Bacteriophages show promise for microbiome engineering, but studying their transmission dynamics in multimember communities and animal hosts is technically challenging. We therefore created 'Phollow', a live imaging-based approach for tracking phage replication and spread in situ with single-virion resolution. Following interbacterial phage transmission is achieved by marking virions with distinct fluorescent proteins during assembly in newly infected cells. In vitro cell virology studies revealed clouds of phage virions dispersing upon bacterial lysis, leading to rampant transmission. Combining Phollow with optically transparent zebrafish, we visualized phage outbreaks within the vertebrate gut. We observed that virions from a zebrafish-derived Plesiomonas strain, but not a human-derived E. coli, rapidly disseminate systemically to the liver and brain. Moreover, antibiotics triggered waves of interbacterial transmission and sudden shifts in gut community ecology. Phollow ultimately empowers multiscale investigations of phage transmission and transkingdom interactions that have the potential to open new avenues for phage-based microbiome therapies.

Bacterial viruses (known as "phages") shape the ecology and evolution of microbial communities, making them promising targets for microbiome engineering. However, knowledge of phage biology is constrained because it remains difficult to study phage transmission dynamics within multi-member communities and living animal hosts. We therefore created "Phollow": a live imaging-based approach for tracking phage replication and spread in situ with single-virion resolution. Combining Phollow with optically transparent zebrafish enabled us to directly visualize phage outbreaks within the vertebrate gut. We observed that virions can be rapidly taken up by intestinal tissues, including by enteroendocrine cells, and quickly disseminate to extraintestinal sites, including the liver and brain. Moreover, antibiotics trigger waves of interbacterial transmission leading to sudden shifts in spatial organization and composition of defined gut communities. Phollow ultimately empowers multiscale investigations connecting phage transmission to transkingdom interactions that have the potential to open new avenues for viral-based microbiome therapies.