Regarding the cdt genes, cdtB
was present in 121 isolates (81.2%), while cdtA and cdtC were present in 102 (67.1%) and 103 (68.7%) isolates, respectively.
Presence / absence of cdtB
in these isolates were confirmed by two different sets of primers, one set (forward5' GTTAAAATCCCCTGCTATCAACCA 3' and reverse- 5' GTTGGCACTTGGAATTTGCAAGGC 3') reported in an earlier study (19) and the other set (forward- 5' GCGTTGATGTAGGAGCTAATCGTG 3' and reverse-5'GGTTGATCGCGTTGAGTTCGTT 3') designed in our laboratory (gene bank accession no DQ882648) using Biosoftware (Primer Premier, PREMIER Biosoft International, Palo Alto, CA, USA).
Both isolates had tcdA, tcdB, and cdtB
and had a deletion in tcdC (data not shown).
albertii strains isolated so far contained the cdtB
gene encoding the cytolethal distending toxin B subunit (8,9), we examined the presence and subtype of the cdtB
gene as described (online Technical Appendix).
It consists of two independent unlinked proteins chains that are encoded by two separate genes, designated cdtA and cdtB
, and according to SCHWAN et al.
A multiplex PCR protocol that amplified the consensus portion of the B subunit of the cytolethal distending toxin gene (cdtB
) as described by Toth (16) was modified by using each of the primer pairs (s1/as1 and s2/as2) individually to screen for cdtB
in all isolates.
Primers used in study of hospitalized patients with Clostridium difficile infection, Canada, 2004-2008 Sequence (5' Primer [right arrow] 3') Specificity tcd3 TGCAATTATAAAAACATCTTTAAAC tcdC PaLoc negative regulator tcd4 TATATCTAATAAAAGGGAGATTG cdtS-F1 TGGACAGGAAGAATAATTCCTTC cdtB
binary toxin subunit B cdtS-R1 TGCAACTAACGGATCTCTTGC E5 CTCAAAACTTTTTAACGAGTG ermB erythromycin/clindamycin resistance E6 CCTCCCGTTAAATAATAGATA GyrAF TTGAAATAGCGGAAGAAATGA gyrA DNA gyrase subunit A GyrAR TTGCAGCTGTAGGGAAATC GyrBF GAAGGTCAAACTAAAACAAA gyrB DNA gyrase subunit B GyrBR GGGCTCCATCTACATCG Table 2.
PCR ribotyping and detection of genes for toxins A (tcdA), B (tcdB), binary toxin (cdtB
), and toxin regulator (tcdC) were performed as previously described (4,8,9).
Presence of tcdA, tcdB, cdtB
(binary toxin), and deletions in tcdC was determined by PCR (2).
PCR was used to detect cdtB
, one of the genes encoding binary toxin.
([double dagger]) Traits that did not show p [less than or equal to] 0.01 but were detected in [greater than or equal to] 1 isolate each include the F7-2, F8, F9, F11, F12, F12, F14, and F15 papA alleles, papC (P fimbriae assembly), papEF (P fimbriae tip pilins), papG alleles I and II (both internal and flanking sequences), afaE8 (variant Dr binding adhesin), gafD (G fimbriae), F17 fimbriae, f1mH (type 1 fimbriae), clpG (adhesin), cdtB
(cytolethal distending toxin B), ireA (siderophore receptor), kpsM III (group 3 capsule), K1 and K2 kpsM II variants, cvaC (microcin V), ibeA (invasion of brain endothelium), and rfc (04 lipopolysaccharide biosynthesis).
PCR ribotyping and gene identification for toxins A (tcdA) and B (tcdB), the binding component of CDT (cdtB
), and the tcdC gene were performed as previously described (4,10).