Directed evolution of bacteriophages: impacts of 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 is to propagate phage on multiple bacterial hosts, pool the lysate, and repeat the propagation process until phage(s) can form plaques on the target host(s). In theory, this propagation process produces a phage lysate that contains input phages and their evolved phage progeny. However, in practice, this phage 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 Pseudomonas aeruginosa phage-host system, in which we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing analysis revealed that this observed host-range expansion was due to a Casadabanvirus prophage that originated from one of the Appelmans hosts. Host-range analysis of the prophage showed that it could infect five of eight bacterial hosts initially used, allowing it to proliferate and persist through the end of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment. This work highlights the impact of prophages in directed evolution experiments and the importance of incorporating sequencing data in analyses to verify output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine presence of prophage.