Comparison of two host-phage interaction methods in Pseudomonas aeruginosa

Goals: The aim of this work is the systematic comparison of double agar overlay plaque assay (DPA) and plankton killing assay (PKA) on a large phage-strain panel to identify strengths and weaknesses of both methods and to initially conclude which method allows for the best in vivo prediction. In addition, predictions concerning a therapy regime (simultaneous / sequential) will be compared for both methods.

Introduction:During phage therapy, usually first a phagogram, similar to an antibiogram, is carried out to determine the suitability of phage(s) of interest. Various methods are described for testing phage efficacy. The two most common are DPA, which tests the host-phage interaction on solid medium, while in PKA host-phage interaction is analysed in liquid. These two methods were compared.

Materials & Methods142 Pseudomonas aeruginosa isolates from different clinical backgrounds (urinary tract, rectal, bronchiectasis, COPD, cystic fibrosis, wounds) were used to determine the host range of 27 phages by DPA and PKA. PKA was also performed simultaneously and sequentially on selected strains using 8 strictly lytic phages.

Results:The host spectra determined using DPA and PKA differed considerably. For Pakpunavirus, Pbunavirus and Litunavirus, the assessment of lytic activity demonstrated great therapeutic potential (15-47 % coverage). Elvirus, Phikzvirus (jumbophages) were underestimated with DPA (23% coverage) but lysed well in PKA (65% coverage). Many siphoviruses (mainly lysogenic) were overestimated by DPA and lysed only a few strains in PKA. Pbunavirus, Phikzvirus and Bruynoghevirus together showed an improvement in bacterial load applied sequentially and simultaneously in some strains.

Summary: Both methods have limitations. We choose PKA as standard method for phage therapy as it is automatable, independent of size/ morphology of the plaque, dependent on lifecycle of phage and comes closest to the situation in the patient. Regarding therapy regime, it is more important which phages are combined than whether it is carried out sequentially or simultaneously.