Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the