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Polymerase chain reaction (PCR) amplification of a segment of the toxin A gene was used to detect toxigenic Clostridium difficile directly from stool specimens of patients with antibiotic-associated diarrhea. Although PCR-inhibitory substances were recognized in DNA prepared from stool specimens, the inhibitory substances were eliminated by using an ion-exchange column after phenol-chloroform extraction. Eventually, 39 stool specimens were evaluated by PCR. PCR results for detection of toxigenic C. difficile were in complete agreement with cell culture assay results; all 12 PCR-positive stool specimens were positive by cytotoxin assay, and all 27 PCR-negative specimens were negative by cytotoxin assay. Toxigenic C. difficile was cultured from all PCR-positive specimens. These results suggest that PCR amplification may be an effective method for laboratory diagnosis of C. difficile-associated diarrhea and colitis.

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

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Polymerase chain reaction (PCR) assay was compared with cell culture assay performed with use of HT29/C1 (human colonic epithelial) cells for identifying strains of enterotoxin-producing Bacteroides fragilis (ETBF) isolated from extraintestinal specimens. A total of 188 unselected strains obtained over 2 years at a central clinical laboratory in Tokyo were tested. Overall, 35 strains (18.6%) were positive by cell culture and PCR assay, 152 strains were negative by both assays, and 1 strain was negative by cell culture assay but positive by the PCR assay; the same results were obtained in repeated assays. Among 64 strains from blood, 18 (28.1%) were ETBF, a rate that was significantly higher (P < .05) than the 17 ETBF (13.7%) among 124 strains from other sites. These results suggest that PCR assay is a simple and reliable tool for detecting ETBF and that enterotoxin may be a virulence factor in bacteremia caused by B. fragilis.

Transfer of imipenem resistance in Bacteroides fragilis was studied. Clinical isolate B. fragilis 10-73 was highly resistant to imipenem. Imipenemresistance was transferred from 10-73 to B. fragilis strain TM4000at a frequency of 10~6/input recipient by a filter mating technique. The resistance could also be retransferred. B. fragilis 10-73 and both primary and secondary transcipients produced an imipenem-hydrolyzing metallo-/Mactamase. Acquisition of imipenemresistance correlated with the appearance of plasmid DNAwith a size (ca. 13.6kb) similar to that of the donor strain. TM4000 transformed by electroporation with purified DNAof the 13.6-kb plasmid pBFUKl produced the metallo-/Mactamase and was resistant to imipenem. Transfer was resistant to DNasetreatment and no transfer was seen with a sterile filtrate of the donor culture. It is suggested that gene transfer in B. fragilis has the properties of a conjugation system rather than those of transformation or transduction.

A new strictly anaerobic, moderate thermophilic (optimum temperature, 45"C), cellulolytic, sporeforming bacterium was isolated from Thai compost. The cells of this organism stained gram positive but became gram negative as cultures reached stationary phase. They were nonmotile rods and formed terminal oval spores which swelled the cells. The deep colonies of this organism were spindle shaped and yellowish white. A variety of carbohydrates, such as cellobiose, esculin, and xylose, served as substrates for growth. Ethanol, acetate, butyrate, hydrogen, and carbon dioxide were produced during growth on cellulose or cellobiose. This organism hydrolyzed crystalline cellulose, rice straw, and other cellulosic materials without any chemical pretreatment. Optimal growth occurred at 45°C and pH 7.0. The deoxyribonucleic acid base composition was 40 mol% guanine plus cytosine. The name Clostridium josui sp. nov. is proposed for this new isolate, and the type strain has been deposited in the Fermentation Research Institute, Tsukuba, Japan, as strain FERM P-9684.Cellulolytic anaerobic bacteria isolated from various sources have been used to convert cellulosic materials into valuable compounds, such as ethanol and organic acids. Some of these bacteria are mesophiles (3, 6 ) , whereas others are thermophiles (2). Usually the thermophiles elaborate enzymes that are more active at high temperatures and more thermostable than enzymes produced by their mesophilic counterparts (21). In addition, they have faster growth rates, as well as higher rates of saccharification, than mesophiles (4, 5). In a previous paper (17), we described a mesophilic cellulolytic anaerobe, Coprococcus species. However, the activity of this organism decreased rapidly when it was grown at high temperatures, such as 45°C. Therefore, it has been our aim to search for a similar organism of the thermophilic type. In this report, we describe a potent cellulolytic, anaerobic, moderately thermophilic organism that was isolated from compost in Thailand. MATERIALS AND METHODSCellulosic materials. Ball-milled cellulose (BMC) was prepared by ball milling a 3% water suspension of pure cellulose (KC flock W-300; Sanyo Kokusaku Pulp Co., Tokyo, Japan) for 3 days. The microcrystalline cellulose used was Avicel (E. Merck AG, Darmstadt, Federal Republic of Germany). Agricultural cellulosic wastes, such as rice straw, rice husks, corncobs, sawdust, and bagasse, were obtained from sources in Thailand and were used without any chemical pretreatment. After drying, these cellulosic materials were ground for 3 min in a vibrating sample mill (model TI-100; Heiko Seisakusho, Ltd., Tokyo, Japan), sieved through a 100-mesh sieve (149 Fm), and used as main carbon sources in media.Other chemicals used were commercial products of the highest purity available.Media and culture techniques. (i) Rumen fluid medium. A rumen fluid medium was prepared by modifying the medium described by Taya et al. (20). The procedure used was as follows. A mixture of 7.5 ml of mineral I (K,HPO,), 7.5 ml of ...

Objective: Prevotella bivia is one of the anaerobic bacteria that resides in the flora of the female genital tract. We studied the pathogenicity of P. bivia in a rat pyometra model.Methods: The experimental animal (rat) model of pyometra was developed to investigate the pathogenicity of P. bivia in a rat pyometra model.Results: In the groups inoculated with aerobes alone, the infection rate was 10% (1/10) in the Staphylococcus aureus-or Staphylococcus agalactiae -inoculated group and 20% (2/10) in the Escherichia coli-inoculated group. Infection Was not established in the groups inoculated with anaerobes alone. High infection rates were observed in all the mixed-infection groups. In the S. agalactiae-and Bacteroides fragilis-, S. agalactiae-and P. bivia -, E. coli-and B. fragilis-, and E. coli-and P. biviainoculated groups, an infection rate of 100% (10/10) was demonstrated. The efficacy of antibiotics such as flomoxef (FMOX) could be determined using a rat pyometra model. In relation to the alteration of vaginal microbial flora during the menstrual cycle, estrogen increased the growth of P. was determined in the association of mixed infection with aerobic bacteria. The adequacy of a rat pyometra model was evaluated for the administration of efficacy of antibiotics. Since P. bivia is predominantly isolated in the vaginal cavity during the follicular phase, the growth of P. bivia might be associated with sex steroid hormones, especially estrogen and progesterone. Additionally, the effects of estrogen and progesterone on the growth of P. bivia were studied using this model.