Abstract
*Correspondence – Stephen Kaye: S.B.Kaye@liverpool.ac.uk
Introduction Pseudomonas aeruginosa injects toxins, ExoS or ExoU, into host cells via the type III secretion system (T3SS) which destroy cells and help evade the immune system. First-line fluoroquinolones demonstrate better in vitro activity against P. aeruginosa but in certain clinical situations aminoglycosides are more effective. We evaluate the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS and toxin expression, and the associated toxicity in corneal epithelial cell infection models.
Methods Expression levels of pcrV (T3SS needle component) from ExoU-expressing PA103 and ExoS-expressing PA76026 after 16h incubation in each antimicrobial was detected using western blotting. qRT PCR detected mRNA levels of ExoU, ExoS, pcrV and ExsA (T3SS activating factor) after PA103 and PA76026 were exposed to tobramycin and moxifloxacin. LIVE/DEAD and LDH assays after 24h evaluated how the antimicrobials influenced acute cytotoxicity in a HCE-T cell scratch and infection model.
Results Tobramycin significantly reduced pcrV in both strains by 50.5–74.0% compared to the fluoroquinolones (p=0.001 and 0.003), even at low concentrations. Fluoroquinolones significantly increased pcrV by 57.0–81.8% (p=0.004 and 0.003). mRNA levels of ExoU, ExoS, pcrV and ExsA were reduced by tobramycin but moxifloxacin increased pcrV, ExsA and ExoS. Tobramycin, despite more bacterial expansion compared to the same relative concentrations of fluoroquinolones, reduced ExoU/ExoS cytotoxicity and allowed complete wound healing.
Discussion Tobramycin downregulates T3SS expression and reduces ExoS/ExoU mediated cytotoxicity which protects infected HCE-T cells even at low concentrations. Fluoroquinolones however upregulated T3SS and do not negate the cytotoxic effects.
