Directed Evolution of a Tequatrovirus Bacteriophage Expands Host Range Against Carbapenem-Resistant Escherichia coli ST131

The global spread of carbapenem-resistant Escherichia coli ST131 strains is a major public health issue, due to their increasing prevalence in community-acquired and nosocomial multidrug-resistant infections, and their high phenotypic and genotypic diversity. In this context, bacteriophage therapy might represent an interesting complementary strategy to control bacterial infections. On the one side, bacteriophages are specific and thus prevent the depletion of commensal bacteria. On the other side, such specificity impair the use of bacteriophages against multiple targeted pathogenic bacteria.

We have thus studied the evolutionary adaptation of a bacteriophage to an environment composed of multiple genetically diverse, non-coevolving clinical pathogenic isolates. The goal is to test the hypothesis: does evolving on a genetically diverse sub-collection enable infection of all isolates in the full collection?

We first isolated a virulent Tequatrovirus and tested its virulence over a collection of 134 bacterial isolates all attributed to 4 clades (A, B, C1 and C2) within the sequence type 131. Susceptible strains are scattered along the phylogenetic tree, but the C clade appears particularly resistant. The correlation between phylogenetic distance and phage susceptibility is currently being analyzed.

Based on Appelmans' evolution protocol (Burrowes 2019), we confronted the Tequatrovirus clonal population over 14 sequential passages to a bacterial sub-population composed of 46 bacterial strains of E. coli selected to be representative of the whole collection genomic diversity, based on the core-genome. This process was repeated over three independant phage populations.

Evolved phage populations show a significant broadening of the host spectrum by infecting 38/46 genotypes (82%) compared to 29/46 (63%) by the ancestral bacteriophage. 2/46 remained completely resistant (4.4%). We then assessed the activity of the evolved phage populations against the 89 additional E. coli ST131 genotypes not used during evolution. Interestingly, the evolved bacteriophages presented a significant larger host range to these genotypes by infecting 66/89 (i.e. 82%) while the ancestral population was infecting only 66/89 (i.e. 74%). 7/89 remained resistant to the evolved phage populations. No phylogeny signal could be found for the resistant isolated.

Our results suggest that using a phage isolated from the wild word lacks of efficacy against various compared to that same phage adapted to a subset of a bacterial genotypes.

directed evolution on a genetically varied bacterial population can significantly improve the efficacy and versatility of bacteriophages against multi-resistant strains of E. coli ST131.