Figure 6.

Antimicrobial activity against P. aeruginosa is reduced in BALF from newborn CF ferrets in a pH- and bicarbonate-independent manner. (A–C) Proliferation of various strains of bacteria in BALF from non-CF and CF newborn ferrets. BALF from non-CF and CF newborn ferrets was concentrated to 2–5 μg/μl by centrifugation as described in Materials and Methods. A 15-μg sample of the BALF was inoculated with 5,000 CFU of Escherichia coli (EC838) (A), S. pseudintermedius (clinical isolate from an adult CF ferret lung) (B), or P. aeruginosa (PA01) (C) and incubated at 37°C for 3 hours. The surviving bacteria were then quantified by CFU assay. (D) Proliferation of PA01 bacteria in BALF at various pH levels. Concentrated non-CF or CF BALF (15 μg; prepared as in A–C) was inoculated with 5,000 CFU PA01 in sodium phosphate buffer at the indicated pH, at 37°C for 3 hours, before CFU quantification. (E) Proliferation of PA01 in the presence or absence of BALF at various bicarbonate concentrations. Concentrated non-CF or CF BALF (15 μg) in sodium phosphate buffer (prepared as in A–C) was supplemented with a vehicle or a concentrated bicarbonate solution to the indicated final concentration. Mock samples (no BALF) were also carried along as controls. These samples were then inoculated with 5,000 CFU PA01 at 37°C for 3 hours before CFU quantification. The data in D and E were log transformed before being plotted. Average input CFU (100%) is denoted with a dashed line on each graph. Error bars represent SEM. ***P < 0.001 by Mann-Whitney test (C); significant differences between genotypes at each pH or bicarbonate concentration are marked as: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Sidak’s multiple comparison posttest (D and E); a significant difference (^†P < 0.05) between non-CF pH 7.2 and non-CF pH 8.3 was also observed (one-way ANOVA, Sidak’s multiple comparison posttest).