Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas aeruginosa in vitro and in wax moth larvae

Abstract

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 against Pseudomonas aeruginosa PAO1 in 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 with P. 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.

Fig 1

Bacterial density and resistance to bacteriophages. (a to c) Density of bacteria in populations treated with single phages, pairs of phages either sequentially (filled circles) or simultaneously (open circles), or all four phages sequentially (filled circles) or simultaneously (open circles). Density is shown as means ± SE for 16 replicate selection lines (four at each dilution factor) after 12 experimental transfers. (d to f) Resistance to selective phages, calculated as the proportion of infection assays that showed no evidence of inhibition by the phages to which bacteria were exposed over the phage therapy experiment; points show means ± SE from four replicates in each treatment. (g and h) Total resistance, measured against all four phages; points show means ± SE for four replicate populations; scores for total resistance in the all-phage treatments are not shown because they are identical to those for resistance to selective phages.

Fig 2

Relative fitness of resistant mutants isolated after a single growth cycle of selection with each phage individually or with all phages applied simultaneously. Points show means from four independently isolated mutants, and the average total resistance (proportion of four phages resisted) is given to the right of each point. Each point shows means ± SE for four resistant mutants, except for PT7, where one resistant mutant failed to grow during resistance assays.

Fig 3

Effects of phage therapy on life spans of infected wax moth larvae. All larvae were infected with PAO1, and some were given phage therapy (x axis). Bars show means ± SE for 18 larvae in each treatment.