Extensive library of Ruminococcus gnavus phages revealing genomic and phenotypic diversity with novel clusters

Ruminococcus gnavus is a prevalent member of human gut microbiota and is associated with inflammatory bowel disease. Phage-based microbiota engineering represents a promising approach for alleviating dysbiosis; however, knowledge regarding R. gnavus phages remains limited. We report the isolation and characterization of 29 novel R. gnavus phages from sewage samples. All isolates exhibited siphovirus morphology, with genome sizes ranging from 32.3 to 46.0 kbp, and represented 18 putative new genera and 24 new species. Whole-proteome clustering tools indicated that R. gnavus phages form a distinct new cluster and are divided into three clusters that could correspond to new subfamilies. Representative phages from each cluster were phenotypically characterized in detail, based on adsorption kinetics, one-step growth, and stress tolerance assays, which revealed diverse infection dynamics and structural features, such as unique globular appendages at distal tail fibers. Collectively, this study established the largest and most diverse R. gnavus phage library to date, providing fundamental insights into phage-host interactions and a valuable resource for the future development of phage-based strategies to modulate R. gnavus in the human gut.IMPORTANCERuminococcus gnavus is a common gut commensal consistently associated with inflammatory bowel disease (IBD). Phage-based microbiota engineering has emerged as a promising strategy for alleviating dysbiosis. However, progress has been hampered by the limited availability of R. gnavus phages. In this study, we established and characterized a library of 29 novel R. gnavus phages. Whole-genome/proteome and phenotypic analyses revealed that these phages may represent a new taxonomic lineage and display variation in their infection strategies. This work substantially expands the known repertoire of R. gnavus phages and provides a critical resource for dissecting phage-host interactions in the human gut. Importantly, our findings lay the foundation for the future development of phage-based approaches to selectively modulate R. gnavus populations and inform therapeutic strategies for IBD.