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Interest in using bacteriophages to treat bacterial infections (phage therapy) is growing, but there have been few experiments comparing the effects of different treatment strategies on both bacterial densities and resistance evolution. While it is established that multiphage therapy is typically more effective than the application of a single phage type, it is not clear if it is best to apply phages simultaneously or sequentially. We tried single- and multiphage therapy againstPseudomonas aeruginosa PAO1in vitro , using different combinations of phages either simultaneously or sequentially. Across different phage combinations, simultaneous application was consistently equal or superior to sequential application in terms of reducing bacterial population density, and there was no difference (on average) in terms of minimizing resistance. Phage-resistant bacteria emerged in all experimental treatments and incurred significant fitness costs, expressed as reduced growth rate in the absence of phages. Finally, phage therapy increased the life span of wax moth larvae infected withP. aeruginosa , and a phage cocktail was the most effective short-term treatment. When the ratio of phages to bacteria was very high, phage cocktails cured otherwise lethal infections. These results suggest that while adding all available phages simultaneously tends to be the most successful short-term strategy, there are sequential strategies that are equally effective and potentially better over longer time scales.

Effects of Sequential and Simultaneous Applications of Bacteriophages on Populations of Pseudomonas aeruginosa In Vitro and in Wax Moth Larvae