Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile

Abstract

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. 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, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. 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 analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, 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 complete, 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, using cutoffs of 30-50 allele differences. The exceptions 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. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

Retrospective Study of the Epidemiology of Clostridioides difficile Infection in the Neurosurgery Department of a Tertiary Hospital in China

“…Although PCR-ribotyping is the gold-standard method for genotyping, it does not provide sufficient discriminatory power to distinguish related strains. 32 Furthermore, it suggests that there was no epidemiologic transmission of C. difficile ST-37/RT017 strains between the departments in our hospital during this period. Comparing the same clones of C. difficile ST-37/RT017 strains isolated from the neurosurgery department, we found that there was no transmission of C. difficile ST-37/RT017 strains on the same floor, as the strains apparently derived from different clones.…”

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

“…These data demonstrate that C. difficile ribotype 151 is polyphyletic with isolates from the same ribotype falling into two different cryptic clades as well as a 'regular' clade, which has not been reported before. Though there is good concordance between PCR ribotype and MLST-based approaches (13,35,36), these findings underscore that PCR ribotyping is insufficiently discriminatory for phylogenetic placement.…”

Make It Less difficile: Understanding Genetic Evolution and Global Spread of Clostridioides difficile

“…Tigecycline, a glycylcycline chemically and structurally similar to tetracyclines, has been proposed for the treatment of CDI due to its ability to inhibit spore formation and reduce the toxin production by the pathogen [ 80 ]. It should be noted that some tet genes (i.e., tet (X3) and tet (X4), encoding a flavin-dependent monooxygenase), which [ 81 , 82 , 83 , 84 , 85 , 86 , 87 ] typically confer tetracycline resistance, also appear to mediate tigecycline resistance in various microorganisms of clinical relevance [ 81 , 82 ], thus challenging the clinical efficacy of the entire family of tetracycline antibiotics, including any derivatives.…”

“…Due to its high discrimination capacity, PCR ribotyping is currently recommended by the European Centre for Disease Prevention and Control (ECDC) for surveillance of the C. difficile spread [ 84 ]. However, this molecular technique has a delivery time of up to one week and often requires in-house protocol optimization, thus not being fully transferable between laboratories [ 85 ]. Another technique in use is pulsed-field gel electrophoresis (PFGE).…”

“…In recent years, with the advancement of whole genome sequencing (WGS) techniques, the scientific community is moving towards these methods instead of standard PCR ribotyping and is trying to develop new methods for the characterization of C. difficile strains [ 85 , 91 ]. The two main approaches to discover genomic variations are single nucleotide variant (SNV) analysis and core genome or whole genome MLST (cgMLST, or wgMLST).…”