Molecular typing & evolution of Clostridium difficile

Abstract

This thesis describes the application of molecular typing methods to study the epidemiology and evolution of Clostridium difficile. C. difficile is a gut bacterium that is a major cause of antibiotic associated diahrrea in the developed world. The emergence of the __hypervirulent__ types causing C. difficile infections (CDI) with more severe complications has driven the demand for rapid molecular assays that can detect these types. We aimed to identify novel molecular markers for __hypervirulent__ C. difficile and use these markers to develop new rapid assays. This thesis describes the development and evaluation of a new (multiplex) real-time PCR assay to diagnose CDI. It also provides an overview of the currently applied methods to study CDI. We report on an internationally-standardized protocol for high resolution, capillary gel-based electrophoresis, PCR-ribotyping. Furthermore, we describe two novel genetic markers that are linked to two C. difficile phylogenetic lineages. We describe the application of multilocus sequence typing to study the population structure of C. difficile. We report on whole genome sequencing (WGS) and the related single nucleotide polymorphism (SNP) analysis, a recently emerged typing method that we used to study transmission of C. difficile type 078 between animals (pigs) and humans (farmers).