Ribotyping

PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.

We have developed a Clostridium difficile PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 C. difficile isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006-2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping.

We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans.

Hundreds of ribotypes of the gastrointestinal pathogen Clostridioides difficile have emerged over the last three decades, yet the factors driving their emergence are poorly understood. Recently, there has been an increase in infections caused by PCR ribotype 023 (RT023) strains in Europe. We profiled the growth of seven RT023 strains in 190 unique carbon sources and found they were able to grow in xylitol, a sugar alcohol used as a food additive in humans and animals. Other ribotypes of C. difficile tested (n = 19) displayed little to no growth in 0.5% xylitol and were growth-inhibited in higher concentrations of xylitol. Genome sequencing identified that RT023 strains acquired a putative xylitol dehydrogenase (xdh) gene in a mobile genetic element (MGE) that is absent from other C. difficile ribotypes. We created a deletion of xdh in the RT023 strain PRB1128 and observed poor growth in xylitol, indicating that the xdh is necessary for xylitol utilization. Complementation of the xdh mutant with a plasmid-based inducible copy of the xdh gene restored growth in xylitol. We performed competition assays in minibioreactor arrays (MBRAs) and observed that PRB1128 outcompeted the non-xylitol-utilizing strain CD2015 (RT027) in the presence of xylitol. These data support that the xdh gene within RT023 strains provides a fitness benefit for growth in xylitol. Interestingly, the chromosomal locus where the MGE inserted appears to be a hotspot for genetic insertions across clades of C. difficile. Together, this work improves our understanding of the molecular basis for niche adaptation of C. difficile.IMPORTANCEGenetic factors aiding in the emergence of the opportunistic pathogen Clostridioides difficile are poorly understood. Infections with clade 3 (PCR ribotype 023) strains causing severe disease have increased since 2008. Here, we show RT023 strains have the unique ability to utilize xylitol, a sugar alcohol used as a food additive in humans and animals, due to the presence of a xylitol dehydrogenase (xdh) gene within a mobile genetic element (MGE). This xylitol utilization ability confers a fitness benefit in competition against other C. difficile ribotypes, as well as in a fecal community in vitro. Research investigating the underlying genetic factors driving the physiology of C. difficile will improve our understanding of colonization and hypervirulence.