Identification of toxigenic Clostridium difficile by the polymerase chain reaction.

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 HindIII-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. difficile. Nontoxigenic C. difficile appeared to lack the C. difficile toxin A gene.

Use of modified norleucine-tyrosine broth in identification of Peptostreptococcus anaerobius.

Gas-liquid chromatography was employed to analyze the volatile and nonvolatile acids produced in modified norleucine-tyrosine (MNT) broth by various gram-positive cocci. The MNT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit, Mich.), 0.2% L-norleucine, and 0.1% L-tyrosine. The microorganisms included reference strains and clinical isolates of Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. indolicus, P. magnus, and P. prevotii), Staphylococcus spp. (S. aureus, S. epidermidis, and S. saccharolyticus), and Streptococcus spp. (S. agalactiae, S. intermedius, S. mutans, S. sanguis I, and S. sanguis II). Only Peptostreptococcus anaerobius strains produced caproic and valeric acids in MNT broth cultures. All 11 P. anaerobius strains produced valeric acid in MNT broth, and only 1 strain failed to produce caproic acid in the medium. This unique feature aids in rapid, reliable identification of P. anaerobius with a minimum number of tests.

C-ring cleavage of flavonoids by human intestinal bacteria.

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 flavonoid-hydrolyzing 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.

Refractory periodontitis: mixed infection with Bacteroides gingivalis and other unusual Bacteroides species. A case report.

Host immune response and the predominant subgingival microflora were evaluated in a 47-year-old male exhibiting severe, recurrent periodontitis. The patient's neutrophils were chemotactically elevated but other functions were within normal limits. Significantly, Bacteroides gingivalis and Bacteroides zoogleoformans constituted 80% of the cultivable microflora and total cell count in subgingival plaque. The remainder of the cultivable microbiota was comprised of Fusobacterium nucleatum and Haemophillis aprophillis. The present study provides additional evidence for an association between B. gingivalis and severe, recurrent periodontal disease.

Bacteremia with Anaerobiospirillum succiniciproducens.

Anaerobiospirillum succiniciproducens is a motile, spiral anaerobic bacterium with bipolar tufts of flagella. Reports of clinical illness due to A. succiniciproducens are rare. In a retrospective review of anaerobic isolates referred to the Centers for Disease Control (CDC) from January 1, 1975, through January 31, 1986, isolates of A. succiniciproducens from the blood of 21 patients were identified. A single patient whose blood isolate had not been received at CDC was included in the review. These 22 patients were from 15 states. Their mean age was 58.6 years. Underlying disorders included alcoholism, atherosclerosis, malignancy, surgery, diabetes mellitus, and dental caries. Clinical features included gastrointestinal tract signs and symptoms in 17 (77%) of 22, fever greater than 38 degrees C in seven (37%) of 19, and leukocytosis of more than 10,000 cells/mm3 in 11 (58%) of 19. Although 16 patients received antimicrobial therapy, its effect on outcome was unclear. A. succiniciproducens was reported to have contributed to the deaths of seven patients. Disorders predisposing patients to anaerobic infections may put them at increased risk for A. succiniciproducens bacteremia. The presence of antecedent gastrointestinal tract signs and symptoms suggests that the gastrointestinal tract might be the primary portal of entry.

Potential role of microorganisms isolated from periodontal lesions in the pathogenesis of inflammatory bowel disease.

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.

Selective medium for the isolation of Bacteroides gingivalis.

Bacteroides gingivalis has been implicated in various forms of periodontal disease and may be responsible for other diseases in humans. The role of B. gingivalis in disease has been difficult to assess, because it is inhibited by most selective media commonly used by clinical laboratories to aid in isolating gram-negative, nonsporeforming anaerobes. We have developed a new medium, Bacteroides gingivalis agar, which contains bacitracin, colistin, and nalidixic acid as selective agents. This medium allowed B. gingivalis to be isolated from oral specimens with little difficulty and also allowed B. gingivalis to be isolated from phenotypically similar Bacteroides species, such as B. asaccharolyticus and B. endodontalis, with which it can easily be confused.

Clostridium difficile. Colonization and toxin production in a cohort of patients with malignant hematologic disorders.

We examined 45 (80%) of 56 consecutive adult patients with malignant hematologic disorders who were hospitalized during a 15-week period at Emory University Hospital, Atlanta. Stool samples for Clostridium difficile culture and cytotoxin assay were obtained on admission and then weekly during each patient's hospitalization. On admission, four patients had detectable C difficile in their stool samples, which was associated with prior antimicrobial use but not with prior cancer chemotherapy. One of the four patients with positive stool samples also had toxin present in the stool sample and was the only one with diarrhea. Eight (36%) of 22 patients hospitalized for one or more weeks had C difficile isolated from at least one stool specimen. The positive cultures showed no clustering in time, and no risk factors were identified for colonization. Only seven of 15 culture-positive stool samples and three of seven toxin-positive samples were associated with diarrhea.

Botulism and tetanus: selected epidemiologic and microbiologic aspects.

Botulism is rare in both developing and developed countries. During 1980 only 89 cases (18 food borne, 68 infant, 2 wound, 1 unspecified) were reported in the United States. Coproexamination is essential for laboratory confirmation of infant botulism. Botulinal antitoxins of equine origin are used for treating food-borne and wound botulism but are usually not recommended for infant cases. Tetanus is much more common in some developing countries than in developed countries. During 1980 only 95 cases of tetanus were reported in the United States; in 68 (72%) of these cases, the patient was 50 years or older, and in only two (2.1%) cases was the patient younger than one year. Tetanus neonatorum is a major problem in some developing countries. Diagnosis of tetanus is based primarily on clinical findings, but laboratory studies can be helpful, especially in epidemiologic investigations. Human hyperimmune immunoglobulin is now used in the treatment of tetanus.

Comparison of three reagents for detecting indole production by anaerobic bacteria in microtest systems.

Three reagents for detecting indole, Kovac, Ehrlich, and p-dimethylaminocinnamaldehyde (DMCA), were evaluated with commercial microtest systems for characterizing and identifying anaerobic bacteria. The DMCA reagent, the most sensitive of the three reagents, gave a positive reaction with 445 of 449 strains of various indole-producing anaerobic bacteria. There was 99.6% agreement between the results obtained with the DMCA in the microtest systems and results using the conventional tube test to detect indole by using xylene extraction and Ehrlich reagent. Ehrlich reagent detected indole in 163 of 176 (92.6%) indole-positive strains when the inoculum was overlaid with mineral oil before incubation. Kovac reagent was the least sensitive of the reagents tested. When the inoculum was overlaid with mineral oil, Kovac reagent detected only 80 of 108 (74.0%) of indole-positive strains. In addition to being the most sensitive reagent for detection indole, DMCA also allowed detection of indole derivatives (skatole, 3-indolepropionic acid, and 3-indolebutyric acid) produced by some clostridia.

Norleucine-tyrosine broth for rapid identification of Clostridium difficile by gas-liquid chromatography.

A new medium, norleucine-tyrosine (NT) broth, was developed for rapid identification of Clostridium difficile on the basis of caproic acid and p-cresol production. The NT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit), 0.2% L-norleucine and 0.2% L-tyrosine (wt/vol; final concentrations), and a mixture of salts. The procedure for demonstrating caproic acid and p-cresol production involves extracting NT broth cultures with ether or chloroform and analyzing the extracts with a gas-liquid chromatograph equipped with a thermal conductivity detector, as is customary for analysis of volatile fatty acids. A total of 120 strains of C. difficile from diverse geographic locations were tested by this procedure, and they all produced caproic acid and p-cresol in NT broth. No other Clostridium species or other microorganisms tested have been found to produce both products in NT broth.

Comparison of media in the Anaerobe-Tek and Presumpto plate systems and evaluation of the Anaerobe-Tek system for identification of commonly encountered anaerobes.

Using a variety of sporeforming and nonsporeforming anaerobic bacteria, we compared 10 differential agar media of the Anaerobe-Tek (A/T) system recently marketed by Flow Laboratories, Inc. (McLean, Va.) with 10 comparable media in Presumpto quadrant plates (Presumpto 1, 2, and 3) developed by the Centers for Disease Control Anaerobic Bacteria Branch. The A/T identification system was evaluated by comparing the species identity of anaerobes determined as recommended by the manufacturer's instruction manual with the identity of the strains obtained by the Centers for Disease Control Anaerobe Reference Laboratory by using conventional procedures. We also compared reactions obtained with the Presumpto plates with a chopped meat glucose broth culture as a source of inoculum with those obtained by using a turbid cell suspension from growth on blood agar as inoculum. The agreement of results for the 16 characteristics compared ranged from 92.8 to 100%. Comparison of test results obtained with 10 media in the Presumpto plate and A/T systems from the examination of 223 strains of anaerobes, representing 54 different taxa, showed the following agreement between A/T and CDC systems: catalase production, esculin hydrolysis, glucose fermentation, and lecithinase production (100%); inhibition of growth by bile agar (99.6%); lipase production (99%); DNase (98.7%); fermentation of lactose and mannitol (98.2%); starch hydrolysis (96.9%); gelatin hydrolysis (96.4%); and casein hydrolysis (94.6%). Of the 204 strains of common anaerobes tested with the A/T system, only 70% were correctly identified to the species level. However, several strains could have been identified correctly with the A/T system if data on certain other characteristics had been included in the A/T data base.

Practical procedure for demonstrating bacterial flagella.

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.

Gas-liquid chromatography technique for detection of hippurate hydrolysis and conversion of fumarate to succinate by microorganisms.

A gas-liquid chromatography technique which allows simultaneous detection of hippuric acid (N-benzoylglycine) hydrolysis and conversion of fumaric acid to succinic acid by microorganisms uses a new medium, hippurate-formate-fumarate broth, and a gas chromatograph equipped with a thermal conductivity detector. This technique gave more reproducible results than other tests used in the study for detecting hippurate hydrolysis and also gave consistent results in detecting succinic acid produced from utilization of fumaric acid.

Gelatin agar medium for detecting gelatinase production by anaerobic bacteria.

A new medium, Lombard-Dowell gelatin agar, was developed for detecting gelatinase activity by anaerobic bacteria. The medium contained: Trypticase (BBL Microbiology Systems), 5.0 g; yeast extract (Difco Laboratories), 5 g; sodium chloride, 2.5 g; sodium sulfite, 0.1 g; L-tryptophan, 0.2 g; L-cystine, 0.4 g; hemin, 10.0 mg; vitamin K1, 10.0 mg; agar, 20.0 g; D-glucose, 1.0 g; gelatin, 4.0 g; and distilled water to 1 liter. The pH was adjusted to 7.5. The medium was dispensed in 100- by 15-mm quadrant plastic dishes (5 ml per quadrant). To test for gelatinase activity, we inoculated the medium with a young enriched thioglycolate or chopped meat glucose broth culture or a turbid cell suspension in Lombard-Dowell broth, using a sterile cotton swab, and incubated it under anaerobic conditions for 48 h at 35 degrees C. The quadrants were then flooded with Frazier solution, and clear zones around the bacterial growth were recorded as positive for gelatinase activity. The new medium was tested with a variety of anaerobic bacteria, and the results were compared with data obtained with the conventional technique for detecting gelatinase activity. Overall, there was satisfactory agreement between the two tests in the detection of gelatinase activity, but the Lombard-Dowell gelatin agar tests was more rapid and somewhat more sensitive than the conventional test.

Antibiotic-associated pseudomembranous colitis: an epidemiologic investigation of a cluster of cases.

Ten cases of antibiotic-associated colitis (AAC) were identified at a hospital in Washington, D.C., from March 17 to May 9, 1979. No geographic clustering of cases was found, nor was an association with increased use of antibiotics demonstrated. Exposure to aminoglycosides, cephalosporins, and clindamycin was associated with AAC, as was a history of enemas in the seven days before the onset of illness (P=0.045). This association was strengthened when gastrointestinal procedures-defined as (1) three or more enemas per week, (2) the insertion of a nasogastric tube for two or more days, or (3) gastrointestinal surgery-were performed within seven days of the onset of illness (P=0.007). Clostridium difficile was not isolated from the hospital environments, nursing personnel, or family members of the patients. C. difficile was isolated from stool specimens of five (36%) of 14 patients who served as controls.

Atypical toxin variant of Clostridium botulinum type B associated with infant botulism.

An atypical toxin variant of Clostridium botulinum (strain 657) was isolated from the feces of a 6-week-old female infant whose symptoms and clinical history were consistent with infant botulism. Toxin detected in the feces and the toxin produced by isolates from the feces and from two rectal swabs could be neutralized by type B botulinal antitoxin only at very high ratios of of antitoxin to toxin in the neutralization mixture. One international unit of type B antitoxin neutralized only about 10 lethal doses of 657 toxin as compared with approximately 10,000 lethal doses of conventional type B toxin from the Beans strain. Antitoxin prepared against 657 toxin was 10 times more effective against the conventional toxin than against the homologous toxin. Toxoid-antitoxin-binding studies indicate that both 657 toxin and type B toxin are heterogeneous and that both toxins may contain the same molecular variants, but that the proportions of the variants are different in each.