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Clostridioides difficile Molecular Epidemiology in Alberta, Canada
- Author / Creator
- Lloyd, Colin D
Clostridioides difficile is a major cause of infectious diarrhea in the hospital setting resulting in significant morbidity among elderly patients with recent antibiotic and healthcare exposure. Antibiotics disrupt the resident microbiota allowing for germination of C. difficile spores conferring susceptibility to C. difficile infection (CDI). In CDI cases, production of C. difficile A and B toxin causes diarrheal symptoms which progress in severity to pseudomembranous colitis, toxic megacolon, and death. In addition to the A and B toxins, some strains also contain a “binary” toxin. Non-toxigenic C. difficile lacking the A, B, and binary toxin genes do not cause disease. Asymptomatic colonization is also observed, particularly in children and infants, in which colonization rates are high. This population represents a potential reservoir of pathogenic strains in the community. There are currently no reports on the C. difficile genotypes colonizing Canadian children. C. difficile genotyping and culture are lengthy processes not routinely performed in the clinical microbiology laboratory, which impairs timely investigation of potential C. difficile outbreaks. C. difficile ribotyping, a genotyping method, requires a C. difficile isolate for genetic characterization. This thesis addresses these deficits by: 1) characterizing the C. difficile strains present in children and adults from Alberta, Canada, and 2) developing a direct from stool C. difficile ribotyping method.
To characterize the C. difficile molecular epidemiology in Alberta, 308 C. difficile isolates recovered from symptomatic (diarrhea and vomiting) and asymptomatic children recruited — through the Alberta Provincial Pediatric EnTeric Infection TEam (APPETITE) study — were genotyped using PCR ribotyping. Ribotypes identified in APPETITE children were compared to ribotypes identified in 79 adult and 18 pediatric C. difficile infection cases. Ribotype 106 was the most prevalent (20.8%) molecular fingerprint identified in the APPETITE children. Similarly, ribotype 106 was predominant in pediatric CDI cases (27.8%) in contrast to ribotype 027 (44.3%) in adult CDI cases. Isolate ribotypes identified in APPETITE study children were also present in adult and pediatric CDI cases. With respect to toxin genes, isolates from APPETITE study children and pediatric CDI cases contained toxin A and B genes (ranging from 88.1-94.1%), whereas 53.2% of isolates from adult CDI cases contained the binary toxin gene in addition to the toxin A and B genes. Of note, the presence of toxin in stool did not significantly differ (p=0.22) between symptomatic and asymptomatic children in the APPETITE study.
Using samples from the APPETITE study and CDI cases with direct PCR ribotyping, a direct from stool C. difficile ribotyping technique and algorithm was developed. A total of 187 stools containing toxigenic C. difficile were subjected to direct ribotyping. The success rate for direct ribotyping from stool was 66.8%; whereas 33.2% of the remaining samples required broth enrichment to produce a ribotype. Direct ribotyping was observed to correlate with the C. difficile bacterial load based on the qPCR cycle threshold (Ct) values targeting the 16S and tcdB genes, as the Ct values were significantly lower (p<0.001) in directly ribotyped stools as compared to enriched stools. Similarly, toxin positive stools were more likely to be ribotyped directly from the stool compared to toxin negative stools (p<0.001). High concordance (94.7%) was observed between direct and isolate ribotypes. Non-matching samples were due to mixed infections with more than one ribotype (4.8%) or inability to recover an isolate (0.5%). Overall, potentially mixed infections were identified in 7.5% of the samples. Additional validation revealed direct ribotypes were highly concordant (87.0%) with isolate ribotypes independently generated at the National Microbiology Laboratory. Additionally, a multiplex qPCR assay detecting triose phosphate isomerase (tpi), an enzyme involved in glycolysis present in toxigenic and non-toxigenic C. difficile, and the toxin B gene (tcdB) was developed for simultaneous detection of toxigenic and non-toxigenic C. difficile.
In conclusion, this thesis identifies the prevailing C. difficile ribotypes circulating in children as well as CDI cases in Alberta, Canada, which have not been previously reported, and describes the development of a novel direct from stool PCR ribotyping method for C. difficile surveillance.
- Graduation date
- Spring 2021
- Type of Item
- Master of Science
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.