Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.

A cytotoxigenic Clostridium difficile strain that fails to produce toxin A but causes hemorrhage and bloody fluid accumulation in ligated ileal loops of rabbits and hemorrhage and diarrhea in hamsters is described. The lack of reaction of DNA from this strain in hybridization studies with a toxin A gene-specific 4.5-kb probe and polymerase chain reaction studies with six toxin A-specific primers indicate the absence of the toxin A gene. The cytotoxin produced by this strain was not responsible for the enterotoxic or hemorrhagic activity and shared characteristics with toxin B, i.e., its cytotoxicity was neutralized by antibodies to toxigenic strains of C. difficile and Clostridium sordellii. Polymerase chain reaction studies with toxin B-specific primers showed that the DNA from this strain produced a 690-bp product in addition to the expected 591-bp product.     

Colonic IgA producing cells and macrophages are reduced in recurrent and non-recurrent Clostridium difficile associated diarrhoea

BACKGROUND: In Clostridium difficile associated diarrhoea (CDAD), histological changes in the colonic mucosa range from minimal inflammation to pseudomembranous colitis (PMC). The disease also recurs in a considerable proportion of patients. AIM: To investigate mucosal immune system cells in colonic biopsies of patients with CDAD. METHODS: Colonic biopsies were obtained from 12 control patients with diarrhoea, six patients with CDAD and minimal inflammation, and 10 patients with CDAD with pseudomembranous colitis (samples obtained from areas with and without inflammatory exudate). Immunohistochemical studies were performed using antibodies to T cells (CD3), macrophages (CD68), B/plasma cells (CD79alpha), and to IgA, IgM, and IgG. Labelled cells in lamina propria were quantified. RESULTS: In contrast to T cells, there were significant reductions in B/plasma cell and macrophage counts in all biopsies from patients with CDAD compared with controls (p<0.001). Studies using anti-immunoglobulin antibodies showed significant reductions in IgA producing cells in CDAD biopsies (p<0.05), with the greatest reduction in samples from patients with PMC. In contrast, there was a significant increase (p<0.05) in IgG producing cells in CDAD biopsies. Only patients with PMC relapsed. In these patients, B/plasma cell and IgA producing cell counts (in biopsies with and without inflammatory exudates) were significantly lower (p<0.01) in mucosal samples from those who subsequently relapsed (five) than those who did not. CONCLUSIONS: A selective reduction in mucosal IgA producing cells and macrophages is associated with colonic disease in C difficile infected patients. Severe reduction in colonic IgA producing cells may predispose to recurrence of CDAD.     

Purification and characterisation of Clostridium difficile toxin A by bovine thyroglobulin affinity chromatography and dissociation in denaturing conditions with or without reduction

Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of greater than 240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with beta-mercaptoethanol.     

An in-vitro model of colonisation resistance to Clostridium difficile infection

To investigate the importance of the normal gut flora in preventing the establishment of Clostridium difficile in vivo we have developed an in-vitro test system based on growth in faecal emulsions. Growth of C. difficile and cytotoxin production are inhibited in faecal emulsions from healthy adults, but not in sterilised emulsions; the importance of viable bacteria in the inhibitory system is evident. Generally, faecal emulsions derived from infants, children and geriatric patients were less inhibitory than those from healthy adults. Those from bottle-fed infants were significantly less inhibitory than those from breast-fed infants. Decreased levels of cytotoxin in the latter group were attributed to the acidic pH of the stools. With the different patient groups studied, faecal samples not inhibitory to C. difficile in vitro were obtained from 21% of patients with antibiotic-associated diarrhoea, 33% of those taking antibiotics but who did not have diarrhoea, 18.7% of those with diarrhoea unassociated with antibiotics, and 79% of those with C. difficile-mediated diarrhoea. In some cases inhibition was due to low faecal pH, as in some infants, and in others to other filterable substances. The degree of inhibition could not be linked to specific volatile fatty acids or enzymes.     

Usefulness of culture in the diagnosis ofClostridium difficile infection

Between January and April 1993, culture forClostridium difficile and a faecal cytotoxin assay were performed on 500 selected specimens. Isolates from culture-positive patients from whom faecal samples were cytotoxin negative were also examined in vitro for cytotoxin production. The significance of a positive culture result in the absence of faecal cytotoxin was assessed. Forty-one of the 500 specimens were toxin positive. In only 25 of these wasClostridium difficile examination specifically requested. Six of nine culture-positive cytotoxin-negative patients (11 specimens) had recently received antibiotics. In four of these,Clostridium difficile was considered to be of possible clinical significance. Culture and in vitro determination of toxin production of isolates may aid in the diagnosis of some additional cases, but cytotoxin detection remains the single optimal routine laboratory method for diagnosis.     

Effect of Clostridium difficile Toxin A on Human Colonic Lamina Propria Cells: Early Loss of Macrophages Followed by T-Cell Apoptosis

We have previously shown that Clostridium difficile toxin A induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis. Because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis. The role of toxin A in the induction of apoptosis therefore remains to be determined. In addition, sensitivities to C. difficile toxin A of lamina propria lymphocytes, macrophages, and eosinophils, which lie below the surface epithelium, are not known. In contrast to epithelial cells, these lamina propria cells do not require adhesion-dependent signalling from the extracellular matrix for survival, and this may allow the mechanisms of toxin A-induced cell death to be further investigated. The aim of this study was to investigate the effect of purified C. difficile toxin A on human colonic lamina propria T cells, macrophages, and eosinophils. We show that C. difficile toxin A induces loss of viability in isolated colonic lamina propria cell preparations containing the three different cell types in a dose- and time-dependent fashion. Exposure to high concentrations of the toxin led to loss of macrophages within 72 h. T-lymphocyte and eosinophil cell death was prominent at later time points and occurred by apoptosis. Exposure to toxin A also induced the production of tumor necrosis factor alpha by the isolated colonic lamina propria cells. However, the presence of neutralizing antibodies to this cytokine did not influence C. difficile toxin A-induced T-cell apoptosis. Moreover, purified T cells also underwent apoptosis following exposure to toxin A, implying that apoptosis occurred as a consequence of a direct interaction between T cells and the toxin. Our studies suggest that C. difficile toxin A is capable of suppressing human colonic mucosal immune responses by inducing early loss of macrophages followed by T-cell apoptosis.     

Plasmid analysis as a means of strain differentiation in Clostridium perfringens.

A total of 114 Clostridium perfringens isolates were serotyped and examined for plasmids. Fifty-two strains were from hospitalized patients with diarrhea or from hospital environments, and 62 epidemiologically unrelated isolates were obtained from food poisoning outbreaks. All strains were screened for bacteriocin production against a common indicator strain of C. perfringens. In the one significant hospital outbreak of C. perfringens diarrhea, three to five plasmid types were found in strains of the predominant serotype, but no similar correlation between serotype and plasmid type was found in random isolates from a variety of sources. All of the strains associated with the diarrhea outbreak produced bacteriocins, whereas 63% of the strains from various sources produced bacteriocins. The typing data suggest a promising differentiating capability for plasmid analysis in the epidemiological study of outbreaks of food poisoning, diarrhea, or infections caused by C. perfringens.     

Analysis of latex agglutination test for Clostridium difficile toxin A (D-1) and differentiation between C difficile toxins A and B and latex reactive protein.

Virulent toxigenic and avirulent non-toxigenic strains of Clostridium difficile gave a positive result in the latex agglutination test (LAT) for C difficile toxin A (D-1). Similar concentrations of latex agglutinating antigen were produced by these strains in vivo. Positive reactions were also given by C sporogenes, proteolytic C botulinum Types A, B, and A/F, and Bacteroides assaccharolyticus. The latex agglutinating antigen was denatured by boiling for 10 minutes, but not by heating at 56 degrees C for 30 minutes. The reaction was abolished by incubation of test material with crude C difficile antitoxin but not with other clostridial antitoxins or specific antitoxin to C difficile toxin A. The latex agglutinating antigen present in C difficile eluted between 0.39% and 0.47% M sodium chloride, and that produced by the other clostridia, between 0.35% and 0.43% M sodium chloride by fast protein liquid chromatography. The latex agglutinating antigen of C difficile was neither cytotoxic nor mouse lethal and was distinct from toxin A and toxin B. In the analysis of faecal specimens from patients with diarrhoea the latex agglutination test correlated better with the presence of C difficile than with toxin B and detected both toxigenic and non-toxigenic strains. The latex agglutination test should only be used in the laboratory as an alternative to culture for C difficile and not as a method for the detection of C difficile toxins.     

Meeting report: Metastasis Research Society–Chinese Tumor Metastasis Society joint conference on metastasis

During September 16th–20th 2016, metastasis experts from around the world convened for the 16th Biennial Congress of the Metastasis Research Society and 12th National Congress of the Chinese Tumor Metastasis Society in Chengdu, China to share most current data covering basic, translational, and clinical metastasis research. Presentations of the more than 40 invited speakers of the main congress and presentations from the associated Young Investigator Satellite Meeting are summarized in this report by session topic. The congress program also included three concurrent short talk sessions, an advocacy forum with Chinese and American metastatic patient advocates, a ‘Meet the Professors Roundtable’ session for young investigators, and a ‘Meet the Editors’ session with editors from Cancer Cell and Nature Cell Biology. The goal of integrating expertise and exchanging the latest findings, ideas, and practices in cancer metastasis research was achieved magnificently, thanks to the excellent contributions of many leaders in the field.