Isolation and Characterization of Clostridioides difficile Bacteriophages

Bacteriophages, or simply phages, are viruses that infect bacteria. They are the most abundant biological entity on Earth and their impact on the physiology and evolution of bacteria is tremendous. Phages have been at the forefront of developments in molecular biology and genetics. With the rise in antibiotic resistance worldwide, they are now also viewed as powerful antimicrobials to fight multidrug-resistant pathogens. Several methods have been developed over decades of fundamental phage research to work with phages. Although some of these methods are readily applicable to most phages, others need to be specifically adapted to a given phage-host ecosystem. For example, Clostridioides difficile is a strict anaerobe and methods that are normally performed on the open bench need to be done inside an anaerobic chamber, which complicates working with phages. Phage propagation in liquid and on solid agar can also be influenced by different parameters such as the concentration of agar or agarose and the presence of divalent cations. Our lab has 20 years of experience working with C. difficile phages and this chapter describes basic methods that we use to isolate, purify, propagate, and characterize phages infecting C. difficile.