Divergent microbial preludes to necrotising enterocolitis defined by gut phages and bacterial resistomes.
BACKGROUND: Translating microbiome correlations into robust predictive features for complex gut disorders remains elusive, partly due to oversimplified models of pathogenesis and neglect of the virome, a key player in microbial ecosystems. Necrotising enterocolitis (NEC), a devastating disease of preterm infants with no reliable clinical predictors, exemplifies this challenge. OBJECTIVE: To determine the predictive potential of the gut prophageome and polymicrobial aetiologies for NEC. DESIGN: We applied integrated metagenomic and metatranscriptomic analyses and machine learning to 1825 longitudinal stool samples from 43 preterm infants who later developed NEC and 86 gestational age-matched and birthweight-matched controls across three US hospitals. We characterised gut prophageome acquisitions and their association with clinical exposures, including antibiotics, diet and pharmacotherapies. To predict NEC risk, we integrated pre-onset prophageome, antibacterial resistome and bacteriome profiles with neonatal pathology, stratifying the cohort by disease onset timing (early: ≤40 days; late: >40 days) for separate analysis. RESULTS: NEC cases exhibited distinct viral diversity trajectories before disease onset. Early-onset NEC was best predicted by phage-bacterial interaction signatures (75% accuracy, 81% sensitivity). Metatranscriptomics revealed increased phage DNA abundance with low gene expression, suggesting a lysogenic lifestyle that may stabilise pathobionts. These phages encode metabolic genes potentially enhancing pathobiont resilience. Late-onset NEC was best predicted by antibacterial resistome profiles (83% accuracy). CONCLUSION: The gut prophageome serves as both a source of pre-symptomatic predictive signals and an active modulator of NEC pathogenesis, with distinct microbial mechanisms driving early-onset and late-onset disease. These polymicrobial etiologies inform strategies for early detection, risk stratification and the development of microbiome-targeted preventive and therapeutic interventions.