Characterization of the clade 4 non-toxigenic C. difficile isolate L-NTCD03 carrying the cfr(B) gene

Nibbering, B.; Nooij, S.; Harmanus, C.; Sanders, I. M. J. G.; Miedema, I. M.; Vossen, R. H. A. M.; Kloet, S.; Ardis, C. K.; Britton, R. A.; Yousefi, F.; Bayne, J.; Charavaryamath, C.; Law, A.; Murphy, M. L.; Sponseller, B.; Burrough, E. R.; Mooyottu, S.; Opriessnig, T.; Kuijper, E. J.; Smits, W. K.

doi:10.64898/2026.01.12.695941

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bioRxiv · 10.64898/2026.01.12.695941

Characterization of the clade 4 non-toxigenic C. difficile isolate L-NTCD03 carrying the cfr(B) gene

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Abstract

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, these are understudied, despite having significant potential impact for our understanding of the colonization process and as therapeutic modalities. Here, we present an in-depth characterization - including the complete genome sequence - of the non-toxigenic C. difficile strain L-NTCD03. This strain belongs to PCR ribotype 416, clade 4 and multilocus sequence type 39. It is resistant to multiple antimicrobials, but not those used for treatment of CDI. We validated the relevance of the cfr(B) gene from this strain in antimicrobial resistance to clindamycin, linezolid, retapamulin and streptogramin A. We found the L-NTCD03 strain to be non-toxic in various assays. Altogether, L-NTCD03 is a promising candidate for developing into a live biotherapeutic product.

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Nibbering, B., Nooij, S., Harmanus, C., Sanders, I. M. J. G., Miedema, I. M., Vossen, R. H. A. M., Kloet, S., Ardis, C. K., Britton, R. A., Yousefi, F., Bayne, J., Charavaryamath, C., Law, A., Murphy, M. L., Sponseller, B., Burrough, E. R., Mooyottu, S., Opriessnig, T., Kuijper, E. J., Smits, W. K.. 2026-01-12. Characterization of the clade 4 non-toxigenic C. difficile isolate L-NTCD03 carrying the cfr(B) gene. https://doi.org/10.64898/2026.01.12.695941

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