Never before has there been a more timely opportunity to investigate the molecular genetics of Clostridium difficile . Over the last few years the perception of C. difficile has changed from an obscure, and often under-researched, bacterium to one of major clinical importance, at least in industrialized nations. Coupled with the increased interest in this organism researchers now have a greater understanding of its genetic content and molecular epidemiology; a direct consequence of the multiple C. difficile genomes which have been, and currently are being, sequenced. Concurrent with the sequencing efforts have been the development of tools to genetically manipulate the organism. We are now in a position to answer fundamental questions about the biology and pathogenicity of the organism. The techniques detailed in this volume should allow researchers to enter a new era of C. difficile research where it is possible, finally, to fulfil Koch’s molecular postulates and determine empirically how this once enigmatic organism is able to cause disease.