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Clostridioides difficile PCR ribotype 151 is polyphyletic and includes pathogenic isolates from cryptic clade C-II with mono-toxin pathogenicity loci that can escape routine diagnostics

We report a patient case with pseudomembranous colitis associated with a mono-toxin producing Clostridioides difficile belonging to the very rarely diagnosed PCR ribotype (RT) 151. The infection was difficult to diagnose, since the isolate and the feces sample tested negative for toxin-encoding genes using a routine commercial test. This prompted us to sequence n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. By including whole genome sequence data from other sources, we could further place these isolates into the phylogenetic tree of C. difficile and assign them to their respective clades. These analyses revealed that 1) RT151s are polyphyletic with isolates falling into clades 1, and cryptic clades C-I and C-II 2) RT151 contains both non-toxigenic and toxigenic isolates and 3) RT151 C-II isolates contained mono-toxin pathogenicity loci (PaLoc). Additional analysis with PacBio circular consensus sequencing revealed that the isolate from our patient case report contains a novel PaLoc insertion site, lacked tcdA and a had significantly divergent tcdB sequence that might explain the failure of the diagnostic test. The study is noteworthy as 1) RT151 encompasses both typical and cryptic clades and 2) conclusive evidence for CDI due to clade C-II isolates was hitherto lacking.