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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 ε2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae ε2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε2-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 ε2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε2-phages are remarkably more suitable than the wild-type phages for phage therapy.

Bacterial vaginosis (BV) is the most frequent vaginal infection in women of reproductive age. It is caused by the overgrowth of anaerobic vaginal pathogens, such as Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, which are vaginal pathogens detected during the early stages of incident BV and have been found to form multi-species biofilms. Treatment of biofilm-associated infections, such as BV, is challenging. In this study, we tested the role of an investigational engineered phage endolysin, PM-477, in the eradication of dual-species biofilms composed of G. vaginalis–F. vaginae or G. vaginalis–P. bivia. Single-species biofilms formed by these species were also analysed as controls. The effect of PM-477 on biomass and culturability of single- and dual-species biofilms was assessed in vitro using a microtiter plate assay, epifluorescence microscopy, confocal laser scanning microscopy, and quantitative PCR. The results showed that PM-477 was particularly effective in the disruption and reduction of culturability of G. vaginalis biofilms. In dual-species biofilms, PM-477 exhibited lower efficiency but was still able to selectively and significantly eliminate G. vaginalis. Since polymicrobial interactions have been shown to strongly affect the activity of various antibiotics, the activity of PM-477 in dual-species biofilms is a potentially promising result that should be further explored, aiming to completely eradicate multi-species biofilms associated with BV.