Tracy D. Wilkins

Clostridium difficile: its disease and toxins.

Clostridium difficile is the etiologic agent of pseudomembranous colitis, a severe, sometimes fatal disease that occurs in adults undergoing antimicrobial therapy. The disease, ironically, has been most effectively treated with antibiotics, although some of the newer methods of treatment such as the replacement of the bowel flora may prove more beneficial for patients who continue to relapse with pseudomembranous colitis. The organism produces two potent exotoxins designated toxin A and toxin B. Toxin A is an enterotoxin believed to be responsible for the diarrhea and mucosal tissue damage which occur during the disease. Toxin B is an extremely potent cytotoxin, but its role in the disease has not been as well studied. There appears to be a cascade of events which result in the expression of the activity of these toxins, and these events, ranging from the recognition of a trisaccharide receptor by toxin A to the synergistic action of the toxins and their possible dissemination in the body, are discussed in this review. The advantages and disadvantages of the various assays, including tissue culture assay, enzyme immunoassay, and latex agglutination, currently used in the clinical diagnosis of the disease also are discussed.

Clostridium difficile Testing: after 20 Years, Still Challenging

More than 20 years ago, as Clostridium difficile was being established as the cause of pseudomembranous colitis and antibiotic-associated diarrhea (AAD), many clinical laboratories were using or beginning to use cycloserine-cefoxitin-fructose agar, a selective medium developed by Lance George and

The Mouse Model of Amebic Colitis Reveals Mouse Strain Susceptibility to Infection and Exacerbation of Disease by CD4+ T Cells

Amebic colitis is an important worldwide parasitic disease for which there is not a well-established animal model. In this work we show that intracecal inoculation of Entamoeba histolytica trophozoites led to established infection in 60% of C3H mice, while C57BL/6 or BALB/c mice were resistant, including mice genetically deficient for IL-12, IFN-γ, or inducible NO synthase. Infection was a chronic and nonhealing cecitis that pathologically mirrored human disease. Characterization of the inflammation by gene chip analysis revealed abundant mast cell activity. Parasite-specific Ab and cellular proliferative responses were robust and marked by IL-4 and IL-13 production. Depletion of CD4+ cells significantly diminished both parasite burden and inflammation and correlated with decreased IL-4 and IL-13 production and loss of mast cell infiltration. This model reveals important immune factors that influence susceptibility to infection and demonstrates for the first time the pathologic contribution of the host immune response in amebiasis.

Medium for Use in Antibiotic Susceptibility Testing of Anaerobic Bacteria

A medium is described which was designed for use in testing the minimal inhibitory concentration of antibiotics for anaerobic bacteria by agar dilution. It contains: Trypticase (1%), Gelysate (1%), yeast extract (0.5%), glucose (0.1%), pyruvate (0.1%), arginine (0.1%), NaCl (0.5%), hemin (5 μg/ml), vitamin K1 (0.5 μg/ml), agar (1.5%). The medium does not require the addition of blood to support growth of most clinical isolates of anaerobic bacteria.

Environmental Response and Autoregulation of Clostridium difficile TxeR, a Sigma Factor for Toxin Gene Expression

TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli.

Mutagens in the feces of 3 South-African populations at different levels of risk for colon cancer.

The incidence of mutagens in the feces of 3 South-African populations at different risk levels for colon cancer has been determined. Lyophilized fecal samples were extracted with ether and the mutagenicity of the extracts determined using the Salmonella/mammalian microsome mutagenicity test. 19% of the samples from urban white South-Africans, a population at a high risk for colon cancer, were mutagenic using Salmonella typhimurium strain TA100. This incidence was significantly greater (p less than 0.001) than the incidence of mutagen excretion in the low-risk populations of urban blacks (2%) and rural blacks (0%). This pattern was also obtained using Salmonella typhimurium strain TA98. The incidence of mutagen excretion for urban whites was 10%, as compared to 5% and 2% for urban and rural blacks, respectively.

A non-toxic lectin for antigen delivery of plant-based mucosal vaccines

RicinB, the non-toxic galactose/N-acetylgalactosamine-binding subunit of ricin, was fused to a model antigen, green fluorescent protein (GFP), and expressed in tobacco plants and hairy root cultures to test for utility in mucosal vaccine delivery/adjuvancy. The fusion protein retained both GFP fluorescence and galactose/galactosamine-binding activity. Intranasal immunization of mice with galactosamine-affinity purified ricinB:GFP recovered from tobacco root cultures triggered significant increases in GFP-specific serum IgGs. This strong humoral response was comparable to that observed following GFP immunization with cholera toxin adjuvant. GFP at the same concentrations but without an adjuvant was non-immunogenic. Induction of higher levels of IgG(1) than IgG(2a) following ricinB:GFP immunization suggested the presence of a Th2 response. Serum and fecal anti-GFP IgA were also induced by immunization with ricinB:GFP. Our data suggest that ricinB can be used as an adjuvant and antigen carrier to the mucosa and is efficient in eliciting systemic and mucosal immune responses.

Vaccination against lethalClostridium difficile enterocolitis with a nontoxic recombinant peptide of toxin A

Toxin A ofClostridium difficile has a complex series of repeating units, each 20 or 30 amino acids in length, located at the COOH-terminus of the molecule. In the following study, we found that antiserum against a nontoxic recombinant peptide comprising 33 of the 38 repeating units neutralized the enterotoxic and cytotoxic activity of the toxin and that hamsters vaccinated with the recombinant peptide were partially protected againstC. difficile disease.

Collaborative Evaluation of a Proposed Reference Dilution Method of Susceptibility Testing of Anaerobic Bacteria

An agar dilution method for susceptibility testing of anaerobic bacteria was evaluated to determine whether results obtained would be consistent enough to recommend it as a reference method. The study was conducted in 10 laboratories where the minimum inhibitory concentrations of six antibiotics (carbenicillin, cefoxitin, chloramphenicol, clindamycin, penicillin G, and tetracycline) were determined against 10 bacterial strains on Wilkins-Chalgren agar prepared by three manufacturers. Minimum inhibitory concentrations falling on the modes varied from 57 to 80% of all determinations and on the mode or within ±1 log2 dilution of the mode from 87 to 100% within each laboratory. When data from all laboratories were pooled, minimum inhibitory concentrations from each laboratory agreed with the overall mode 48 to 71% of the time, with an overall agreement at ±1 log2 dilution of 96%. This degree of reproducibility allows for recommendation of the procedure as a reference method. Results with three of the test strains were very consistent, and these strains are recommended as control strains: Clostridium perfringens ATCC 13124, Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741. The minimum inhibitory concentrations for these strains were on the mode or within ±1 log2 dilution of the mode 98, 99, and 99% of the time, respectively. The remaining anaerobic bacteria are recommended as reference strains.

Fibrinolytic activity of oral anaerobic bacteria

Abstract We assayed 13 species of anaerobic microorganisms found in the human oral cavity for fibrinolytic activity. Activity was detected in 6 of 6 strains of Bacteroides melaninogenicus subsp. asaccharolyticus , 3 of 3 strains of Bacteroides oralis , and 11 of 12 strains of Treponema denticola . The activity observed in Bact. oralis was variable and was the only species that required plasminogen. The fibrinolytic activity of T. denticola was extracellular. The culture supernatant was concentrated by ultrafiltration, fractionated with ammonium sulphate, then applied to a column of 2 per cent agarose. This resulted in a 1750-fold purification. The purified T. denticola fibrinolysin had only one major band on polyacrylamide gel electrophoresis, and an apparent molecular weight near 1,000,000. It was not inactivated by heating at 75 °C for 10 min and was stable from pH 5.5 to 9.5. The enzyme appeared to be an aggregation of smaller molecules. The fibrinolytic enzyme of Bact. melaninogenicus subsp. asacch . was bound to the cell membrane and shared many properties with the collagenase reported by others. The activity of this enzyme was enhanced by treatment with SDS.