Clostridium difficile in association with sporadic diarrhoea.

A total of 154 patients admitted to an infectious diseases unit were included in a year''s prospective survey of sporadic diarrhoeal disease. Stools from 19 of them yielded Clostridium difficile, generally on more than one occasion. Twelve of these patients were assessed as having a severe or moderately severe gastrointestinal illness: Cl difficile was the only pathogen isolated from 10 of them, and two had an associated salmonella infection. Seven had had a recent course of antibiotics, but five had not taken antibiotics. Faeces from seven patients with moderate or mild gastrointestinal illness yielded Cl difficile, and two of these patients also had an associated salmonella infection. Two patients in this group had no antibiotic history. From these findings, the occurrence of C difficile in faeces could not be described as antibiotic-associated. Faecal Cl difficile cytotoxin was detected in only six patients, and generally at low levels. In such patients a more relevant pathogenic index might take account of the numbers of Cl difficile present and of their toxigenic potential.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Occurrence of Clostridium difficile in feces of children with dysfunction of the digestive tract and other disorders

Thousand and six hundred ninety two fecal samples from children of few weeks old up to over ten years were tested for the presence of Clostridium difficile. Most of them were treated with antibiotics and showed diarrhea symptoms. Hundred and twenty three strains of C-difficile were submitted to serological typing and their sensitivity to 10 selected antibiotics and chemotherapeutic agents was determined. Among 109 strains of C. difficile tested for enterotoxin production by latex test 82 strains (75.2%) were positive. Almost half (48%) of the isolated strains belonged to serotype C. Most of the strains were resistant to cefoxitin (88%) and clindamycin (76%). Over 90% of strains were sensitive to vancomycin and azlocillin and 86% to chloramphenicol and metronidazole. In the majority of patients with positive C. difficile cultures diarrhea was present, however, it was difficult to find a direct link between these symptoms and antibiotic therapy.     

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.     

Colonization by Clostridium difficile of neonates in a hospital, and infants and children in three day-care facilities of Kanazawa, Japan.

The intestinal-carriage rates of Clostridium difficile in neonates hospitalized in the University Hospital's Center for Perinatal and Reproductive Health and in infants and children enrolled in two day-nurseries and a kindergarten were examined. Swab samples from the floors of these facilities were also analyzed to determine the extent of environmental contamination by this organism. C. difficile was found in the stool of only one of 40 neonates during the normal 1-week stay in the hospital after delivery. The isolate from the neonate was identical to that of her mother, as determined by PCR ribotyping, pulsed-field gel electrophoresis analysis, and toxin gene type, suggesting that the C. difficile-positive neonate acquired the organism from her mother rather than from the environment. By contrast, 47 (48.0%) of the 98 infants and children, comprising 50 enrolled in two day-nurseries who were >= 3 years old and 48 enrolled in a kindergarten who were 2-5 years old, carried C. difficile. The carriage rate in infants under 2 years of age was much higher (84.4%) than in children 2 years old and older (30.3%). When analyzed according to age group, the carriage rates were 100, 75.0, 45.5, 24.0, 38.5, and 23.5% in infants and children 0, 1, 2, 3, 4, and 5 years old, respectively. The observation that several children were colonized with the same type of C. difficile strain in each day-care facility, and that the floors of day-nursery A and kindergarten C were contaminated with C. difficile strains identical to those colonizing the intestines of children enrolled in those facilities suggests that cross-infection of C. difficile among children occurs through C. difficile-carrying children or their contaminated environments.     

Variation in nuclear DNA content in an ex-type isolate of Penicillium measured by continuous flow microfluorimetry

Abstract The effect of purified enterotoxin produced by Clostridium difficile on Chinese hamster ovary (CHO) cells was examined. In a certain concentration range (0.9–3.6 μ g/ml), purified toxin caused CHO elongation, namely a cytotonic effect, which is similar to a typical morphological change in CHO cells induced by cholera enterotoxin. At a higher concentration, purified enterotoxin had a cytotoxic effect on CHO cells which was neutralized by anti- C. difficile enterotoxin, but not by anti- C. difficile cytotoxin. Thus, enterotoxin had both cytotonic and cytotoxic effects on CHO cells. About 60 and 180 min were required for binding of enterotoxin to CHO cells, and its internalization, respectively, both times being much longer than those for C. difficile cytotoxin.     

Modulation of cytotoxin production by Clostridium difficile in the intestinal tracts of gnotobiotic mice inoculated with various human intestinal bacteria

Gnotobiotic mice died 2 days after inoculation of a cytotoxigenic Clostridium difficile strain. Protection occurred when mice were previously inoculated with a strain of Escherichia coli or Bifidobacterium bifidum. Intestinal cytotoxin production was highly reduced in the surviving mice, whereas the C. difficile population level did not decrease to a great extent.     

Modulation of cytotoxin production by Clostridium difficile in the intestinal tracts of gnotobiotic mice inoculated with various human intestinal bacteria.

Gnotobiotic mice died 2 days after inoculation of a cytotoxigenic Clostridium difficile strain. Protection occurred when mice were previously inoculated with a strain of Escherichia coli or Bifidobacterium bifidum. Intestinal cytotoxin production was highly reduced in the surviving mice, whereas the C. difficile population level did not decrease to a great extent.     

Clostridium difficile-associated diarrhea from a general hospital in Argentina

Clostridium difficile is responsible for 15-25% of all cases of antibiotic associated diarrhea. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Although the clinical and financial impact of nosocomial C. difficile infection is believed to be significant, only limited information is available on the importance of C. difficile as a cause of diarrhea in Argentina. The aim of the study was to evaluate the impact and diagnosis methods of CDAD from symptomatic patients in a general hospital from Argentina. Consecutive diarrheal stool samples from symptomatic patients from a General Hospital in Argentina were screened for toxigenic C. difficile between April 2000 and April 2001. Toxins were detected in stools by the Premier Cytoclone A+B EIA. Each specimen was examined for toxigenic C. difficile strains by culture. From 104 specimens, 40 (38.5%) [32 of 87 patients (36.8%)] were positive and 64 (61.5%) [55 of 87 patients (63.2%)] were negative by stool toxin assay and/or toxigenic culture. In 11 of 40 positives samples C. difficile toxins were detected only by toxigenic culture. Five (15.6%) patients presented with symptomatic recurrences. Toxin-negative strains were not isolated. This data indicates that the high prevalence of toxigenic strains of C. difficile is of concern in routine diagnostic testing for C. difficile toxins in our study population. Detection of toxins in stools by EIA, coupled with testing strains for toxigenicity only in those cases in which direct toxin assay produces negative results, may be a satisfactory strategy. CDAD is an emerging nosocomial problem in our hospital. It will be necessary to evaluate the epidemiology and measures to control nosocomial spread.     

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.     

Occurrence of Clostridium difficile in fecal samples of HIV-infected children in Poland

The prevalence of Clostridium difficile and its toxins (A and B) in HIV-positive children in Poland was investigated in a group of 18 children, aged 6 months to 8 1/2 years. Stool samples were tested using an antigen detection method for toxin A/B, cytotoxicity-neutralization and culture. In 3 cases (17%) C. difficile toxins were detected in both stool samples and strains recovered from culture. The three strains isolated were shown by PCR methods to contain toxins A and B genes. All children had been treated previously with antimicrobial and antiviral agents. All three C. difficile-positive children had mild diarrhea that resolved without specific therapy. Further studies involving a large number of children and molecular analyses of isolated C. difficile strains are necessary to determine the frequency and rate of carriage of C. difficile strains among HIV-positive children in Poland.     

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。     

兰州市婴幼儿艰难梭菌带菌状况

在兰州市随机采集的２１６名健康婴幼儿粪标本经分离并对其培养特性、菌落菌体形态特征、生化反应特性以及毒素原性等进行一系列检查,检出了３７人的粪便含有艰难梭菌（１０４─１０８／ｇ）,检出总阳性率为１７．１％,新生儿、婴儿及幼儿各年龄组的检出阳性率分别为１３．５％（１９／４１）、３３．３％（１３／３９）、１３．９％（５／３６）。３７株分离菌中毒素原性阳性者仅有８株（２１．６％）,均分布于婴儿与幼儿两组。新生儿１４１人中２７人的粪标本为胎粪,只有１人（３．７％）含艰难梭菌（２×１０６／ｇ）,但不产毒素。所有婴幼儿中有２２人因各种原因曾用过抗菌药物,但仅有２人的粪便含艰难梭菌,而且均为非产毒株,表明艰难梭菌检出率与药物服用之间似无显著的相关性。     

Nutritional aspects of cytotoxin production by Clostridium difficile.

Arginine was the only amino acid used by Clostridium difficile that permitted cytotoxin synthesis in a peptone-based medium. Synthesis of cytotoxin was delayed when glucose was used as the substrate. Addition of rifampin or puromycin to cultures prior to release of cytotoxin inhibited the release of cytotoxin, suggesting that a protein essential for cytotoxin release is synthesized after cytotoxin is synthesized.     

Effect of toxins produced by various Clostridium difficile strains on cecum size reduction in gnotobiotic mice

Inoculation of axenic mice with Clostridium difficile strains induced a significant reduction in ceca weight (dry or wet), whereas a nontoxinogenic strain led to a partial reduction. A strain, which produces cytotoxin and no enterotoxin in vivo, caused a reduction similar to that observed with a nontoxinogenic strain. Simultaneous cytotoxin and enterotoxin production by various C. difficile strains caused the cecum size to diminish to that observed for conventional control mice.     

Evaluation of the effect of medicines on biological properties of Clostridium difficile

The presence of the persistence factors (anti-lysozyme and anti-complement activity) in the vegetative forms of C. difficile was experimentally proved. The effect of different medicines (vitamins B1, B6 and C, prebiotic inulin, probiotics Bifidumbacterin and Enterol) on the persistence factors of C. difficile and microbial resistance to vancomycin, thienam, lincomycin, clindamycin was evaluated. The anti-lysozyme and anti-complement activity of C. difficile was found to decrease under the influence of vitamins B1, B6, C, inulin, exometabolites of bifidobacteria. Under the impact of the preparations used in this study changes in the sensitivity of C. difficile to antibiotics of the lincoamide, carbapenem, glycopeptide groups were found to occur. The data obtained reveal one of the possible mechanisms of the corrective action of the medicines under study on the intestinal microbiocenosis in patients with antibiotic-associated colitis.     

Prevention of Clostridium difficile induced mortality in gnotobiotic mice by Saccharomyces boulardii

Oral preventive treatment of gnotobiotic mice by Saccharomyces boulardii significantly decreased mortality following Clostridium difficile infection. A single S. boulardii ingestion protected 16% of mice, whereas 56% were protected when S. boulardii was given continuously in the drinking water. No direct antagonistic effect of the yeast on C. difficile numbers was detected, whereas a modulation of fecal cytotoxin production was demonstrated.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Pseudomembranous colitis: isolation of two species of cytotoxic clostridia and successful treatment with vancomycin.

Lincomycin-resistant Clostridium sporogenes obtained from the stools of a patient with lincomycin-associated pseudomembranous colitis produced a heat-stable cytotoxin in low titre when grown in chopped meat medium. Vancomycin eradicated this strain and all other clostridia, and controlled the symptoms. When diarrhea recurred 7 days after treatment with vancomycin was stopped, clostridia including C. sporogenes and C. difficile were again isolated. The C. difficile produced a heat-labile cytotoxin in high titre that was unaffected by growth in various media and induced colitis in hamsters. Treatment with vancomycin, to which all the clostridia were sensitive, eradicated both toxic species and controlled the diarrhea. Antibiotic-induced pseudomembranous colitis may be associated with more than one species of toxin-producing clostridia. Vancomycin therapy should be continued for 10 days or more in patients with severe disease to eradicate the responsible organism.     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。