Two Phages of the Genera Felixunavirus Subjected to 12 Hour Challenge on Salmonella Infantis Showed Distinct Genotypic and Phenotypic Changes

Abstract

<i>Salmonella</i> Infantis is considered in recent years an emerging <i>Salmonella</i> serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control <i>Salmonella</i> Infantis (<i>S</i>I). The aims of this work were to characterize two phages of the <i>Felixounavirus</i> genus, isolated using the same strain of <i>S</i>I, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of <i>S</i>I, (2) proportion of <i>S</i>I survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected <i>S</i>I decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for <i>S</i>I infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 10¹¹ PFU/mL), and a lower proportion of <i>S</i>I survivor was obtained from the challenge assay. In <i>S</i>I that interacted with phages, we identified SNPs in two genes (<i>rfaK</i> and <i>rfaB</i>), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of <i>Salmonella</i> phages (WHR and NHR) with <i>S</i>I for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.