Outer membrane permeability and polymyxin B tolerance of P. aeruginosa PAO1 incubated in water.

The baseline of outer membrane permeability was measured as a function of 1-N-phenylnaphthylamine (NPN) uptake and subsequent fluorescence in relative light units (RLU). Log phase cultures of PAO1 were prepared and inoculated into water for incubation. After 1, 7, 21, and 28 days incubation, cells were treated with sodium azide, an active efflux inhibitor. After NPN addition, polymyxin B (time = 0) was added to disrupt the outer membrane and increase NPN uptake into the hydrophobic environment of the envelope. The tolerance to polymyxin B treatment was compared between mid-log LB cultures and cells incubated in water for up to 4 weeks. Values shown are the average and standard error of triplicate samples. Significant differences for the final time points were determined using one-way ANOVA with Bonferroni post-tests when compared to the LB control (* P<0.05, *** P<0/001, ****P<0.0001).