Evolutionary and coevolutionary phage training approaches enhance bacterial suppression and delay the emergence of phage resistance

Abstract The development of phage resistance by bacteria is a major barrier that impedes the therapeutic use of phages. Phage training has been proposed as a novel tool that harnesses the evolutionary potential of phages to improve phage infectivity. Both evolutionary and co-evolutionary phage training models have been previously reported to train phages. However, both of these phage training models have been reported able to effectively suppress the emergence of phage-resistant bacteria mutants, thus presenting a contradictory phenomenon. Therefore, in this study, we set out to systematically compare the effectiveness of both evolutionary and co-evolutionary phage training models with regard to phage physiology, infectivity, and genotype. To this end, a natural lytic phage capable of infecting a Klebsiella pneumonia strain was isolated from wastewater and subjected to evolutionary and co-evolutionary phage training for 30 days. After the phage training, the physiology and genomic characteristics of evolved and co-evolved phages were assessed. Our results demonstrated that both evolved and co-evolved phages exhibit improved bacterial suppression activity and are able to delay the emergence of phage resistance. Furthermore, both phages harbored unique genome mutational changes in different functionally associated phage proteins. Similarly, evolved and co-evolved phage-resistant bacteria mutants that arose post phage infection displayed varying phage resistance sensitivities, which may be correlated to the unique genome mutational change identified in cell membrane structure. In particular, co-evolved phage-resistant bacteria mutants exhibited less phage resistance compared to evolved phage-resistant bacteria mutants. These results highlighted the finding that the co-evolutionary phage training model serves as a better phage training model as it endows phage with improved infectivity, but also selects for phage-resistant bacteria with a lower phage resistance when compared to evolutionary phage training.