WRTs AI AI and AI showed the same ST as
WRTs AI-82/1, AI-9-1 and AI-60 showed the same ST as was published by Dingle et al. in RTS 103, 013 and 097 (Dingle et al., 2011). WRTs 015 and 002 were assigned as WRTs 015-like and 002-like due to slight changes in their CE-ribotyping profiles; however, the ST of WRT 002-like (ST8) was identical to that of RT 002 in the studies of Knetsch et al. and Dingle et al. (Knetsch et al., 2012, Dingle et al., 2011). The ST of WRT 015-like (ST44) corresponds with the findings of Dingle et al., who identified two STs in RT 015 isolates: ST44-tcdC wild type and ST10, similarly to Knetsch et al. (Knetsch et al., 2012), with the presence of 18bp 4EGI-1 in the tcdC gene (Dingle et al., 2011). The distribution of isolates depending on the age of the patients revealed the highest ratio of non-toxigenic to toxigenic ribotypes (64:18) and low presence and absence of two predominant toxigenic RTs 001 (1.2%) and 176 (0%) in patients two years old and younger. In other age groups (3–18 years, 19–64 years and ≥65 years), the non-toxigenic and toxigenic isolates ratio decreases (27:76, 33:476 and 53: 1454), while the occurrence of RTs 001 and 176 increases (11.7%, 35.5%, to 56.4%), respectively. The predominant occurrence of RTs 001 and 027 in older population was also found in the study of authors von Müller et al., where RT 027 was not present and RT 001 was present in 9.6% in the group of patients 0–17 years and these ratios increased to 30.7% for RT 027 and 38.6% for RT 001 in the oldest group of patients (>85years), (von Müller et al., 2015). The application of the new CE-ribotyping protocol (Fawley et al., 2015) changed the CE-ribotyping profile in 7.5% of profiles (n=4) with a subsequent change of identification by the WEBRIBO database in two profiles. The WEBRIBO database provides a broad spectrum of available CE-ribotyping profiles, but the raw data are obtained by different protocols (primer design, polymer type) and some of the CE-ribotyping profiles are designated only by a WEBRIBO number or by a combination of letter and number. This stresses the importance of the use of a standardized protocol and also the standardisation of an appropriate dataset of reference C. difficile strains uploaded to the WEBRIBO database. The MLST of seven housekeeping genes of 53 ribotypes revealed 40 different STs clustering to 5 clades. Although the MLST was performed only in one isolate of each identified CE-ribotyping profile, we found the correlation with STs identified in ribotypes represented in the Leeds-Leiden C. difficile reference strain collection published by Knetsch et al. (Table 1, marked with b) (Knetsch et al., 2012). The most heterogeneous was MLST clade 1, which included 44 CE-ribotyping profiles of 53 CE-ribotyping profiles. MLST clade 1 heterogeneity was also observed in the study by Stabler et al., who found that this clade contained 106 STs of the 141 studied STs (Stabler et al., 2012). Knetsch et al. typed 35 STs out of 56 as belonging to clade 1 (Knetsch et al., 2012), whereas Griffiths et al. concluded that 31 STs out of 40 belonged to clade 1 (Griffiths et al., 2010). Similarly, Dingle et al. found 60 STs out of 69 belonging to clade 1 (Dingle et al., 2011). Several isolates belonging to a different RT or WTR revealed the same ST (clade) and the specific deletion in tcdC gene that suggests their phylogenetic relationship. RTs 027 and 176 revealed ST1 (clade 2), as was published by Knetsch et al. (Knetsch et al., 2012), as well as the presence of one base pair deletion at nucleotide position 117, which is a target site for commercial molecular systems (Krutova et al., 2014a, Mentula et al., 2015), and 18bp deletions in the tcdC gene. RT 023 and WRT 438 revealed ST5 (clade 3) and had 54bp deletions in the tcdC gene. RTs 078, 126 and WRT 413 showed ST11 and 39bp deletions in the tcdC gene. Isolates harbouring 54bp and 39bp deletions (except WRT 413) as previously described above revealed a nonsense mutation C184T (Spigaglia and Mastrantonio, 2002, Curry et al., 2007). All these isolates (RT 023, 027, 126, 176 and WRTs 413 and 438) revealed the presence of binary toxin genes, another important C. difficile virulence factor (Gerding et al., 2014).