International typing study of toxin A-negative, toxin B-positive Clostridium difficile variants

Abstract

Clinically important strains of Clostridium difficile that do not produce toxin A but produce toxin B and are cytotoxic (A(-)/B(+)) have been reported from multiple countries. In order to compare the relatedness of these strains, we typed 23 A(-)/B(+) C. difficile isolates from the United Kingdom (6 isolates), Belgium (11 isolates), and the United States (6 isolates) by three well-described typing methods. Restriction endonuclease analysis (REA), PCR ribotyping, and serogrouping differentiated 11, 4, and 3 different strain types, respectively. Twenty-one of the 23 A(-)/B(+) variants had a 1.8-kb truncation of the toxin A gene characteristic of toxinotype VIII strains; 20 of the 21 toxinotype VIII-like strains were PCR type 17. PCR type 17 isolates could be differentiated into two separate strain groups by serogrouping and by REA. REA further discriminated these isolates into eight subgroups (REA types). PCR type 17-serogroup F-REA group CF isolates were recovered from all three countries, and one specific REA type, CF4, was recovered from patients with C. difficile disease in the United Kingdom and the United States. C. difficile A(-)/B(+) variants of apparent clonal origin are widely distributed in Europe and North America.

FIG. 1.

Relationships of PCR ribotype (PCR Type), serogroup, REA group, and REA type analyses for 23 A⁻/B⁺ C.difficile toxin variants. The type designations for individual isolates are shown on the same horizontal lines. Isolates with the same designation were grouped, and the numbers of isolates are indicated in parentheses. The toxin A gene (tcdA) deletions determined by PCR amplification are shown for comparison with the typing results. One serogroup F-REA group CF isolate was differentiated from the top cluster by PCR ribotype and included separately (PCR type 47-serogroup F-REA type CF4).

FIG. 2.

REA of A⁻/B⁺ C.difficile toxin variants from the United Kingdom and Belgium. HindIII REA patterns are shown for study isolates 1 to 6 from the United Kingdom (A) and study isolates 7 to 17 from Belgium (B). The HindIII-restricted lambda phage molecular size marker is shown in the far left lane of each gel. The study isolate numbers and the REA type designations are shown above the gel lanes.

FIG. 3.

PCR amplification and RFLP analysis of selected A⁻/B⁺ C.difficile toxin variants. Results of PCR amplification of the tcdA-tcdC gene (A) and the tcdB gene (B) are shown, with the corresponding restriction enzyme digestion patterns of the amplified products below: Pst-I-restricted tcdA-tcdC amplicons (C) and HincII-restricted tcdB amplicons (D). The 1-kb Plus DNA ladder molecular size marker is shown in the far left lane of each gel. The study isolate numbers are shown above the gel lanes. The standard toxigenic strain VPI 10463 and the nontoxigenic REA type M3 isolate 1413 are also indicated above the lanes as the positive (A⁺/B⁺) and negative (A⁻/B⁻) control, respectively.