Transition from PCR-ribotyping to Whole Genome Sequencing based typing of Clostridioides difficile

Clostridioides difficile is associated with severe and even life-threatening colitis and nosocomial outbreaks. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, since the introduction of whole genome sequencing (WGS) for molecular epidemiology, PCR-ribotyping capacities have decreased. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of genome analysis granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from our laboratory, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not exclusive, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters. The exception are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. With regard to these questions, PCR-ribotyping alone provides insufficient information for certainty. We discuss the associated challenges and opportunities between these technologies.