Growth of P. aeruginosa cultures infected with CK4 phages and phenotypic traits of phage-resistant mutants. (A) PAO1 cultures growing in LB at 37°C were infected at m.o.i. = 2.5 with CK4 or CK4− components at time = 0 and the OD₆₀₀ measured at intervals. The red arrow indicates the OD₆₀₀ reached by the cultures at the end of the experiment. The last time point of the growth curves of E215-infected PAO1 overlaps with that of PAO1 infected with DEV, PYO2, and E217. n.i., non-infected. Similar results were obtained in replicate experiments. (B) Growth of CK4, DEV, and E217 on cross-resistant mutants (CRmut) and mutants specifically resistant to E217 (ERmut). Four microliters of phage lysates at ca. 10⁸ pfu/mL were spotted on LB agar plates topped with soft agar inoculated with 0.4 mL of PAO1 or PAO1 mutants’ cultures. (C) Phenotype of ERmut mutants. Patch on LB agar formed by non-pigmented (ERmut-a and CR-mut) and brown-pigmented (ERmut-b) mutants.

Phage eop on LPS-defective mutants. E217 or DEV lysates (indicated on the left of the panels) were serially diluted (×10) in 96-well plates and replicated as explained in Materials and Methods on PAO1 and PAO1 mutants carrying the indicated plasmids in the presence of 0.2% arabinose in the top agar. The plates were incubated overnight at 37°C.

Altered LPS of phage-resistant mutants and defective adsorption of the cross-resistant wzy mutant. (A) LPS extracted from PAO1, PaPh24, PADR6 (wzy⁻), PAER10b (algC⁻), PAER6b (galU⁻), and PAER5b (wapH⁻) was run in a 15% Bis-Tris (upper panel) or a 18% tricine (lower panel) gel and silver stained. LPS extracted from 0.05 or 0.01 OD₆₀₀ of cells were loaded in the upper and lower panel. Migration of molecular weight (MW) markers is reported on the right in kDa. Different forms of LPS (39) are indicated on the left. vl/l and m, very long/long- and medium-length O-specific antigen (OSA) capped LPS; cpa, common polysaccharide antigen; C + 1, LOS capped with a single O-antigen repeat; u, LOS with uncapped core; t, LOS with truncated core. (B) The E217-resistant 4a mutant is cross-resistant to DEV and complemented by wzy. (C) DEV phage has defective adsorption to the 4a wzy mutant. % ADS, adsorbed vs. input phage. The average (N = 3) with standard deviation is reported. Significance of difference was estimated with t-test. ***P < 0.001.

Phage-resistant mutants have attenuated virulence in zebrafish. Left panel. Survival of zebrafish embryos (N = 60) infected with the indicated P. aeruginosa strains. Kaplan-Meyer curves represent results deriving from two independent experiments in which groups of 30 embryos were injected with the indicated strains. hpi, hours post-infection. Significance of the difference with respect to survival upon PAO1 infection was estimated with the Gehan-Breslow-Wilcoxon test. ***P < 0.001. ns, not significant. Right panel, bacterial burden estimated as described on four groups of five embryos for each condition. Significance of the difference with respect to the average bacterial burden in PAO1 set to 1 (dashed line) was estimated with t-test. ***P < 0.001, and **P < 0.01; ns, not significant. wapH⁻, PAER5b; galU⁻, PAER6b; wzy⁻, PADR6; algC⁻, PAER10b; ΔpilQ, PAMO302.

Twitching motility assay. Twitching motility of the indicated strains was assayed as described in Materials and Methods. The results of a representative experiment are shown on the left. The red lines indicate the diameter of the twitching halos formed by PAO1 and Δwzy. The other strains did not form visible halos. On the right, the areas of twitching halos formed in replicate experiments (N = 5) are reported. Significance of the difference was evaluated with one-way ANOVA (F, 295.7; P, 9.8 × 10^-21). Δwzy, PAMO301; wzy⁻ pilS⁻, PADR2; wzy⁻ pilQ⁻, PADR3; ΔpilQ, PAMO302.

Phage growth on different P. aeruginosa strains. (A and B) Plating on PAO1 mutants. Phage lysates were diluted (×10) in 96-well plates and replicated on PAO1 and the indicated PAO1 mutants. The plates were incubated overnight at 37°C. wapH⁻, PAER5b; galU⁻, PAER6b; wzy⁻, PADR6; algC⁻, PAER10b, ΔpilQ, PAMO302; Δwzy, PAMO301. The CK4 components are reported in red. (C) Different RAPD PCR band pattern obtained with primers 4010 and 4031 on the indicated phages. The same patterns were obtained in a replicate experiment. MW marker migration is indicated on the left (kb). (D) Percentage of the P. aeruginosa strains listed in Table S4 susceptible to all phages listed in Table 4 but E221, for which only a subset of clinical strains was analyzed. Each dot represents the host range of a different phage. LPS, phages not growing on wzy mutants; T4P, phages not growing on the ΔpilQ mutant.