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Alternative treatments for  Escherichia coli  infections are urgently needed, and phage therapy is a promising option where antibiotics fail, especially for urinary tract infections (UTI). We used wastewater-isolated phages to test their lytic activity against a panel of 47  E. coli  strains reflecting the diversity of strains found in UTI, including sequence type 131, 73 and 69. The plaquing host range (PHR) was between 13 and 63%. In contrast, the kinetic host range (KHR), describing the percentage of strains for which growth in suspension was suppressed for 24 h, was between 0% and 19%, substantially lower than the PHR. To improve the phage host range and their efficacy, we bred the phages by mixing and propagating cocktails on a subset of  E. coli  strains. The bred phages, which we termed evolution-squared ε 2 -phages, of a mixture of  Myoviridae  have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations,  Myoviridae  ε 2 -phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε 2 -phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε 2 -phages with the broadest host range reveals that they are mosaic intercrossings of 2–3 ancestor phages. The recombination sites are distributed over the whole length of the genome. All ε 2 -phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε 2 -phages are remarkably more suitable than the wild-type phages for phage therapy.

Natural Bred ε2-Phages Have an Improved Host Range and Virulence against Uropathogenic Escherichia coli over Their Ancestor Phages