ABSTRACT
Western blotting (immunoblotting) with antisera against each of 10 reference serogroups was evaluated as a means of typing Clostridium difficile. A total of 164 clinical isolates of C. difficile were tested. Variations in band profiles in each serogroup were used to type isolates into subserogroups. This technique was useful for an epidemiological investigation.
Subject(s)
Bacterial Typing Techniques , Blotting, Western/methods , Clostridioides difficile/classification , Antibodies, Bacterial , Bacterial Typing Techniques/statistics & numerical data , Blotting, Western/statistics & numerical data , Clostridioides difficile/immunology , Clostridioides difficile/isolation & purification , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Epidemiologic Methods , Evaluation Studies as Topic , Humans , Sensitivity and Specificity , SerotypingABSTRACT
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.
Subject(s)
Bacterial Toxins/genetics , Clostridioides difficile/isolation & purification , Enterocolitis, Pseudomembranous/microbiology , Enterotoxins/genetics , Feces/microbiology , Polymerase Chain Reaction , Bacterial Toxins/analysis , Bacterial Toxins/biosynthesis , Base Sequence , Clostridioides difficile/genetics , DNA, Bacterial/analysis , DNA, Bacterial/chemistry , Enterotoxins/analysis , Enterotoxins/biosynthesis , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Bacterial Proteins , Bacterial Toxins/genetics , Clostridioides difficile/classification , Cytotoxins/genetics , Genes, Bacterial , Polymerase Chain Reaction , Base Sequence , Blotting, Southern , Clostridioides difficile/genetics , Cytotoxicity Tests, Immunologic , DNA, Bacterial/biosynthesis , DNA, Bacterial/chemistry , Molecular Sequence Data , Repetitive Sequences, Nucleic AcidABSTRACT
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.
