New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection

Abstract

Isolates of Clostridium difficile from 159 hospitalized Danish patients (2005) were analysed by a new 5-plex PCR method targeting the toxin genes tcdA, tcdB, cdtA and cdtB, and 16S rDNA as an internal positive control. Additionally, the toxin-regulating gene tcdC was partially sequenced by a new sequencing-based method that revealed genetic changes that may render the gene product inactive. Finally tcdA was analysed using a previously published method for the detection of internal deletions. The 5-plex PCR revealed four different toxin gene profiles: 36 tcdA+, tcdB+, cdtA+/cdtB+; one tcdA+, tcdB-, cdtA+/cdtB+; 98 tcdA+, tcdB+, cdtA-/cdtB-; and 24 non-toxigenic tcdA-, tcdB-, cdtA-/cdtB-. Deletion studies revealed that 26 strains contained a c. 700-bp deletion in tcdA, and 39 strains contained at least one possible inactivation feature in tcdC. The prevalence of the binary toxin genes was 23%. All strains with the tcdA+, tcdB+, cdtA+/cdtB+ profile were investigated by PCR ribotyping, and this revealed eight different ribotypes, none of which were 027. The 5-plex PCR method offers a one-step, rapid and specific screening method for C. difficile toxin genes. This toxin gene profiling, together with deletion studies in tcdA and tcdC, may allow an evaluation of the pathogenic potential of C. difficile.

Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil

“…AVBERSEK et al (2009) also detected binary toxin genes in C. difficile strains isolated from piglets, but the CDT + rate was not reported. Also, the absence of non-toxigenic strains positive for binary toxin corroborates previous studies (ARROYO et al, 2007;PERSSON et al, 2008;STUBBS et al, 2000). According to STUBBS et al (2000), binary toxin genes are commonly observed in strains that possess some part of the pathogenicity locus containing the genes for toxins A and B.…”

“…The tcdA-specific primers were designed to flank the smallest of the three deletions in the 3´ region of the tcdA gene characterized in A -B + variant strains and generated a 369-bp fragment for A + B + strains and a 110-bp fragment for A -B + strains (LEMEE et al, 2004). The cdtB-specific primers were previously described by PERSSON et al (2008) and were designed from a conserved region of the cdtB gene, which is responsible for the codification of the binding component of the binary toxin from C. difficile. The final reaction volume was 25µl.…”

“…The presence of binary toxin (CDT + ) observed in the present study was much higher (50%) than in previously reported studies. PERSSON et al (2008), working with samples isolated from humans, reported that the prevalence of CDT + strains was 26%, 97.3% of which were A + B + . ARROYO et al (2007) found only 4% of strains were CDT + in a study with diarrheic horses.…”

An Unusual Case of Clostridium difficile Infection in Trinidad and Tobago

“…Clostridium difficile-associated diarrhea seems to be on the increase and is implicated in 20-30% of all antibiotic associated diarrheal cases [21]. Fatal cases caused by toxin A and B producing Clostridium difficile strains, have been reported in countries such as, Canada, United States and Europe [7,11,14,22]. However not much data has been published on the extent to which psuedomembranous colitis caused by Clostridium difficile is associated with fatality.…”

“…Psuedomembranous colitis is a mucosal disease, recognizable by numerous yellow plaques which are 0.2 -2.0 cm in diameter; these attach themselves to varying lengths of the colon [22]. Patients with psuedomembranous colitis are normally treated based on the stage of the disease.…”