Fig. 1: Pf4 and Pf6 are coordinately produced in MPAO1 biofilms and contribute to host antibiotic resistance and virulence.

a Schematic of the flow cell biofilm system used in this study. Images in bottom left show representative crystal violet-stained catheter sections (purple staining indicates biofilm formation). Planktonic cells are free-living cells grown in liquid culture. b Phage titers (left) and transmission electron microscopy (TEM, right) of biofilm effluents from day 6 MPAO1 biofilms. c Fluorescence microscopy of day 6 biofilms in MPAO1 (left) or PAO1 (right) strains expressing GFP-tagged Pf4. d Genome comparisons of de novo sequenced MPAO1, PAO1, and the first NCBI-deposited MPAO1 and PAO1 genomes. MPAO1 sequenced in this study was used to annotate the Pf4 and Pf6 prophages (shown at right with core Pf regions marked). e Representative phage titers of day 6 biofilm effluents from the indicated MPAO1 derivatives plated on lawns of the MPAO1 strain. f Percentages of Pf4 and Pf6 phages in biofilm effluents from the MPAO1 WT and mutant strains quantified by qPCR. Data are shown as the mean ± SD. Two-sided Student’s t Test was used for comparisons between Pf4 and Pf6 in each strain (n = 3). P < 0.05 was considered statistically significant. g Gentamicin (Gm) sensitivity of various strains in varying biofilm states. Experimental design is shown in top panel. ND: not detected. Data are shown as the mean ± SD. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used (n = 3), and P < 0.05 was considered statistically significant. h Survival of C57BL/6J mice infected with planktonic or biofilm-state MPAO1 P. aeruginosa WT (n = 11) and ΔPf4ΔPf6core (n = 10 and 12 for planktonic and biofilm-state, respectively). The log-rank (Mantel-Cox) test with 95% confidence interval was used, and P < 0.05 was considered statistically significant.