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2023 OMIG Abstract

Intracellular Lifestyle of P. Aeruginosa and Its Implications for Ofloxacin Resistance Phenotypes

Naren G. Kumar, Orneika Flandrin, Yujia Yang, Daniel Schator, David J Evans, and Suzanne Fleiszig

Department of Optometry and Vision Science, University of California Berkeley, Berkley, CA

Purpose: Determine how the intracellular lifestyle of P. aeruginosa in corneal epithelial cells reduces the efficacy of fluoroquinolones, a host cell permeable antibiotic commonly used to treat infectious keratitis.

Methods: Live time-lapse microscopy was used to study the intracellular persistence of P. aeruginosa in cultured human corneal epithelial cells in vitro and the murine cornea in vivo. Bacterial gene knockout mutants were utilized to determine the involvement of biofilm-associated genes in the persistence of intracellular bacteria post-antibiotic treatment and its influence on host cell death. 2-D proteomics was performed on infected cells in vitro to identify novel targets involved in the persistence of intracellular bacteria during ofloxacin treatment. The corneal scarification model and confocal microscopy were used to study the fate of intracellular bacteria in vivo after treatment with 0.3% ofloxacin solution for 6 h.

Results: In the corneal scratch infection model P. aeruginosa persisted after treatment with a clinical dose of ofloxacin and ciprofloxacin (0.3% solution) for 6 h. While replicating bacteria in the cornea were susceptible to fluoroquinolones, bacterial aggregates persisted in vivo. Interestingly, persisting bacteria in vivo appeared to localize only to the corneal epithelium, not the stroma. These in vivo results recapitulate the findings from our in vitro study showing the intracellular persistence of P. aeruginosa in up to 10% of cells at doses up to 100 X MIC. In-vitro experiments showed that intracellular biofilm gene expression was not required for antibiotic tolerance, however, deletion of genes encoding biofilm components (cdrA and exopolysaccharide) was associated with increased intracellular bacterial load and reduced host cell death by ~70%. 2-D proteomics performed on cultured human corneal epithelial cells infected with P. aeruginosa identified cell membrane-associated proteins, and other small substrate-specific porins as putative targets involved in the intracellular persistence of P. aeruginosa in epithelial cells after ofloxacin treatment.

Conclusions: Corneal epithelial cells harbor persistent intracellular bacteria that are refractory to antibiotic treatment in vitro and in vivo. Intracellular biofilm gene expression though not involved in tolerance to high-dose fluoroquinolones alters the host response (cell death) during infection. These findings may help explain the prolonged course of antibiotic treatment required for some forms of Pseudomonas keratitis.

Disclosure: S

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