The development of targeted phage metagenomics

Bacteriophages drive microbial ecology, gene transfer, and host evolution, yet linking them to hosts remains a challenge with no clear answer to “who infects who.” Culture based methods detect only plaque-forming phages, underestimating diversity, while metagenomics, though powerful for assessing richness, cannot resolve host specificity. To address this gap, we developed a culture-independent approach using a nucleated bacterial minicells with metagenomics, enabling selective enrichment and characterisation of host-specific phages within complex microbial communities. Minicells were purified from three representative Gram-negative hosts, modified to produce minicells (Escherichia coli PB114, Salmonella enterica serovar Typhimurium STM14-2193, and Klebsiella pneumoniae KP06714). Minicell preparations yielded high concentrations of cells (>107/mL), with intact surface receptors required for phage adsorption. Furthermore, preparations contained minimal parental DNA. As a proof of principle, in known mixtures of host-specific and non-host-specific phages, minicells selectively enriched host-specific phages with adsorption efficiencies mostly above 98%, while depleting non-host-specific phages. Selectivity was confirmed by plaque assays, electron microscopy, and sequencing, demonstrating minicells could serve as functional, label-free capture systems for host-phage interactions. Application to sewage-derived viromes demonstrated, for the first time, minicell enrichment of a natural viral community. E. coli minicells reliably enriched coliphages with < 2% host DNA contamination, recovering novel species and genera. Over 25% of viral operational taxonomic units were complete or high quality, exceeding typical viromics reports. In contrast, S. enterica serovar Typhimurium, minicells showed variable outcomes, underscoring host-specific differences in enrichment performance. This study established the use of minicells as a reproducible platform for enriching host specific phages from both defined mixtures and environmental samples. By combining the biological specificity of adsorption with the resolution of metagenomics, minicell enrichment bridges the gap between culture-based and sequencing-based approaches. This proof of principle advances phage discovery pipelines and provides a foundation for ecological studies and virome characterisation.