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

ABSTRACT 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. IMPORTANCE Enterobacter species are emerging opportunistic pathogens increasingly implicated in hospital-acquired infections. Although prophages play a pivotal role in bacterial genome evolution and host adaptation, their diversity and distribution across Enterobacter species remain largely uncharacterized. In this study, we performed a large-scale genomic analysis of 3,661 prophages from 20 Enterobacter species and uncovered an extensive and previously hidden prophage diversity. Our analysis revealed significant differences in prophage species richness across both host species and isolation sources, with E. kobei and environmental isolates exhibiting the highest richness. Prophage populations were strongly structured by host species and geography, showing limited cross-species transmission and a high degree of congruence between phage and host phylogenies. These findings highlight the structured and lineage-specific nature of prophage populations in Enterobacter and provide valuable insights into phage–host coevolution, microbial biogeography, and the design of targeted phage therapy strategies.