Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Background: Bacteriophages (phages) are viruses that infect and kill bacteria. The antimicrobial resistance crisis has driven renewed interest in phage therapy, including the use of phages to treat chronic orthopaedic infections. Methods: Here, we present the results of the first three orthopaedic patients treated with phage therapy in the United Kingdom. Results: The first patient was treated in May 2023 and received phages active against Staphylococcus aureus. At nine months follow-up, the patient’s wound remained healed, the C-reactive protein normal and the patient was walking independently. The second patient received phages active against Klebsiella pneumoniae and S. aureus; the infection remained unresolved. The third patient received phages active against Staphylococcus epidermidis; at six months follow-up, the patient was free of infection. Endotoxin was considered at least partially responsible for mild self-limiting adverse effects in two cases. Conclusions: These promising results hint at the potential for phage therapy to transform the care of chronic orthopaedic infections.

Bacteriophages are crucial components of the human microbiome and hold promise as therapeutic agents. Yet, their physical interactions with mammalian cells remain poorly understood. Here, we developed a high-throughput platform to identify phages that adhere to epithelial layers and the proteins that mediate this interaction. The identified phages encode immunoglobulin (Ig)-like domain-containing proteins that, when displayed on a non-adherent phage, confer epithelial binding and internalization in vitro, and increased phage retention in the mouse gut in vivo . Phages encoding these adhesins are among the most abundant and prevalent human gut phages, including crAss-like phages and a distinct cluster of uncharacterized myophages. Domain sequence variation alters epithelial interaction profiles, and internalized phages traffic to the endoplasmic reticulum through the Golgi apparatus, suggesting access to non-degradative internalization pathways. These findings reveal widespread phage-human interactions in the human virome, with potential impacts on health and implications for next-generation phage therapeutics.

The narrow host range of bacteriophages limits their application against genetically diverse bacteria, motivating rational host-range modification. Progress in programmable engineering depends on establishing design principles that determine the compatibility of possible host-recognition modules with phage scaffolds. Here, we establish a genomics-guided framework that systematically identifies compatibility-determining adapter domains in receptor-binding proteins for the rational engineering of phages to target clinically relevant pathogen populations. Applying this approach to 1,270 phage genomes infecting Acinetobacter baumannii and Klebsiella pneumoniae , we show that viral diversity is highly structured: 60% of the 2,313 receptor-binding proteins group into only 19 major clusters sharing conserved N-terminal compatibility adapters. The structurally most conserved adapters in Autographivirales are associated with diverse capsule-degrading depolymerases. Known host-specificities within single-adapter clusters target capsule types that represent up to 29% of A. baumannii and 44% of K. pneumoniae carbapenem-resistant populations. Overall, we define a global repertoire of modular host-recognition components for programmable configuration of phage therapeutics.

Bacteriophages are crucial components of the human microbiome and hold promise as therapeutic agents. Yet, their physical interactions with mammalian cells remain poorly understood. Here, we developed a high-throughput platform to identify phages that adhere to epithelial layers and the proteins that mediate this interaction. The identified phages encode immunoglobulin (Ig)-like domain-containing proteins that, when displayed on a non-adherent phage, confer epithelial binding and internalization in vitro, and increased phage retention in the mouse gut in vivo. Phages encoding these adhesins are among the most abundant and prevalent human gut phages, including crAss-like phages and myoviruses closely related to the recently proposed Flandersviridae family. Domain sequence variation alters epithelial interaction profiles, and internalized phages traffic to the endoplasmic reticulum through the Golgi apparatus, suggesting access to non-degradative internalization pathways. These findings reveal widespread phage-human interactions in the human viral community, with potential impacts on health and implications for next-generation phage therapeutics.

Phage therapy is gaining increasing interest in the fight against critically resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here we combine large-scale phylogeographical analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within six years. As we demonstrate in Eastern Europe, this spatio-temporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of a four-phage cocktail against the most prevalent strain type in both in vitro and in vivo animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy. Highlights: A few carbapenem-resistant Acinetobacter baumannii types dominate infections worldwide Phylogeography reveals stable strain composition of individual countries over a six-year period This spatio-temporal distribution allows preemptive preparation of region-specific phage collections A four-phage cocktail is efficacious against the most prevalent strain type in Europe.