Glutamate dehydrogenase is highly conserved among Clostridium difficile ribotypes.

gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.

Clostridium difficile prevalence rates in a large healthcare system stratified according to patient population, age, gender, and specimen consistency.

We evaluated Clostridium difficile prevalence rates in 2,807 clinically indicated stool specimens stratified by inpatient (IP), nursing home patient (NH), outpatient (OP), age, gender, and specimen consistency using bacterial culture, toxin detection, and polymerase chain reaction (PCR) ribotyping. Rates were determined based on the detection of toxigenic C. difficile isolates. We identified significant differences in the rates between patient populations and with age. Specimens from NH had a higher rate (46%) for toxigenic C. difficile than specimens from IP (18%) and OP (17%). There were no gender-related differences in the rates. Liquid specimens had a lower rate (15%) than partially formed and soft specimens (25%) and formed specimens (18%) for the isolation of toxigenic C. difficile. The nontoxigenic rate was lowest for NH (4%) and highest for patients<20 years of age (23%). We identified 31 different toxigenic ribotypes from a sampling of 190 isolates that showed the lowest diversity in NH. Fluoroquinolone resistance was observed in 93% of the 027 isolates, all of the 053 isolates, and in four other ribotypes. We observed different rates for toxigenic C. difficile in stratified patient populations, with the highest rate for NH, a low overall nontoxigenic rate, and fluoroquinolone resistance.

Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.

The intestinal effects of Clostridium difficile toxin A are inidated by toxin binding to luminal enterocyte receptors. We reported previously that the rabbit ileal brush border (BB) receptor is a glycoprotein with an alpha-d-galactose containing trisaccharide in the toxin-binding domain (1991. J. Clin. Invest. 88:119-125). In this study we characterized the rabbit ileal BB receptor for this toxin. Purified toxin receptor peptides of 19 and 24 amino acids showed 100% homology with rabbit sucrase-isomaltase (SI). Guinea pig receptor antiserum reacted in Western blots with rabbit SI and with the purified toxin receptor. Antireceptor IgG blocked in vitro binding of toxin A to rabbit ileal villus cell BB. Furthermore, anti-SI IgG inhibited toxin A-induced secretion (by 78.1%, P < 0.01), intestinal permeability (by 80.8%, P < 0.01), and histologic injury (P < 0.01) in rabbit ileal loops in vivo. Chinese hamster ovary cells transfected with SI cDNA showed increased intracellular calcium increase in response to native toxin (holotoxin) or to a recombinant 873-amino acid peptide representing the receptor binding domain of toxin A. These data suggest that toxin A binds specifically to carbohydrate domains on rabbit ileal SI, and that such binding is relevant to signal transduction mechanisms that mediate in vitro and in vivo toxicity.

Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines.

The use of bacterial exotoxins may constitute novel adjuncts to treatment of gastrointestinal tract malignancies. Clostridium difficile toxin A was evaluated for its cytotoxic effect in vitro on 24 human cell lines and strains including carcinomas of the colon, pancreas, prostate, lung, breast, and lymphoid malignancies, as well as nonmalignant tissues. All nine colon and five pancreas cell lines were extraordinarily sensitive to the cytotoxic effect of Clostridium difficile toxin A at very low concentrations. This effect, which occurred rapidly and was dose dependent, was observed in all cells of seven colon and two pancreas cell lines at concentrations as low as 1-5 ng/ml (10(-12) to 10(-11) M), whereas cells derived from other sites required 60 to greater than 500 ng/ml to achieve an equivalent effect. The data suggest that Clostridium difficile toxin A may have potential therapeutic value in the treatment of some gastrointestinal tract cancers.

Fecal neutral steroids in two Japanese populations with different colon cancer risks.

Several workers have associated fecal neutral steroids with colon cancer frequency. They suggested that the risk for colon cancer increases with a rise in the level of total and certain neutral steroids. The Japanese in Hawaii, who are at high risk for this cancer, had a higher concentration of cholesterol and total animal steroids in their fecal specimens than did the people in Akita, Japan, who are at low risk. However, the rest of the findings on neutral steroids were unremarkable or inconsistent in comparison with those of other studies. These data are suggestive but not strongly supportive of a relationship between fecal neutral steroid patterns and colon cancer risk.

Cephalosporin-associated colitis and Clostridium difficile.

A case of cephalosporin-associated colitis occurred in which a tissue-cultured morphologic-altering activity was demonstrated in the patient's feces during the active episode. Neutralization of the tissue culture activity by antiserum directed against a partially purified toxin of Clostridium difficile provided a more suggestive link between the colitis and this clostridial species.

Degradation of polysaccharides by intestinal bacterial enzymes.

Polysaccharides with structures resembling components of dietary fiber were fermented by a number of species of anaerobic bacteria from the human colon. Some strains also fermented glycoprotein mucins. The strains that fermented the widest range of polysaccharide substrates were in the two genera Bacteroides and Bifidobacterium. Polysaccharide degrading enzymes from several Bacteroides species have been studied, and in most cases the enzyme activities were cell bound rather than extracellular. In all cases, the polysaccharide degrading enzymes were inducible rather than constitutive. Thus the metabolic activity of the flora could be altered considerably by the amount and type of fiber in the diet, even though the composition of the flora itself remained unchanged. The products of enzyme action included monosaccharides and oligosaccharides of varying chain lengths.

Complex carbohydrate breakdown in the human colon.

Concentrations of water-soluble high- and low-molecular-weight carbohydrates were determined in the intestinal contents of four human subjects who had died accidentally. In all four subjects, concentrations of high-molecular-weight carbohydrate were lower throughout the colon than in the ileum. In some subjects, similar but less dramatic differences were also found with low-molecular-weight carbohydrate. Components of both high- and low-molecular-weight peaks included neutral sugars such as rhamnose, galactose, mannose, xylose, and arabinose, which are found in many plant polysaccharides. Components characteristic of mucins (fucose, hexosamines, and sialic acids) were also detected. These results indicate that dietary fiber and mucin are degraded in the human colon.

Characterization of a mutagenic bacterial product in human feces.

Mutagens detectable with the Ames assay have been found in the feces of apparently healthy individuals and the incidence of this mutagenic activity was found to be greater in a population at high risk for colon cancer than in a population at low risk. A compound accounting for the mutagenic activity has been isolated by high performance liquid chromatography. Two closely related forms which behave identically chemically could be resolved. The compound was active on Salmonella typhimurium TA98 and TA100, had a characteristic ultraviolet absorption spectrum with maxima at about 320, 340, and 365 nm, fluoresced green in long wavelength ultraviolet light, and had the same mobility on the thin-layer chromatography as the mutagenic activity in a direct ether extract of feces. The compound was unstable in air but could be stabilized in the presence of butylated hydroxytoluene. Upon oxidation the compound lost its mutagenicity and its ultraviolet absorption spectrum underwent a blue shift so that the absorption maxima were at 295, 310, and 325 nm. Determination of the structure of the mutagen has been difficult since the compound was not volatile and production of a volatile derivative has not been successful. On thin-layer chromatography plates the compound reacted with reagents that detect chlorinated compounds. By thermal energy analysis it did not appear to contain a nitroso group. The compound increased in concentration upon anaerobic incubation of feces at 37 C and this increase was prevented by cold, air, and antimicrobial agents. This suggests to us that the fecal flora produces the compound.

On distribution of different fecapentaenes, the fecal mutagens, in the human population.

It has been shown by an HPLC analysis using a quarternary solvent mixture in an isocratic mode that human excretors of these fecal mutagens excrete both fecapentaene -12 and -14 but the ratios vary greatly between individuals. Since these mutagens are produced by the bacterial flora of the colon, this may indicate differences in the flora between these individuals or differences in the availability of different precursor molecules in their colons. Any relationship of these findings to the etiology of colonic cancer is not clear.

Carcinogenicity tests of fecapentaene-12 in mice and rats.

Fecapentaenes, a class of direct-acting bacterial mutagens, have been isolated from the feces and intestinal tract of humans on a Western meat-containing diet. Two bioassays to test pure fecapentaene-12 (FP-12) for carcinogenicity were performed. FP-12 in dimethylsulfoxide (DMSO) solution was injected i.p. into newborn ICR/MA mice on days 1, 3, 7, 10, 14 and 21. The mice killed after 21 months had neoplasms in liver, lung, glandular stomach and subcutaneous fibrosarcoma. Intrarectal (i.r.) infusion of FP-12 in an aqueous vehicle into male F344 rats for 71 weeks, and killing the rats after 21 weeks more, displayed no evidence of neoplasia associated with FP-12 exposure. The positive control, N-nitrosomethylurea (NMU), given i.r. as 4 2-mg doses in 2 weeks, as expected, yielded multiple colonic neoplasms in less than 11 months. Fecapentaene may exert its effect in bacteria and in newborn mice through the generation of hydroxy radicals. However, adult rodent and human colon may have adequate biochemical defense mechanisms against low level, even continuous exposures to chemicals like FP-12, and thus be at low risk of neoplasia, as was found.

Clostridium difficile toxins A and B can alter epithelial permeability and promote bacterial paracellular migration through HT-29 enterocytes.

Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis. We hypothesized that C. difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier. Experiments were designed to clarify the effects of C. difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions. In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL). To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles. Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers. To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers. Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes. Thus intestinal colonization with toxigenic C. difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.

Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium.

BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium. HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria. DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration. CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

Comparison of culture, cytotoxicity assays, and enzyme-linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease.

Clostridium difficile culture, test tube, and microtiter cytotoxicity assays, and enzyme-linked immunosorbent assays (ELISAs) for toxin A and toxin B, were simultaneously performed on 113 fresh diarrheal stool specimens randomly selected from those submitted to our clinical laboratory for routine C. difficile testing. The performance of these tests in diagnosing C. difficile-related enteric disease (CDRED) was based on a clinical assessment of the likelihood of CDRED as determined by a systematic review of case histories blinded from the test results. Among 61 antibiotic recipients, both the microtiter cytotoxicity assay and the toxin A ELISA were highly specific for CDRED (95% and 100%, respectively). Specificities for the other procedures were much lower (tube cytotoxicity assay, 79%; culture, 74%; and toxin B ELISA, 56%). The high sensitivities of the culture (89%) and toxin B ELISA (83%) were somewhat negated by their low specificities. The only test that was both specific and had acceptable sensitivity (78%) was the microtiter cytotoxicity assay. This study indicates that ELISAs for detection of C. difficile toxins are not as reliable as the cytotoxicity assay in the laboratory diagnosis of CDRED, and that clinical correlation is essential in the evaluation of any new test for CDRED.

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis.

The Bacteroides fragilis enterotoxin is an extracellular zinc metalloprotease that has been implicated in diarrheal disease of humans and animals. This toxin causes fluid accumulation in intestinal loops and is cytotoxic for HT-29 cells, an intestinal carcinoma cell line. Here we report the cloning and sequencing of the toxin gene (bftP). bftP is 1191 nucleotides coding for a 397 amino acid protein of 44.4 kDa. The toxin has a signal peptide of 18 amino acids that is typical of many lipoproteins followed by a 379 amino acid protoxin. The portion of the protoxin found in culture filtrates and stools begins at amino acid 212. An additional open reading frame located immediately upstream shows some sequence identity with cobra cytotoxins. If expressed, the ORF protein product could also play a role in the virulence of B. fragilis.

Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C. difficile.

Clostridium sordellii produces two toxins, designated HT (haemorrhagic toxin) and LT (lethal toxin), that are similar to toxins A and B of C. difficile. The physicochemical properties of toxins HT and A were remarkably similar. The specific biological activities of toxin HT were almost the same as those of toxin A, and their NH2-terminal sequences shared close homology. The properties of toxins LT and B were similar, as were their NH2-terminal sequences, but toxin B was much more cytotoxic than toxin LT. Immunodiffusion analysis with specific antibodies showed that although toxins B and LT shared major antigenic determinants, each had unique epitopes. The results suggest that toxins B and LT have diverged more than toxins A and HT. Immunoblotting with antibodies to the toxins of C. difficile showed that toxins HT and LT had common antigenic determinants.

Metabolism of 1,4-dinitro-2-methylpyrrole, a mutagen formed by a sorbic acid-nitrite reaction, by intestinal bacteria.

1,4-Dinitro-2-methylpyrrole (DNMP), a mutagenic product formed by the interaction of two common food additives, sorbic acid and sodium nitrite, was transformed to 1-nitro-2-methyl-4-aminopyrrole (NMAP) by human fecal mixtures and various intestinal bacterial strains. Under anaerobic conditions the cell suspensions of Actinomyces, Bacteroides, Clostridium, Eubacterium, Fusobacterium, and Peptostreptococcus spp. demonstrated the nitroreduction activity. Under aerobic conditions, only Actinomyces and Bacteroides spp. showed activity, and this was at a decreased level. In cell suspensions of Bacteroides thetaiotaomicron VPI 5482, NAD(P)H and glucose accelerated the reduction rate, whereas dicoumarol and heat significantly inhibited the rate, and flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) did not affect the rate. With cell-free preparations of the same strain, reduction required NAD(P)H as a cofactor in a dose-dependent fashion and was inactivated by air and heat.

Clostridium perfringens iota toxin: synergism between two proteins.

The iota toxin of Clostridium perfringens type E is a guinea pig dermonecrotic, mouse lethal toxin which cross-reacts with the iota-like toxin of Clostridium spiroforme. Antiserum raised against C. spiroforme or C. perfringens type E neutralizes the toxin from both species. By using C. spiroforme antiserum and crossed immunoelectrophoresis, we have found that there are two cross-reacting proteins, designated iota a (ia) and iota b (ib) in the culture filtrate of C. perfringens type E. Both proteins of C. perfringens were separated by preparative isoelectric focusing and had very little toxic activity when tested alone. However, when they were recombined there were 8- and 25-fold increases in bioactivity as determined by mouse lethal and guinea pig dermonecrotic assays, respectively. These results demonstrate that the iota toxin of C. perfringens requires two immunologically and biochemically different proteins for maximum activity.

Production of monoclonal antibodies against Clostridium difficile cytotoxin using immunosorbent binding bioassay procedure.

In the following study, a novel screening approach was used to develop monoclonal antibodies specific for toxin B of Clostridium difficile. The approach, which consisted of an immunosorbent binding bioassay (ISBBA), is based on antigen immunocapture by monoclonal antibodies and detection of biological activity. Our results showed ISBBA, which uses unpurified antigen, to be more sensitive than the neutralization assay and ELISA for the detection of toxin B antibody.

Stimulation of enzyme secretion from isolated pancreatic acini by Clostridium difficile toxin B.

Exposure of isolated rat pancreatic acini to increasing concentrations (10 ng - 800 ng/ml) of toxin B from Clostridium difficile produced a biphasic effect on the rate of secretion of amylase, trypsinogen, and chymotrypsinogen. Whereas doses of toxin B from 10-30 ng/ml increased enzyme secretion by 15-20%, doses between 30 ng and 60 ng/ml showed a regression of this effect, whereafter the rate of secretion of amylase, trypsinogen, and chymotrypsinogen increased with increasing concentrations of the toxin. Toxin B concentration of 800 ng/ml enhanced amylase, trypsinogen and chymotrypsinogen secretion by 119%, 185% and 195%, respectively, when compared with the basal level. Stimulation of enzyme secretion by toxin B was not affected by the presence of either actinomycin-D or cycloheximide, at a concentration which inhibited acinar RNA or protein synthesis by 80-90%. Although toxin B as well as CCK8, carbachol and secretin by themselves caused significant stimulation in amylase, trypsinogen and chymotrypsinogen secretion from isolated pancreatic acini, toxin B together with either CCK8, carbachol or secretin produced no further augmentation in enzyme secretion than what was observed with the secretagogues alone. It is concluded that toxin B of Cl. difficile exerts a direct effect on pancreatic acinar cells as evidenced by stimulation of enzyme secretion.