Fig 1.
In vitro data and schematic of each ODE model.
(A) P. aeruginosa PAO1 growth without the addition of phage. (B) P. aeruginosa PAO1 growth after single phage treatment with either phage LUZ19, PYO2, or E215, and after simultaneous double-phage cocktail treatment with a pair of phages from LUZ19, PYO2, and E215. (C) P. aeruginosa PAO1 bacteriophage insensitive mutants (BIM) growth. (D) Schematic of a single phage interaction model. Bacteria (B) replicate at the rate (rn). In the presence of phages (P) that decay at rate (p), sensitive bacteria either mutate (a) into phage resistant single-mutant bacteria (BR) or are bound to and infected by phage (b). Infected bacteria are subsequently moved to the infected class (BI). New phages are released when the infected bacteria cell is lysed (hs). (E) Schematic of a two-phage interaction model with and without collateral phage resistance. In the presence of phages (P1 and P2), bacteria (B) mutate to either receptor-specific single-mutant bacteria (,
) which a phage using an alternate receptor (Pn) can still adsorb to, or become double-mutant bacteria (
) resistant to both receptors. Adsorbed bacteria are subsequently moved to the infected class,
). If the two phages use the same receptor, collateral resistance occurs and the double-mutant is no different than the single mutant, that is BR = BR12 and no additional mutational outcomes emerge and BR1 and BR2 are omitted.
Table 1.
Phage and bacteria parameters used in the ODE models.
Table 2.
Phage and bacteria parameters new to the two-phage treatment models.
Fig 2.
(A) Heatmap representation of PAO1 treated with single phage strains LUZ19, PYO2, or E215 (MOI 1) and combination strains LUZ19+E215, LUZ19+PYO2, or PYO2+E215 (MOI 0.5 per phage). (B) Bacterial colony counts (CFU) post treatment with LUZ19 (solid) or PYO2 (dashed). (C) Area under the curve (AUC) estimated the amount of bacterial cell density after 28 h of treatment. Treatments denoted as “a” and “b” were significant to each other and all others. *p < 0.05
Fig 3.
In vitro and bioinformatic analyses of phage resistant PAO1 mutants.
(A) Heatmap representation of mutations identified using BRESEQ post phage treatment. Phage resistant mutations in genes besides LPS or pili are denoted as “other”. (B) Efficacy of plating comparison of wild-type PAO1 and phage resistant PAO1 strains. (C) Diagram of phage receptor binding mechanisms for each of the three phages. LUZ19 binds to type IV pili while phages PYO2 and E215 both bind to the LPS O-antigen.
Fig 4.
In vitro and in silico time-kill kinetics.
(A-C) In vitro curves (blue) of phages (A) LUZ19, (B) PYO2, and (C) E215 treatment at MOI 1. Data shown as mean ± standard error of mean (SEM) of replicate wells (n>11). The curves flatten at the limit of detection (LOD) OD600 ~0.2. The logistic growth rate of phage infected cells BI (orange) and resistant cells BR (green) were fitted according to Eq 1. 95% confidence interval of model fitting is included as shaded area. (D-F) In vitro growth curve (blue) of cocktails (D) LUZ19+PYO2, (E) LUZ19+E215, or (F) PYO2+E215. Logistic growth rate of phage infected cells BI (orange), single-mutant resistant cells BR1 and BR2 (green), and double-mutant resistant cells BR12 (red) were fitted according to Eq 4 (D-E) and 5 (F).
Fig 5.
In silico single phage modeling and parameter sensitivities.
(A-C) Single phage model numerical simulations: (A) LUZ19 only treatment, (B) PYO2 only treatment, and (C) E215 only treatment. For all the simulations, B(0) = 0.2, BI(0) = 0, BR(0) = 0, P(0) = 0.2, b = 382, and rn = 0.88. For each simulation, phage specific parameter values are in Table 1. (D-F) Sensitivity analysis for the single phage model of each phage parameter settings. The relative sensitivities of (D) LUZ19, (E) PYO2, and (F) E215 were analyzed as outlined in Appendix A.
Fig 6.
In silico determination of optimal phage infection characteristics.
Different combinations of phage lysis rate (s) and burst size (h) and their corresponding time for the resistant bacteria to become detectable. The marked points denote our experimental phage strains.
Fig 7.
Double phage model numerical simulations.
(A-C) Double-phage simulations at MOI 0.5 per phage for: (A) LUZ19+PYO2, (B) LUZ19+E215, and (C) PYO2+E215. Phage concentrations are denoted with solid lines with LUZ19 (blue), PYO2 (green), and E215 (purple). For all the simulations, the black dashed line denotes the sensitive bacteria and the red solid line denotes total bacteria. Infected bacterial strains and single-phage resistant mutants are denoted by tall dashed lines and wide dashed lines respectively in colors corresponding to the target phages. The double-phage double-mutant strain is denoted with an orange dashed line.
Fig 8.
Per capita growth rate of phage resistant variants.
Relative growth rates (with 95% confidence intervals) of PAO1 resistant to different single and double phage treatments. Fitness was determined relative to phage sensitive untreated PAO1.
Fig 9.
In silico cocktail simulations using hypothetical phages.
Comparison of two-phage cocktail treatments under decreased phage binding rate b. (A) Cocktail treatment of Pa+Pb where phage Pa has a low lysis rate (s) and high burst size (h) and Pb inversely has a high lysis rate (potency) and low burst size. (B) Cocktail treatment of Pa+Pb where phage Pa has both high potency and burst size while Pb inversely has low potency and burst size.
Fig 10.
In silico experiments for two-phage sequential treatments.
Each curve simulates the total bacteria density (solid line) after adding one phage strain at MOI 1 at time 0, and a different phage strain at MOI 1 at time 8 hours. (A) LUZ19→PYO2 (pink); (B) PYO2→LUZ19 (blue); (C) LUZ19→E215 (yellow); (D) E215 →LUZ19 (green); (E) PYO2→E215 (orange); (F) E215→PYO2 (purple). (G-H) Comparison of double phage sequential treatments with different phage combinations, application order, and second dose timings. (G) The total bacterial density at 15 hours was used to measure the treatment’s ability to control the bacterial population density. (H) The proportion of the bacteria at 15 hours that are resistant to all treatment phages.