In a first step, 3 of 10 PCR fragments covering the entire PaLoc (fragments B1, B3, and A3) were selected to screen for the variations in toxin genes (Fig. A). These parts of the genes were chosen because they had been shown before to be variable and had been characterized to some extent in two variant strains (strain 8864 and the reference strain of serogroup F, ATCC 53598) (21
). Additionally, the repetitive sequences covered by PCR fragments A3 and B3 are prone to homologous recombination and are predestined for deletions or insertions. Fragments B1, B3, and A3 were amplified for all 219 strains studied, and among them, fragment A3 of the tcdA
gene was the only one in which deletions were observed. The different types of deletions by comparison to the sequence of strain VPI 10463 are shown in Fig. . There was no A3 amplicon in strain 8864 (data not shown). Strains of serogroup F displayed a typical major deletion which was also observed in 12 other strains belonging to serogroup X (type 7; Fig. ). Four strains shared a similar 800-bp deletion (type 5; Fig. ). The type 6 deletion was even more extended but was found in only a single isolate.
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 (more ...)
After comparing the amplicon length with that of control strain VPI 10463, the fragments were digested with several restriction enzymes: B1 with HincII, AccI, and EcoRI, B3 with HindIII, RsaI, and HincII, and A3 with EcoRI and SpeI. The restriction patterns were similar to those of the control strain for 172 isolates (79%). Differences were observed in 47 strains. Digestion of A3 amplicons with EcoRI enabled us to identify two additional deletions: one located upstream of the EcoRI restriction site (type 3; Fig. ) and the other located downstream (type 4). These deletions were found in one and two strains, respectively. Type 2 of the EcoRI restriction was observed in 11 strains. Type 8 was found in only one isolate and represents a fragment of normal length which could not be restricted with EcoRI (data not shown in Fig. ).
As shown in Fig. , HincII and AccI distinguished four restriction patterns different from those of the reference strain (type 1) among the B1 fragments; they concerned 4 (type 2), 6 (type 3), 6 (type 4), and 28 isolates (type 5), respectively. Digestion of B1 with EcoRI resulted in only two different patterns (data not shown). Restriction of B3 with HindIII and RsaI gave three patterns different from that of the reference strain for four (type 2), six (type 3), and nine isolates (type 4), respectively (Fig. ). Three different restriction patterns were obtained with Sau3AI and two were obtained with HincII (data not shown).
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 HincII (Hc) and AccI (A) restriction patterns are differentiated, and for B3 four different HindIII (H) and Rsa (more ...)
In a second step, the 47 strains which had shown differences by comparison with the VPI 10463 strain were studied further; the whole PaLoc regions were amplified with 10 overlapping fragments (fragments B1 to B3, A1 to A3, and PL1 to PL4), and up to four restriction sites per fragment were checked as described in Materials and Methods. Deletions were detected only in the A3 region, and insertions were concentrated outside the toxin genes of PaLoc (Fig. B). Among 47 strains that were checked by PL PCRs, 38 had an insertion upstream of tcdD. These were detected in the PL1 PCR or the PL2 PCR. One strain (strain 8864) had a large insertion between tcdB and tcdA (an enlarged PL3 fragment) (data not shown). Among the PL fragments, restriction fragment length polymorphisms (RFLPs) were detected only in PL2 after digestion with NsiI, whereas HindIII in PL1, EcoRV in PL3, and HincII and SpeI in PL4 were conserved.
The B2 fragment was polymorphic when it was tested with four different restriction enzymes. Five restriction patterns were obtained with RsaI (Fig. ), four were obtained with HindIII, and two were obtained with NsiI and EcoRV (data not shown).
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 RsaI restriction sites in the B2 fragment are very polymorphic, and five (more ...)
In fragments A1 and A2 digested with PstI, NsiI, and NcoI (fragment A1) or EcoRV, XbaI, and HaeIII (fragment A2), the restriction fragments were either the same as those obtained with the standard strain VPI 10463 or changed to a second pattern for all other strains (Fig. ). Restriction of A1 with RsaI and restriction of A2 with AccI showed no differences in any of the 47 strains.
Definition of toxinotypes.
Among the 219 isolates studied, 47 showed variations in toxin genes when compared with those of the VPI 10463 reference strain. According to the changes in their PaLoc, 10 toxinotypes could be established and were designated by Roman numerals I to X (Fig. B). As demonstrated above, the major differences between groups were observed in the B1, B3, and A3 domains of the toxin genes and in the segment upstream of tcdD. Changes characteristic for each toxinotype are summarized in Table . Reference strain VPI 10463 was defined as toxinotype 0. A similar toxinotype was observed in the majority of strains (172 of 219). Formerly described variant strains of serogroup F belonged to toxinotype VIII, and isolate 8864 belonged to toxinotype X. For the majority of toxinotypes, more than one isolate was found. The variant strains and their epidemiological data and toxin status are summarized in Table .
TABLE 1 Characteristic changes used for typing of toxingenesa
TABLE 2 C. difficile strains with variant toxingenes
All toxinotypes except types I and II had changes in both the tcdB and the tcdA toxin genes. Toxinotypes IV to X had an insertion upstream of tcdD, and for toxinotype X another insertion upstream of tcdA was characteristic. The same tcdB type was observed in conjunction with different tcdA genes, like in toxinotypes V, VI, and VII or 0, I, and II. Toxinotypes IX and X had very similar tcdB genes as well. On the other hand, the identical type of tcdA gene was found in toxinotypes IX and III, together with different tcdB genes. These similarities among toxin genes or domains in various toxinotypes probably reflect phylogenetic differences or relationships.
PFGE typing of variant strains.
Twenty-five isolates were subjected to PFGE typing. Two belonged to toxinotype 0 (one of them was VPI 10463 and other was strain 34084, one of a few strains belonging to serogroup X which was not of toxinotype VIII), 2 belonged to toxinotype VIII, 5 belonged to toxinotype III, and the remaining 16 were all variant strains of toxinotypes I, II, IV, V, VI, VII, and IX.
Among 25 strains belonging to 11 different toxinotypes, we could differentiate 15 PFGE patterns (Fig. ), and isolate 38544 was nontypeable by PFGE because of DNA degradation (data not shown in Fig. ). Strains belonging to the same toxinotype usually had very similar or identical PFGE patterns. Strains from different toxinotypes could also show identical PFGE profiles. On the other hand, two or three different PFGE types could be recognized in some toxinotypes (toxinotypes IV and VI; Table and Fig. ).
FIG. 5 PFGE of SmaI 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 (more ...) Prevalence of variant strains in serogroups.
Polymorphic toxin genes were found in strains of 9 of the 22 serogroups studied and in one strain of an unknown serogroup (isolate 8864; Table ). Toxinotypes II, III, and IX tend to predominate in certain serogroups. Some other toxinotypes (toxinotypes IV, VI, and VIII) are distributed among two different serogroups.
TABLE 3 Correlation of toxinotypes andserotypes
With regard to the type of variant strains that they include, serogroups are homogeneous or heterogeneous. In a majority of serogroups (serogroups A2 to A4, A6, A8, A9, A11, A13, G, H, K, S1, and S3) only strains of toxinotype 0 are found. In some serogroups (serogroups C, A14, and A5) variant strains could be found, but they are not common. Other serogroups, like E, F, A15, A1, and X, are more likely to include variant strains. Serogroup F is the only one in which isolates of a single toxinotype and no VPI-like strains were found. A single toxinotype was also found in serogroup A16, but it is a new serogroup and only two isolates were studied. Serogroups A1, A15, and E were heterogeneous because they included VPI-like strains and strains of two additional toxinotypes.