The 'Appelmans protocol' is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three Pseudomonas phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive Pseudomona aeruginosa strains. After 30 rounds of selection, phages were isolated from the evolved cocktail with greatly increased host range. Control experiments with individual phages showed little host-range expansion, and genomic analysis of one of the broad-host-range output phages showed its recombinatorial origin, suggesting that the protocol works predominantly via recombination between phages. The Appelmans protocol may be useful for evolving therapeutic phage cocktails as required from well-defined precursor phages.
Keywords: Appelmans; Pseudomonas aeruginosa; antibiotic resistance; bacteriophage evolution; bacteriophage recombination; phage therapy.