Appelmans Protocol forin vitro Klebsiella pneumoniaephage host range expansion leads to induction of a novel temperate linear plasmid prophagevB_KpnS-KpLi5

Abstract Adjuvant therapy with bacteriophage (phage) cocktails in combination with antibiotics is a therapeutic approach currently considered for treatment of infections with encapsulated, biofilm forming, and multidrug-resistant Klebsiella pneumoniae (Kp). Klebsiella phage are highly selective in targeting a bacterial capsule type. Considering the numerous Kp capsule types and other Kp host restriction factors, phage treatment could be facilitated when generating phages with a broad host range A modified ‘Appelmans protocol’ was used to create phages with an extended host range via in vitro forced DNA recombination. Three T7-like Kp phages with highly colinear genomes were subjected to successive propagation on their susceptible host strains representing the capsule types K64, K27, and K23, and five Kp isolates of the same capsule types initially unsusceptible for phage lysis. After 30 propagation cycles, five phages were isolated via plaque assay. Four output phages represented the original input phages, while the fifth lysed a previously non-permissible Kp isolate, which was not lysed by any of the input phages. Surprisingly, sequence analysis revealed a novel N15/phiKO2-like phage genome ( vB_KpnS_KpLi5 ) lacking substantial homologies to any of the used T7-like phages. This temperate phage was only induced in the presence of all input phages (cocktail), but not by any of them individually. Induction of temperate phages may be a stress response caused by using multiple phages simultaneously. Successive use of different phages for therapeutic purposes may be preferable over simultaneous application in cocktail formulations to avoid undesired induction of temperate phages. (243)