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This second edition provides 21 new chapters on methods used in laboratories for investigating the physiology and molecular genetics of the pathogen Clostridium difficile. Chapters detail up-to -date experimental techniques for gene editing and transcriptional analysis which are used to investigate the fundamental biology of the organism and its virulence factors. Additional chapters describe development of potential new treatments including vaccines, bacteriophage and faecal transplantation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Clostridium difficile: Methods and Protocols, Second Edition provides a comprehensive catalogue of molecular tools and techniques authored by the researchers who have developed them.