Human tear fluid protects against Pseudomonas aeruginosa keratitis in a murine experimental model

Abstract

Pseudomonas aeruginosa keratitis is an acute sight-threatening infection. We previously reported that human tear fluid could protect individual human corneal epithelial cells in vitro against invasion by and cytotoxicity due to clinical and laboratory isolates of P. aeruginosa and that the protective mechanism was independent of bacteriostatic activity. In the present study, we examined the effects of human tear fluid in vivo. Tears were collected from healthy human volunteers and were studied in vivo in mice. The effects on the virulence of both invasive and cytotoxic clinical isolates of P. aeruginosa were examined. Tear fluid was found to reduce the severity of disease when corneas were challenged with cytotoxic bacteria immediately after scratch injury, and it completely protected against susceptibility to infection by a cytotoxic strain in a model in which corneas were infected during the healing process 6 h after scratching. Visible protection correlated with the inhibition of bacterial colonization 1, 4, and 48 h postinoculation. Tear fluid also significantly reduced the severity of infections caused by invasive P. aeruginosa in the 6-h-healing model. This result also coincided with significantly reduced bacterial colonization at 48 h. In vitro, human tear fluid significantly reduced the ability of invasive and cytotoxic bacteria to translocate across corneal epithelia and increased transepithelial resistance with or without bacterial inoculation. These data show that human tear fluid can protect against P. aeruginosa corneal infection in vivo and that the mechanism likely involves enhanced epithelial barrier function in addition to protection of individual epithelial cells against bacterial internalization and cytotoxicity.

FIG. 1.

P. aeruginosa keratitis at 48 h postinoculation with cytotoxic strain 6206 using the 6-h-healing murine model of infection. (A and C) Controls. Bacteria were added in MEM. (B and D) Effect of human tear fluid. Bacteria were added in tears (diluted 1:2 in MEM). (A and B) Inoculum, ∼1 × 10³ CFU in 5 μl. (C and D) Inoculum, ∼1 × 10⁴ CFU in 5 μl.

FIG. 2.

Effect of human tear fluid on 48-h corneal colonization rates (means and standard deviations). The scarification infection model and the 6-h-healing infection model were both used, and cytotoxic P. aeruginosa strain 6206 was added to the cornea at a concentration of ∼1 × 10³ CFU in 5 μl of MEM (control) or human tear fluid (diluted 1:2 in MEM).

FIG. 3.

Effect of human tear fluid on 1- and 4-h corneal colonization rates (means and standard deviations). The 6-h-healing infection model was used, and cytotoxic P. aeruginosa strain 6206 was added at a concentration of ∼7 × 10⁴ CFU in 5 μl in either MEM (control) or human tear fluid (diluted 1:2 in MEM). There was a significant difference between the groups (P = 0.0001, as determined by ANOVA). The asterisks indicate that Fisher PLSD and Scheffe F-test posthoc analysis showed that there was a significant difference between tear fluid-treated eyes and controls at each time (P < 0.05).

FIG. 4.

Translocation of P. aeruginosa strains 6206 (A) and 6294 (B) across cultured rabbit corneal epithelial cells grown in vitro on Transwell filter membranes. The viable bacterial counts (means ± standard deviations) in the lower (basal) compartment were determined each hour after bacteria were added to the apical surface in HBSS (control) or with undiluted human tear fluid. The asterisks indicate that for strain 6206 significantly less translocation was observed with tear-treated samples than with controls at 5 h (P = 0.027, as determined by a t test) and at 6 h (P = 0.0006, as determined by a t test) and that similar results were obtained for strain 6294 at 5 h (P = 0.049, as determined by a t test) and at 6 h (P = 0.0009, as determined by a t test).

FIG. 5.

TER (means ± standard deviations) across cultured rabbit corneal epithelial cells in vitro in the presence of the invasive P. aeruginosa strain 6294 (A) or the cytotoxic P. aeruginosa strains 6206 (B) and 19660 (C) with and without undiluted human tear fluid. The asterisk indicates that in cells infected with 6294, tear fluid significantly increased the TER compared to the TER observed with cells infected with bacteria in HBSS alone (P = 0.0006 at inoculation and P = 0.0001 at each time from 1 to 6 h, as determined by a t test).