The aim of this study was to use molecular methods
The aim of this study was to use molecular methods to characterise C. difficile isolates circulating in the Czech Republic from 2013 to 2015.
Material and methods
Discussion During a three-year period (2013–2015) a total of 32 hospitals voluntarily participated in this project, but only 11 hospitals sent isolates for molecular characterisation in each year of the study. Eight percent of C. difficile isolates were non-toxigenic although they were cultured from patients suspected of CDI. These isolates were sent from hospitals with a suboptimal CDI diagnostic algorithm, which means the absence of a confirmatory test for GDH-tested positive only samples (Debast et al., 2014), or they were cultured in our laboratory, where all cultured C. difficile isolates are ribotyped and tested for the presence of genes for toxin production. In our study, 12 hospitals did not confirm the production of toxins by C. difficile isolates cultured from GDH-tested positive only stool samples. Additionally, one laboratory did not test for the presence of toxins in stool samples because they use the nucleic Berberine Sulfate pathway amplification technique (NAAT) as the first diagnostic step. CE-ribotyping of 2201 Czech isolates revealed 166 different CE-ribotyping profiles. Of these, 113 CE-ribotyping profiles (5.1%) were represented by only a single isolate, and its clinical and/or epidemiological significance is unclear. Fifty-three different CE-ribotyping profiles contained at least two isolates per profile. Of the 53 CE-ribotyping profiles, 29 profiles comprising 83.7% of all isolates were recognized identically by two large, frequently used databases. The spectrum of the most frequently found toxigenic RTs found in our study is similar to the most frequently found toxigenic RTs in the European hospital-based survey (Bauer et al., 2011). The exception is RT 176, with its specifically geographic-epidemiological occurrence in the Czech Republic (Krutova et al., 2014b) and Poland (Pituch et al., 2015). RT 176 belongs to the RT 027 “family” (Valiente et al., 2012). Data on CDI patients infected by RT 176 outcomes have recently been published in two single-centre studies, including 30 and 111 patients, respectively. The results showed a higher rate of severe CDI (11/7 and 13/3) and mortality (5/2 and 16/8) in patients infected by RT 176 compared with patients infected by non-176 ribotypes (Drabek et al., 2015, Polivkova et al., 2016). While RT 027 is distributed worldwide (He et al., 2013), its occurrence is rare to date in the Czech Republic (Krutova et al., 2014b). We identified only five isolates in four different hospitals over three years; however, hospitals from border areas with Germany and Poland (Fig. 1) did not participate in this study, and both countries have high prevalence rates of RT 027 (Arvand et al., 2014, Pituch et al., 2015). The second most common CE-ribotyping profile was RT 001 (n=456). In contrast with RT 176, RT 001 is frequently found in many European countries (Bauer et al., 2011, Wiuff et al., 2011, Arvand et al., 2014, Taori et al., 2014, Nyc et al., 2015, Freeman et al., 2015). In our study, the simultaneous presence of ribotypes 001 and 176 was detected in 28 of the 32 hospitals. Of 53 CE-ribotyping profiles, 24 were recognized only by the WEBRIBO database and these isolates comprised 11.2% (n=247) of our collection. The occurrence of several WRTs identified in our study (209, 220, 404, 416, 438, 500, 555, AI-12, AI-20, AI-21, AI-75, AI-9-1) has been reported as human clinical isolates (Novak et al., 2015, Indra et al., 2015, Fang et al., 2014, Rafila et al., 2014, Hell et al., 2011, Indra et al., 2008) or as animal isolates WRTs 203, 209, 413, 446, 596, AI-12, AI-60, AI-8/1, AI-9-1 (Janezic et al., 2014, Schneeberg et al., 2013, Indra et al., 2009, Goldová et al., 2012, Indra et al., 2008). Four of these WRTs (AI-82/1, AI-9-1, AI-60, AI-12) have recently been identified in the UK Ribotyping Reference Laboratory (Leeds, UK) as RTs 103, 013, 097 and 150 respectively (Janezic et al., 2014).