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Yvonne Wu, Connie Tam, David Evans, Suzanne Fleiszig; Human tear fluid triggers virulence expression by Pseudomonas aeruginosa biofilms growing on soft contact lenses. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2893.
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Pseudomonas aeruginosa, a leading cause of contact lens related corneal infections, has many genes devoted to survival, regulation of virulence, and environmental adaptation. Our laboratory previously showed that P. aeruginosa biofilms on contact lenses removed from infected rat eyes caused faster corneal infections than naïve P. aeruginosa. In addition, exposure of P. aeruginosa biofilms on contact lenses to human tear fluid and human corneal epithelial cells in vitro, allowed bacteria to penetrate the corneal epithelium in vivo whereas lens-grown biofilms alone did not. Since the type three secretion system (T3SS) is a major virulence mechanism of P. aeruginosa, we tested the hypothesis that exposure to tear fluid triggers T3SS expression in lens-grown P. aeruginosa biofilms.
To form biofilms, human contact lenses were incubated in P. aeruginosa PAO1 (~1000 cfu/ml) in tryptic soy broth (TSB) at 37oC in stationary conditions for seven days. Controls included P. aeruginosa grown in shaken tryptic soy broth (TSB) overnight (first comparison), or a sub-set of biofilm-coated contact lenses further exposed to PBS (second comparison) or human tear fluid overnight. T3SS gene expression (exsA and exoS) was quantified by real time q-PCR to compare planktonic/biofilm and biofilm exposed to PBS/tears. The internal control for q-PCR was ribosomal 16s.
T3SS gene expression in biofilms was upregulated compared to planktonic cells; for exsA 5.46 ± 0.24 fold increase (95% CI: 4.43 - 6.50, p = 0.02); for exoS 3.76 ± 0.36-fold increase, (95% CI: 2.61 - 4.90, p = 0.01). For biofilms exposed to tears overnight, expression of both exsA and exoS was further upregulated compared to biofilms without tear exposure. For exsA 2.10 ± 0.38 fold higher (95 % CI: 1.03 - 3.15) (p < 0.04); for exoS 1.90 ± 0.26 fold (95 % CI: 1.47 - 2.31) (p < 0.001).
These data show that biofilm growth on a contact lens, and subsequent exposure to human tear fluid, upregulate expression of the T3SS in P. aeruginosa. Since this pathogen can form biofilms on contact lenses in vivo, and T3SS is a known virulence determinant in the cornea, these data suggest a potential mechanism for initiation of contact lens-related infectious keratitis.
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