Clonal relatedness and emerging linezolid resistance in Clostridioides difficile ribotypes 001 and 176 in the Czech Republic

Background and aims Clostridioides difficile ribotypes (RTs) 001 and 176 dominate the epidemiology of C. difficile infection (CDI) in the Czech Republic. We applied whole-genome sequencing (WGS) and broad-range antimicrobial susceptibility testing (AST) to provide detailed characterization of these lineages. Methods Between October and December 2019, 22 hospitals participated. CDI isolates were characterized by PCR ribotyping, toxin gene detection and AST to metronidazole, vancomycin and moxifloxacin. WGS and broad-range AST were performed on selected RT001 and RT176 isolates. Results The mean CDI incidence was 5.1 cases/10,000 patient-days. Isolates and epidemiological data were available for 495/524 (94.5%) CDIs. RT001 ( n = 166, 33.5%), RT014 ( n = 59, 11.9%) RT176 ( n = 51, 10.3%) were the most prevalent. Among 21 RT001 isolates, wgMLST (3745 loci) revealed 0-120 allele differences and 21 genomic inserts, eight of which carried antimicrobial resistance (AMR) genes, including cfrB , which encodes linezolid resistance. In 13 RT176 isolates, wgMLST (3298 loci) showed 0-9 allele differences and 11 inserts, eight with AMR genes. The wgMLST confirmed clonal relatedness of RT001 and RT176 isolates from different hospitals (0-3 allelic differences), yet with variation in acquired AMR gene content. Differences between genotype and expected phenotype were observed in PnimB G and metronidazole, cfrE and linezolid and tet- genes and tetracycline. Conclusion RT001 and RT176 predominate in the Czech Republic, and WGS confirmed their inter-hospital clonal relatedness. Importantly, the emergence of linezolid-resistant RT001 strains was detected in nine hospitals. The inclusion of AMR genes in genetic relatedness analysis showed higher discriminatory power compared to cgMLST or wgMLST alone.