Stable co-existence of Citrobacter rodentium with a lytic bacteriophage during in vivo murine infection

Bacteriophages are ubiquitously present in bacterial communities; however, phage-bacteria interactions in complex environments like the gut remain poorly understood. Although antibiotic resistance is driving a renewed interest in phage therapy, most studies have been conducted in in vitro systems, offering limited insight into the complexity of such dynamics in physiological contexts. Here, we use the mouse-restricted enteric pathogen Citrobacter rodentium (CR), a well-established model for human enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) infections, to investigate phage-pathogen interactions in a murine model with a complex microbiota. We isolate and characterize Eifel2, a novel lytic phage infecting CR, and generate anti-phage-specific antibodies that enable the visualization of phage infections in vitro. In a murine model of CR infection, oral administration of Eifel2 led to robust phage replication in the gut without reducing the bacterial burden or infection-associated inflammation, confirming the establishment of a stable co-existence in the gut. Despite the emergence of a sub-population of phage-resistant CR mutants in vivo, they did not undergo clonal expansion, indicating that additional selective pressures impaired their widespread dissemination in the gut. Together, our findings demonstrate that imaging approaches can capture key infection stages in vitro, although in vivo models are essential for capturing the complexity of phage-bacteria interactions. This work highlights the importance of studying phage therapy in host-pathogen contexts that include a normal microbiota and a suitable host environment, where dynamic co-existence rather than eradication may define therapeutic outcomes.IMPORTANCEBacteriophages, or phages, are viruses that can either kill or persist inside bacteria. Current interests in phage biology are in part ignited by the fact that they could be used to treat infections caused by antibiotic-resistant bacteria. However, most of our understanding of phage-bacterial interactions comes from in vitro models and/or in vivo gut models relying on altering the endogenous microbiota. Here, we report the finding of a novel phage, Eifel2, which specifically targets Citrobacter rodentium (CR), the mouse equivalent of human diarrheagenic E. coli pathogens. Despite effectively killing CR in vitro, CR and Eifel2 develop a co-existence relationship in mice with an intact microbiota. Although CR phage-resistant mutants emerge, host and microbial factors constrain their expansion. This work highlights the importance of studying phage therapy in host-pathogen contexts that include the complete microbiota, where therapeutic outcomes may rely on dynamic co-existence and containment rather than eradication.