Four hitherto undescribed Clostridium strains capable of cleaving the C ring of quercetin, kaempferol, and naringenin at C-3-C-4 were isolated from the fecal flora of humans. None of the strains cleaved catechin. C-ring fission occurred when the substrate was either in solution or in suspension. Mixed cultures of flavonoidhydrolyzing bacteria, flavonoid-cleaving bacteria, and Escherichia coli, which was used to provide the anaerobic environment, rapidly metabolized rutin to 3,4-dihydroxyphenylacetic acid, indicating that the intestinal half-life of the biologically active aglycone is short. The cleaving strains shared many phenotypic characteristics, including their inability to ferment sugars, but they differed sufficiently to indicate that they represent different species.

A total of 20 patients with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis) were evaluated with regard to the role of infectious agents and host response. Patients were selected based upon oral manifestations of their disease, 10 with periodontal disease and 10 without. Microbiologic studies of the periodontal flora of IBD-affected patients revealed a unique microflora composed predominantly of small, motile, gram-negative rods, which were most consistent with the genus Wolinella. Further studies of the host response of these patients revealed a serum-mediated defect in neutrophil chemotaxis in all 10 patients with periodontal disease. Neutrophil phagocytosis was normal. In vitro studies of neutrophil function in response to Wolinella extracts and culture supernatants revealed inhibition of neutrophil chemotaxis in a dose-response fashion. The organism was chemokinetic for neutrophils but not chemotactic. The data suggest that unusual microorganisms colonizing the oral cavity of IBD patients potentially play a role in the pathogenesis of the disease as infectious agents or modifiers of the host response or both.

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We developed a simple, rapid method for demonstrating bacterial flagella with Ryu staining solution that gave satisfactory results for numerous motile and nonmotile bacteria. Two major advantages of this method are that the staining solution, ready for use, is stable at ambient temperature indefinitely and that microscopic examination of bacteria in the stained drop preparations can be performed rapidly.

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Toxigenic strains of Clostridium difficile are causative agents of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis. The toxigenicity is routinely assayed by using highly sensitive cell cultures. We used a simple and rapid polymerase chain reaction (PCR) assay to differentiate toxigenic and nontoxigenic strains of C. difficile. Two sets of oligonucleotide primer pairs derived from nonrepeating sequences of the toxin A gene were used to amplify 546-and 252-bp DNA fragments. A primer pair derived from repeating sequences of the toxin A gene was used to amplify a 1,266-bp DNA product. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 35 cytotoxic strains of C. difficile tested generated the expected amplified DNA. In contrast, none of the 26 noncytotoxic strains tested gave positive results. Although the toxins of C. difficile have been demonstrated to cross-react serologically with the toxins of Clostridium sordellii, we did not detect any amplified DNA in two cytotoxic strains or seven noncytotoxic strains of C. sordellii. PCR was negative in all 30 strains of 20 other Clostridium species. Southern hybridization of HindIll-digested genomic DNA by use of subgenomic probes showed a single hybridization band in toxigenic strains but not in nontoxigenic strains. PCR appears to be a sensitive and specific assay for the rapid identification of toxigenic C. difflcile. Nontoxigenic C. difficile appeared to lack the C. difflcile toxin A gene. Clostridium difficile is known as a major cause of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis (2, 13), although many infants and hospitalized patients can be asymptomatically colonized with C. difficile (3, 16). The organism produces at least two toxins: toxin A (a potent enterotoxin) and toxin B (a potent cytotoxin) (1, 24, 25). These toxins are thought to play a major role in the diarrhea and colitis caused by C. difficile. C. difficile-induced diarrhea or colitis is suspected in the patients who develop diarrhea or colitis during or after treatment with antimicrobial agents and can be confirmed by the detection of the toxin(s) or the isolation of toxigenic C. difficile from the stool specimen. The cell culture assay is preferably used to detect the cytotoxin produced by toxigenic C. difficile strains because of its high sensitivity and * Corresponding author. from nontoxigenic strains. Negative PCR results were demonstrated in DNAs of C. sordellii and other Clostridium spp. MATERIALS AND METHODS Bacteria. The bacteria used in this study are listed in Table 1. The toxigenic strain C. difficile VPI 10463 was provided by

When mixed cultures containing sporeforming bacteria were treated with heat or with ethanol, the latter consistently resulted in better recovery of Clostridium and Bacillus species. Both techniques were effective in eliminating vegetative cells. An ethanol concentration greater than 25% and exposure for 45 min or

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