Human Tear Fluid Reduces Culturability of Contact Lens-Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence-Associated Type III Secretion System

Abstract

Purpose The type III secretion system (T3SS) is a significant virulence determinant for Pseudomonas aeruginosa. Using a rodent model, we found that contact lens (CL)-related corneal infections were associated with lens surface biofilms. Here, we studied the impact of human tear fluid on CL-associated biofilm growth and T3SS expression. Methods P. aeruginosa biofilms were formed on contact lenses for up to 7 days with or without human tear fluid, then exposed to tear fluid for 5 or 24 h. Biofilms were imaged using confocal microscopy. Bacterial culturability was quantified by viable counts, and T3SS gene expression measured by RT-qPCR. Controls included trypticase soy broth, PBS and planktonic bacteria. Results With or without tear fluid, biofilms grew to ~108 cfu viable bacteria by 24 h. Exposing biofilms to tear fluid after they had formed without it on lenses reduced bacterial culturability ~180-fold (p<.001). CL growth increased T3SS gene expression versus planktonic bacteria [5.46 ± 0.24-fold for T3SS transcriptional activitor exsA (p=.02), and 3.76 ± 0.36-fold for T3SS effector toxin exoS (p=.01)]. Tear fluid further enhanced exsA and exoS expression in CL-grown biofilms, but not planktonic bacteria, by 2.09 ± 0.38-fold (p = 0.04) and 1.89 ± 0.26-fold (p<.001), respectively. Conclusions Considering the pivitol role of the T3SS in P. aeruginosa infections, its induction in CL-grown P. aeruginosa biofilms by tear fluid might contribute to the pathogenesis of CL-related P. aeruginosa keratitis.

Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa

“…Non-bacteriostatic activities of tear fluid also include its capacity to disperse P . aeruginosa biofilms [ 16 ], which are thought to be key to the pathogenesis of device-related infections.…”

The Antibiofilm efficacy of nitric oxide on soft contact lenses

“…The difference between experimental and physiological conditions should be also considered for interpretation of our results. The dynamic tear clearance and lens movement were absent in our experimental settings and these two factors might change the biofilm kinetics in human ocular surface [ 46 ]. Finally, the effect of NO on multispecies biofilms was not investigated even though multispecies-associated keratitis is common with contact lens wear [ 27 ].…”

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

“…Studies have suggested the presence of bacterial biofilms on the infected ocular surface and have demonstrated an enhanced biofilm formation capability of ocular bacterial clinical isolates in vitro ( 7 – 11 ). Microscopic inspections have demonstrated the presence of bacterial aggregates and biofilm matrix in experimental infections of the mouse cornea ( 12 – 15 ).…”

“…Evidence has been presented that the P. aeruginosa type III secretion system (T3SS) contributes to the pathogenesis of P. aeruginosa keratitis. Thus, the T3SS activity of P. aeruginosa was shown to prevent reactive oxygen species (reactive oxygen species) production by neutrophils as well as to cause neutrophil apoptosis during corneal infection ( 11 , 17 – 19 ). However, previous in vitro studies have demonstrated an inverse correlation between the expression of biofilm factors and T3SS in P. aeruginosa ( 20 , 21 ), and more evidence is needed for a role of both biofilm formation and T3SS in corneal infection.…”