Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile - PubMed (original) (raw)

Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile

Carina Gonçalves et al. J Clin Microbiol. 2004 May.

Abstract

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.

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Figures

FIG. 1.

FIG. 1.

Detection of CDTa (94 kDa) and CDTb (48 kDa) components by Western blotting using antisera against Ia and Ib from C. perfringens. Lanes: PM, Rainbow molecular marker RPN 800 (Amersham Biosciences); 1, strain 22270; 2, strain 37078.

FIG. 2.

FIG. 2.

ADP-ribosyltransferase assay using a thymidine-containing brain extract as a source of actin. Lanes: 1, strain 8549 (negative control); 2, strain CD196 (positive control); 3, strain 13376; 4, strain 17153; 5, 11419; 6, strain 9459. The two bands visible on the figure represent the two isoforms of actin G.

FIG. 3.

FIG. 3.

Cytotoxic effect of TcdB on MRC-5 monolayers. (A) Negative control. (B) Reference strain ATCC 43596 (serogroup C). (C) Strain 37078.

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