行艰难梭菌的快速培养;富集芽孢培养法杂菌少,适合大量艰难梭菌培养便于结果的观察及进一步的处理。     

Effect of various sodium taurocholate preparations on the recovery of Clostridium difficile spores

The effect of four sodium taurocholate preparations, which are easily available in Japan, on recovery of Clostridium difficile spores was examined. All preparations, except for one, enabled the recovery of nearly all spores counted microscopically. Moreover, by using 69 toxigenic and 34 nontoxigenic C. difficile strains, the relationship between the recovery of spores in the medium with sodium taurocholate and toxigenicity of C. difficile was analyzed. It was noted that the number of strains with recovery rate of more than 70% was greater in toxigenic strains than in nontoxigenic strains, suggesting a more abundant recovery of toxigenic C. difficile strains in the presence of sodium taurocholate.     

Separate isolation of Clostridium difficile spores and vegetative cells from the feces of newborn infants.

A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty-one samples (75%) of C. difficile-positive Twenty-eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.     

123例腹泻患儿粪便中艰难梭菌毒素基因特征及感染危险因素分析

探讨腹泻患儿粪便中艰难梭菌毒素基因特征, 并分析产毒艰难梭菌感染的危险因素。

采集2019年1月至2021年3月嘉兴市第二医院儿科收治的腹泻患儿的粪便标本共123份, 其中社区获得性腹泻患儿60例, 医院获得性腹泻患儿63例。标本进行厌氧培养, 采用实时荧光PCR鉴定艰难梭菌tpi基因, 并检测tcd A、tcd B毒素基因。收集患儿临床资料, 采用Logistic回归分析产毒艰难梭菌感染的危险因素。

123例粪便标本共检出艰难梭菌tpi基因阳性35例(28.46%), 毒素基因中tcd A⁺ B⁺为19例(15.45%), tcd A⁺B^-为3例(2.44%), tcd A^-B⁺为2例(1.63%), tcd A^-B^-为11例(8.94%)。社区获得性腹泻患儿中tcd A/B产毒艰难梭菌感染率为28.33%, 高于医院获得性腹泻患儿的11.11%(P < 0.05)。产毒艰难梭菌感染腹泻患儿与非产毒艰难梭菌感染腹泻患儿的性别、年龄、居住地、是否早产、有无先天性疾病、既往有无胃肠道手术、抗生素应用种类、近1个月内有无机械通气、临床症状、血液白细胞计数、血红蛋白、粪便白细胞计数、血清C反应蛋白、血清白细胞介素-6、血清白蛋白、血肌酐、血清总胆红素水平比较差异无统计学意义(均P > 0.05)。产毒艰难梭菌感染腹泻患儿中的非母乳喂养、近1个月内抗生素用药史、抗生素持续用药时间 > 7 d、近1个月内糖皮质激素用药史、近1个月内抑酸药用药史、近1个月内肠内营养者的比例显著高于非产毒艰难梭菌感染腹泻患儿(均P < 0.05)。非母乳喂养(OR=2.433)、近1个月内抗生素用药史(OR=3.040)、近1个月内肠内营养(OR=2.330)是腹泻患儿产毒艰难梭菌感染的独立危险因素(均P < 0.05)。

嘉兴地区儿童艰难梭菌毒素基因主要为tcd A/B基因, 患儿以社区获得性腹泻为主, 非母乳喂养、抗生素用药史、肠内营养的儿童更容易感染艰难梭菌。

     

Analysis of proline reduction in the nosocomial pathogen Clostridium difficile

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.     

Cross reactivity of Clostridium glycolicum with the latex particle slide agglutination reagent for Clostridium difficile identification

Three strains of unidentified clostridia and 15 known strains of Clostridium glycolicum were examined to investigate cross reactions with the latex particle agglutination reagent used to identify Clostridium difficile. The unknown strains were identified as Cl. glycolicum and cross reacting agglutination occurred in 15/15 (100%) stock Cl. glycolicum strains. Characteristics such as volatile fatty acid profile, propylene glycol fermentation, u.v. fluorescence and the production of p -cresol are required to distinguish between the two species.     

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

ABSTRACT: BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.     

Preparation of antibacterial and antitoxic Clostridium difficile sera.

Preparation of Clostridium difficile antibacterial and antitoxic sera is presented. Fifty one strains (72%) were typeable within Delmee scheme. Twenty strains (28%) belonged to new Polish serogroups designated 18, 27, 70, 71, 72, 88, 89 and NICH. Supernatants of all toxigenic Clostridium difficile strains were neutralized by gamma-globulin fraction of goat Clostridium difficile antitoxin in neutralization assay when it was performed on McCoy cell line. Only 8 toxigenic strains (21%) were positive in counterimmunoelectrophoresis.     

艰难梭菌感染与大肠癌及大肠腺瘤的相关性分析

目的 分析艰难梭菌感染与大肠癌及大肠腺瘤的相关性,为临床预防及治疗肠道肿瘤提供新思路。方法 收集2016年1月至2017年6月于甘肃省人民医院就诊的134例大肠癌患者、52例大肠腺瘤患者及100例健康对照者的粪便标本及临床资料,对粪便标本进行细胞毒性试验检测,分析大肠癌及大肠腺瘤与艰难梭菌感染的相关性。同时分析艰难梭菌感染与大肠癌临床特征及部分样本免疫组化（Her-2、P53、CDX2、CD56及Ki-67）结果的相关性。结果 （1）结直肠癌组检出13例艰难梭菌感染者（9.70%）,大肠腺瘤组检出2例艰难梭菌感染者（3.85%）,对照组未检出艰难梭菌感染者。结直肠癌组艰难梭菌感染率明显高于大肠腺瘤组及对照组（2=11.0953,P0.05）。（3）CDX2的表达与艰难梭菌感染具有相关性,CDX2阴性结直肠癌患者艰难梭菌感染率明显高于CDX2阳性患者（20.8% vs 2.8%,2=8.5224,P0.05）。结论 艰难梭菌感染与大肠癌的发生具有相关性,特别是与大肠癌病理分期及CDX2表达存在相关性。艰难梭菌感染与大肠腺瘤之间无显著相关性。     

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。     

艰难梭菌毒素A 对胆管癌细胞株FRH-0201 增殖和凋亡的影响

目的:研究艰难梭菌毒素A(TcdA)对人胆管癌细胞株FRH-0201细胞凋亡影响及作用机制。方法:采用MTT法检测细胞增殖抑制率,荧光染色检测TcdA作用后细胞形态学变化,流式细胞术检测细胞凋亡,免疫细胞化学法检测Bcl-2表达水平,Caspase3活性检测试剂盒检测Caspase-3的活性。结果:TcdA能抑制胆管癌细胞株FRH-0201细胞增殖且呈时间、剂量依赖性,荧光染色和流式细胞仪检测到细胞凋亡,与对照组相比,TcdA作用后Bcl-2蛋白表达下降,差异有统计学意义(P<0.05),Caspase-3活性增强,差异有统计学意义(P<0.05)。结论:TcdA可以通过下调Bcl-2蛋白表达表达,激活Caspase-3而诱导FRH-0201细胞凋亡。     

3种检测艰难梭菌方法的临床应用评估

本研究旨在对3种检测粪便样本中艰难梭菌的方法进行临床应用评估,为艰难梭菌的实验室检测提供参考。采用艰难梭菌毒素A/B(Clostridium difficile toxins A and B,CDAB)酶联免疫荧光检测法、环丝氨酸-头孢西丁-果糖琼脂(cycloserin-cefoxitin-fructose agar,CCFA)常规培养法和显色培养法(chromID~(TM))同步检测粪便样本中的艰难梭菌,并对培养所得菌株进行tcdB基因扩增以验证其产毒性。以艰难梭菌培养联合tcdB基因扩增为参考方法,分别计算上述3种方法的灵敏度、特异度、阳性预测值和阴性预测值等参数,分析其与参考方法的一致性。研究共收集临床粪便样本164份,参考方法检测结果为58份阳性,106份阴性。经统计分析,艰难梭菌毒素A/B法的灵敏度、特异度、阳性预测值和阴性预测值分别为51.7%、95.3%、85.7%和78.3%,CCFA常规培养法分别为72.4%、98.1%、95.5%和86.7%,而艰难梭菌显色培养法分别为94.8%、92.5%、87.3%和97.0%。3种方法中,艰难梭菌显色培养法与参考方法的一致性最高(Kappa=0.856)。采用该方法检测粪便样本中的艰难梭菌操作简便,成本经济,结果判断直观、准确,具有较好的临床应用价值。     

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。     

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。