Task 4.1 - Phage isolation (m1-42): Upper respiratory secretion samples will be received from subjects of the CURE cohort. These will be seeded in lawns of bacteria identified as relevant from the preliminary experiments, as well as prospectively accrued from the metagenomic analyses. Following enrichment, isolated phages will be characterised functionally and genetically (Milestone 4). Selected phages, as assessed by the downstream analyses, will be further typified and purified. Phage isolation and characterisation will be performed by ELIAVA; ELIBIO will construct
prototypes of phage mixtures and small-scale expansion.

Task 4.2 - Phage-bacteria network analysis (m13-36): Bacterial panels, whose relevance is established in WP3, will be cross-infected with phages from T4.1, using spot assays. The resulting cross-infection matrix will be represented as a network and interactions will be evaluated. Experiments will be performed by ELIAVA and the analysis will be done in collaboration with UMAN.

Task 4.3 - In-vitro phage-bacteria interactions culture system (m24-48): A microfluidics system comprising of parallel microbial and epithelial chambers, will be constructed (milestone 3, m36). Primary human epithelial cells from existing collections (NKUA) or cell lines (ELIBIO) will be grown at air-liquid interface and bacteria introduced initially independently and subsequently in various combinations. With growing conditions established, phage preparations will be added; microbial growth and epithelial inflammatory responses will be monitored by microscopy, Luminex and PCR arrays.