Infection of zebrafish embryos with PAO1. (a) PAO1-GFP injected bacteria were visualized in different districts of the embryo at 20 hpi. Cns: central nervous system. Scale bar indicates 100 μm. (b) Injection of the tol2(mpeg1:mcherry) plasmid allowed the visualization of red macrophages migrating towards green PAO-GFP bacteria. Starting from 10 hpi, different images of the same region of the trunk were taken every 15 minutes. Scale bar indicates 20 μm.

Validation of CF embryos. (a,b) Impaired internal organ position in CF injected embryos. (a) The normal left-looping of the heart (ventral view of the embryo, visualized by means of cmlc2 in-situhybridization techniques), and (b) the normal left-position of the liver (arrows, dorsal view of the embryo visualized by means of prox1a in-situ hybridization techniques), were impaired in CF injected embryos in comparison to controls. Scale bars indicate 100 μm. liv: liver; p: pancreas. (c) Induction of TNF-α and IL-β. Expression levels, measured by RT-qPCR analyses at 8 hpi, in 48 hpf WT and CF embryos injected with PAO1 (+PAO1) are given relative to the average levels measured in mock injected controls. One tailed t-test: t_[4] = 7.62, p = 7.9E10⁻⁴ (TNF-α); t_[4] = 15.26, p = 5.4E10⁻⁵ (IL-β). (d) PAO1 microcolony formation in the hindbrain ventricle of WT and CF zebrafish embryos. The amount of microcolonies in the CF background at 18 hpi was higher compared to the WT siblings injected with the same amount of PAO1 (100 cfu). Scale bar indicates 100 μm. hv: hindbrain ventricle.

Efficacy of phage therapy in zebrafish. (a) Lethality at 20 hpi in WT and CF zebrafish embryos infected with PAO1 (+PAO1) and treated with the phage cocktail (+PAO1 + ϕ). The mean and SD reported are from six and four experiments, respectively, each with 25–40 embryos. Angular transformation was applied to percentage of lethality and one-way ANOVA followed by Duncan’s test was used to test for significance. (WT + PAO1) vs (WT + PAO1 + ϕ) p = 0.004; (WT + PAO1) vs (CF + PAO1) p = 0.0003; (CF + PAO1) vs (CF + PAO1 + ϕ) p = 0.018. (b) Bacterial burden in WT or CF embryos infected with PAO1 or PAO1 + ϕ. The relative percentage of cfu/embryo in PAO1 + ϕ vs PAO1 embryos are given. The mean and SD of three independent experiments is reported. (c,d) Progression of the infection in WT (c) and CF (d) embryos following PAO1 injection (upper embryo) and efficacy of the phage therapy in PAO1 + ϕ injected embryos (bottom embryo) at 4, 9, 14 and 18 hpi. (e) Expression levels of the TNF-α and IL-β genes measured by RT-qPCR at 8 hpi in WT (e) and CF (f) embryos injected at 48 hpf. The mean and SD of four experiments are reported. Statistical significance was assessed by ANOVA followed by Duncan’s test: (e) for TNF-α, (WT) vs (WT + PAO1) p = 0.0018; (WT) vs (WT + PAO1 + ϕ) p = 0.007; (WT + PAO1) vs (WT + PAO1 + ϕ) p = 0.22 n.s.; for IL-β, (WT) vs (WT + PAO1) p = 0.0002; (WT) vs (WT + PAO1 + ϕ) p = 0.0001; (WT + PAO1) vs (WT + PAO1 + ϕ) p = 0.94 n.s.; (f) for TNF-α, (CF) vs (CF + PAO1) p = 0.015; (CF) vs (CF + PAO1 + ϕ) p = 0.019; (CF + PAO1) vs (CF + PAO1 + ϕ) p = 0.77 n.s.; for IL-β, (CF) vs (CF + PAO1) p = 0.00014; (CF) vs (CF + PAO1 + ϕ) p = 0.0007; (CF + PAO1) vs (CF + PAO1 + ϕ) p = 0.031. For simplicity sake, in the figures *p < 0.05; **p < 0.01, ***p < 0.001.