A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates - PubMed (original) (raw)

A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates

M Rupnik et al. J Clin Microbiol. 1998 Aug.

Abstract

Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10,463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortened tcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability in C. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

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Figures

FIG. 1

FIG. 1

Representation of five open reading frames, two toxin genes (tcdA and tcdB), and three additional genes (tcdC, tcdD, and tcdE) of the PaLoc in strain VPI (toxinotype 0) in comparison to those of variant toxinotypes (toxinotypes I to X). The hatched areas represent the repetitive regions of both toxin genes. (A) Relative positions and sizes of 10 overlapping PCR fragments used to amplify the whole PaLoc sequence. (B) Schematic representation of length and restriction site polymorphisms found in different toxinotypes. Gray regions are those in which length polymorphisms were detected: deletions in the A3 fragments of toxinotypes I, II, VI, VII, and VIII or insertions in toxinotypes IV to X. Δ, relative positions and sizes (not in scale) of the deletions. Only some of the restriction sites tested in each PCR fragment are shown, and not all restriction sites of the same enzymes are shown (known for VPI 10463 sequence). A, _Acc_I; Ec, _Eco_RV; E, _Eco_RI; H, _Hin_dIII; Hc, _Hin_cII; h, _Hae_III; N, _Nsi_I; Nc, _Nco_I; P, _Pst_I; R, _Rsa_I; S, _Spe_I.

FIG. 2

FIG. 2

Typical length polymorphisms and RFLPs found in repetitive regions of tcdA (A3 PCR). The lane numbers indicate the type of fragment. Type 1 represents strain VPI 10463, type 7 represents the deletion described for serogroup F strains. Two other large deletions are observed in the uncut PCR fragment (types 5 and 6). With _Eco_RI further differences in this PCR fragment are obtained: the RFLPs in types 3 and 4 are probably due to minor deletions, and in type 2, the restriction fragments are polymorphic because of one missing _Eco_RI site. Types 5, 6, and 7 were not restrictable with _Eco_RI. In all A3 fragments the _Spe_I restriction sites are conserved.

FIG. 3

FIG. 3

Types of polymorphic restriction patterns by B1 PCR and B3 PCR used for toxinotyping. For B1 five patterns (lanes 1 to 5, respectively) of _Hin_cII (Hc) and _Acc_I (A) restriction patterns are differentiated, and for B3 four different _Hin_dIII (H) and _Rsa_I (R) patterns (lanes 1 to 4, respectively) are obtained. Lane M, 100-bp marker.

FIG. 4

FIG. 4

Restriction patterns obtained with B2, A1, and A2 PCR fragments for the different toxinotypes indicated by Roman numerals. Toxinotype 0 represents reference strain VPI 10463. The _Rsa_I restriction sites in the B2 fragment are very polymorphic, and five different patterns (indicated with Arabic numerals over the lanes) were found. When the same strains were checked for differences by A1 and A2 PCRs, typically only two different patterns were found; here the examples of _Nco_I digestion of A1 and _Hae_III digestion of A2 are shown. For A1 patterns 2 and 2a differ because the A1 fragment is shorter in strain 8864 (see text), but the _Nco_I restriction site is conserved. Lane M, marker.

FIG. 5

FIG. 5

PFGE of _Sma_I macrorestriction patterns of representative strains from all toxinotypes. Strains grouped together in one toxinotype have identical or closely related PFGE patterns (lanes 2 to 6 or 11 to 13), but in a few cases they show different patterns on PFGE (lanes 7 to 10 and 14). The same PFGE pattern is rarely observed in strains from different toxinotypes (lanes 8, 10, and 18 and lanes 16 and 17).

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