2.3. Strain characterization
DNA extraction was performed on 48 hours blood agar culture
using MagMAXTM Total Nucleic Acid Isolation Kit (Ambion, Life
Tecnologies) and the MICROLAB STARLET automatic extractor
(Hamilton Robotics). All isolates included in this study were
screened by PCRs for the toxin genes tcdA,tcdB, the binary toxin
genes cdtA and cdtB and the tcdC regulatory gene deletions as
previously described (Stubbs et al., 2000; Lemee et al., 2004;
Antikainen et al., 2009). Toxinotyping and PCR-ribotyping were
performed according to the protocol described by Rupnik et al.
(1998) and Bidet et al. (1999) respectively. For PCR-ribotyping, DNA
fingerprint patterns were compared to those of the predominant
PCR-ribotypes circulating in Europe (the collection was kindly
provided by the European Centre for Disease Prevention and
Control-ECDC). Strains with a pattern different from the reference
strain collection were tested by capillary gel electrophoresis–based
PCR-ribotyping and the resulting patterns were classified according
to the WEBRIBO-database