Convergent evolution during host range expansion and virulence increase in a Salmonella bacteriophage

Viral host range expansion is predicted to evolve at the cost of reduced mean fitness. We investigated the adaptive walks of a virulent phage ( Tequintavirus ) in a spatially variable environment composed of four susceptible bacterial isolates and four resistant ones ( Salmonella enterica serotype Tennessee, sequence types ST5018 and ST319 respectively). Starting from a single ancestral phage, we evolved multiple independent populations through serial passages on non-coevolving bacteria, following the Appelmans protocol. The phage populations evolved an expanded host range and increased virulence. Whole-genome sequencing revealed recurrent parallel mutations across populations (i.e. convergent evolution), particularly in genes encoding exo- and endo-nucleases, dUTPase, and caudal proteins. Notably, two parallel mutations in the gene coding for the Long Tail Fibre became fixed early in the evolutionary trajectories. Reverse-genetics experiments introducing these mutations into the ancestral genome expanded the host range but yielded only marginal increases in virulence, highlighting the effect of compensatory mutations. Abstract Figure Highlights A phage ( Tequintavirus ) was evolved on susceptible and resistant Salmonella enterica strains Experimentally evolved phage populations displayed expanded host range and increased virulence Convergent evolution revealed adaptive mutations modifying receptor recognition in caudal proteins Reverse-genetic showed implication of two Long Tail Fibre mutations in host range expansion In Brief Generalism is traditionally predicted to evolve at the cost of lower mean fitness. Contrary to this textbook view, we demonstrate that generalist phages with expanded host range and increased virulence can readily evolve in vitro and be purposely optimized for phage therapy applications.