Identification of toxigenic Clostridium difficile by the polymerase chain reaction

Abstract

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

High prevalence ofClostridium difficileon retail root vegetables, Western Australia

“…Briefly, a multiplex PCR was used to detect tcdA. The primers included NK2 and NK3 from Kato et al (1991) to detect the A1 region and novel primers (B. Elliott et al unpublished data), tcdA-1 (CAGT-CACTGGATGGAGAATT) and tcdA-2 (AAGGCAA-TAGCGGTATCAG), to detect the A3 region. Each reaction consisted of 4 ll template DNA, 2 mmol l À1 MgCl 2 , 19 buffer II, 0Á01% (w/v) BSA, 200 lmol l À1 each dNTP, 0Á75 U of Taq polymerase and 0Á2 lmol l À1 of each primer in a final volume of 20 ll.…”

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S–23S rRNA intergenic spacer region and its flanking 23S rRNA

“…A primer pair, 16 and 23-10C (Table 2) [9,10], corresponding to positions of 1526^1543 of the 16S rDNA and positions of 456^474 of the 23S rDNA of Escherichia coli, was used to amplify the 16S^23S rRNA ISRs and its £anking 23S rRNA genes of 17 reference strains of the genus Lactobacillus (see strains with the accession numbers in Table 1), which represent the Lactobacillus species commonly found in human intestinal micro£ora and their closely related species. PCR ampli¢cation was performed as previously described [11]. Brie£y, one or two colonies of bacterial strains on an agar plate were suspended in 50 Wl of Tris^HCl^EDTA^saline (pH 8.0).…”

Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains

“…PCR ampli¢cation was performed as described previously [16,17]. The thermal pro¢le was 35 cycles comprising 95³C for 20 s for denature and 55³C for 2 min for annealing and extension.…”