a. Schematic showing biotin/streptavidin linking S. aureus to gold grids that were placed on the airway surface. After removal, bacteria were exposed to fluorescent live/dead stain (SYTO 9/propidium iodide), imaged, and counted.
b. Scanning electron photomicrographs of bacteria-coated grid (top), grid bar (middle), and individual bacteria (bottom).
c. Image of bacteria-coated grid (green=live, red=dead) after placement for 5min on tracheal surface of 1-month-old, wild-type pig. Bottom shows percentage of bacteria that were dead after immersion in saline, water, 70% ethanol, or placement on tracheal surface. n=3 each. Here and elsewhere, bars are SEM.
d. S. aureus-coated grids were placed on tracheal surface of newborn CF and non-CF pigs for indicated times. Data are percentage dead bacteria. Each set of 3 time-points is from a single animal. Letters (a–f) indicate littermates; there is no 1min time point for CF pig in litter c due to experimental error. For each pig, 2–3 grids were used at each time point, 5–16 fields were counted per grid, each field contained ~100–1000 bacteria (), and data from each field were averaged. Operators were blinded to genotype. CF was different from non-CF at all time points, P<0.01.
e. S. aureus-coated grids were placed for 30sec on tracheal surface before and ~5min after methacholine stimulated secretion. n=6/genotype. *P<0.02. For each genotype, data with and without methacholine do not differ significantly.
f. ASL was removed from methacholine-stimulated pigs, bacteria (1×106 CFU/ml) were incubated in 1µl ASL for 10min. in a micro CFU assay, and bacteria were counted by dilution plating. n=6/genotype. *P<0.05.
g. S. aureus-coated grids were placed on surface of primary epithelial cultures for indicated times, and percentage dead bacteria was determined. Each set of data points represents mean from epithelia from a different animal. CF was different from non-CF at all time points, P<0.001.
h. P. aeruginosa-coated grids were placed on surface of cultured epithelia for 5min. n=5 cultures from different pigs per genotype. *P<0.001.
i. S. aureus in 100nl H2O were applied to apical surface of cultured epithelia. Apical surface was washed 24hr later and bacteria counted by dilution plating. Data are % of epithelia that showed no bacterial growth. n=18/genotype. *P<0.005, Fisher's exact test.