Novel ribotype/sequence type associations and diverse CRISPR-Cas systems in environmental Clostridioides difficile strains from Northern Iraq

The environment is a natural reservoir of Clostridioides difficile and here, we aimed to isolate the pathogen from seven locations in Northern Iraq. Four of the sites yielded thirty-one isolates (ten from soils, twenty-one from sediments) which together represent ribotypes 001 (five), 010 (five), 011 (two), 035 (two), 091 (eight) and 604 (nine). Twenty-five of the isolates (∼81%) are non-toxigenic while 6 (∼19%) encode the toxin A and B genes. The genomes of eleven selected isolates represent six sequence types: ST-3 (two), ST-15 (one), ST-107 (five), ST-137 (one), ST-177 (one) and ST-181 (one). Five novel ribotype/sequence type (RT/ST) associations: RT011/ST-137, RT035/ST-107, RT091/ST-107, RT604/ST-177 and RT604/ST-181 were identified and the first three are linked to ribotypes previously uncharacterised by multilocus sequence typing (MLST). Nine of the genomes belong to Clade 1 and two are closely related to the cryptic C-I clade. Diverse multiple prophages and CRISPR-Cas systems (class 1 subtype I-B1 and class 2 type V CRISPR-Cas systems) with spacers identical to other C. difficile phages and plasmids were detected in the genomes. Our data show the broader diversity that exists within environmental C. difficile strains from a much less studied location and their potential role in evolution and emergence of new strains.