Bactericidal phage vectors

A method for preparing a bactericidal phage vector, comprising the following steps:

• (1) Labelling phages,
• (2) Contacting the labelled phages of (1) with bacterial cells, for which a bactericidal effect of said bactericidal phage vector is desired, under conditions which allow infection of said bacterial cells with said labelled phages,
• (3) Identifying and separating infected bacterial cells with labelled phages,
• (4) Metagenomic sequencing of the phages in infected bacterial cells of (3), including bioinformatics analysis to map the genetic relatedness of phages of (3) according to i) their nucleic acid similarity or identity levels;
• or ii) their amino acid sequence similarity or identity levels;
• or iii) a combination of i) and ii), and allocate phages of (3) into genetic clusters based on i) said nucleic acid similarity or identity levels;
• or ii) said amino acid sequence similarity or identity levels;
• or iii) a combination of i) and ii), wherein the phages in one of the genetic clusters show a sequence identity of >75% with other phages in the genetic cluster,
• (5) 4-thiouridine (4-sU)-and total metatranscriptomic sequencing of RNA of early, middle and late expressed phage genes in the phages in infected bacterial cells of (3), and mapping RNA reads to the sequences of (4),
• (6) Selecting two or more suitable marker genes allowing phage isolation from infected bacterial cells with labelled phages of (3), wherein:
• (a) the suitable marker genes are open reading frames (ORFs) showing >bout 95% average nucleotide identity (ANI) or average amino acid identity (AAI) within one genetic cluster of (4) and <about 75% ANI or AAI compared to one or more other genetic clusters of (4);
• and
• (b) the suitable marker genes are ORFs exhibiting at least 3-fold higher expression in early and late infection stages compared to the average expression level of all RNAs from the early, middle, and late expressed phage genes of (5), optionally followed by further bioinformatic analysis for potential further functions of the marker gene,
• (7) Targeted phage isolation based on said two or more selected suitable marker genes of (6),
• (8) Identification of candidate marker genes, being considered said selected suitable marker genes of (6), which exhibit bactericidal activity on the infected host cell by applying i) gene-by-gene or protein-by-protein laboratory evaluation on bactericidal effect;
• ii) bioinformatics predictions of the function of each candidate marker gene based on annotations;
• or iii) a combination of i) and ii), and
• (9) Preparing a phage vector comprising one or more candidate marker genes of (8).