Quantification of bacteria within corneal epithelial cells (intracellular bacteria) during traversal assay at 3 hours (A) and 8 hours (B) after inoculation. Cells were grown as multilayers on semipermeable filters and incubated with 106 CFU of P. aeruginosa strain PAK or its pilA or pilU mutants. (A) Invasion of twitching motility mutants (pilA and pilU) was similar to that of wild-type PAK after 3 hours (*P > 0.05, t-test, for each comparison). (B) After 8 hours, more intracellular bacteria were recovered from within corneal epithelial cells infected with twitching motility mutants (pilA and pilU) than from cells infected with wild-type PAK (*P < 0.01, t-test, for each mutant vs. wild-type PAK). Data are expressed as mean ± SD. Three semipermeable filters were used for each sample.