Extensive Hidden Prophage Diversity in Enterobacter Species Reveals Host Specificity and Local Distribution

Bacteriophages are key drivers of bacterial evolution, particularly through their integration as prophages within host genomes. However, the diversity and host specificity of prophages in clinically relevant pathogens such as Enterobacter species remain poorly characterized. In this study, we revealed the diversity of prophages, mapped their distribution, and explored their relationships with their bacterial hosts. We analyzed 3,661 prophage sequences identified from the genomes of 20 different Enterobacter species. This analysis uncovered an extensive hidden diversity, comprising 1,617 phage genera and 2,423 phage species—nearly 80% of which were singletons—highlighting an exceptionally rich prophage landscape. We found substantial variation in prophage species richness across host species and isolation sources, with E. kobei and environmental isolates exhibiting the highest richness. Prophage populations showed strong host specificity and limited cross-species transmission. Moreover, these prophages exhibited geographic structuring and significant congruence between host and prophage phylogenies, as well as with the ecological lifestyles of their bacterial hosts. Although some interspecies transmission events were detected, they were infrequent. Overall, this study provides new insights into the diversity of Enterobacter prophages and underscores their ecological and clinical relevance in shaping host adaptation and phage–host dynamics.