Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins

Abstract

Pathogenic strains of Clostridium difficile commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for C. difficile disease. Some strains have been reported to produce an additional toxin, a binary toxin designated CDT. Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined. In this study we examined the frequency of binary toxin genes (cdtB and cdtA) among C. difficile isolates that do not produce LCTs (A(-) B(-)) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing. Of 58 strains tested, 9 (15.5%) were cdtB and cdtA positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection. Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc). We describe two new binary toxin-positive variants, one without any remnant of the LCT genes. This previously unknown variation was found in three isolates that were unrelated by REA typing. LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.

FIG. 1.

Schematic representation of the C. difficile PaLoc region coding for the LCTs TcdA and TcdB in reference toxinotype 0 and toxinotypes V and XI. One binary toxin gene-positive isolate was defined as toxinotype V. Whereas previously described toxinotype V isolates produce both LCTs (A⁺ B⁺), this isolate (isolate 4380, REA type AA2) was shown to represent a new type of A⁻ B⁺ C. difficile variant. Three A⁻ B⁻ isolates belonged to toxinotype XIb, and two additional A⁻ B⁻ isolates were typed as toxinotype XIa (not shown), which differs from type XIb within the A3 PCR fragment, where a deletion is present in XIa but not in XIb. Note that toxinotypes XI and V have similar restriction sites. The upstream deletion in toxinotype XI is not yet completely defined, but the remainder of the PaLoc cannot be detected with specific PCRs. PCR results from three of the binary toxin gene-positive strains were consistent with typical “PaLoc-negative” (A⁻ B⁻) strains that contain a 115-bp segment in place of the PaLoc. The 115-bp segment was detected with the Lok1-Lok3 PCR (115-bp segment is not drawn to scale). The positions of primers Lok3 and Lok1 and PCR fragments B1, A3, and Lok1-Lok3 are shown on top of and below the figure. Restriction sites are indicated below each PaLoc schematic: A, AccI; Ec, EcoRV; E, EcoRI; Ha, HaeIII; H, HindIII; Hc, HincII; N, NsiI; P, PstI; R, RsaI; S, SpeI.

FIG. 2.

Comparison of length and restriction site polymorphisms found in PCR fragment A3 of toxinotype XI strains. Toxinotypes XIa and XIb show differences in A3 fragment length (a deletion is present in toxinotype XIa), but neither XIa nor XIb has the EcoRI restriction sites typical for toxinotype 0. Lanes 1, isolate 5943; lanes 2, isolate 4680; lanes 3, isolate 3126 (all three isolates are described in this study); lanes 4, control strain R11402 (type strain of toxinotype XIb); lanes 5, control strain VPI 10463 (type strain of toxinotype 0); lanes M, DNA ladder, 100 bp.

FIG. 3.

Restriction patterns of PCR fragment A2 showing similarities between toxinotypes V and XI. Isolates 4380, 5943, and 3126 are described in this study. Strains R11402 and VPI 10463 are the previously described representative strains for toxinotypes XIb and 0, respectively. Ec, EcoRV; Ha, HaeIII; M, DNA ladder, 100 bp.