High Fluence Increases the Antibacterial Efficacy of PACK Cross-Linking

Purpose: Photoactivated chromophore for keratitis cross-linking (PACK-CXL) is used as an adjunct therapy to antibiotic medication in infectious keratitis. This experimental study aimed at quantifying the PACK-CXL efficacy as a function of UV fluence using several bacterial strains and irradiated volumes. Methods: Six distinct bacterial strains, including standardized strains and clinically isolated strains from patients with keratitis, were analyzed. Bacterial concentrations between 10(2)and 10(8)cells/mL were used (simulating small corneal ulcers). Volumes of either 11 mu L (approximate to 285 mu m stromal thickness) or 40 mu L (approximate to 1000 mu m stromal thickness) were irradiated within a microtiter plate at different fluences (5.4-27 J/cm(2)) and irradiances (3, 9 and 18 mW/cm(2)). The ratio of bacterial killing (B dagger) was determined to evaluate the antimicrobial efficacy of PACK-CXL. Results: B dagger was similar (51 +/- 11%) in bacterial concentrations between 10(3)and 10(5)per ml. In 11 mu L volume,Staphylococcus aureus(SA) 8325-4 ATCC 29213,Bacillus subtilis(BS) 212901, andPseudomonas aeruginosa(PA) 2016-866624 were most sensitive to PACK-CXL at 5.4 J/cm(2)(on average B dagger = 49 +/- 8%), whereasKlebsiella oxytoca(KO) 2016-86624 (B dagger = 25%) was least sensitive. When irradiating a larger volume, B dagger was on average lower in 40 mu L (19 +/- 18%), compared with 11 mu L (45 +/- 17%,P< 0.001). By contrast, applying a higher UV fluence increased B dagger of SA ATCC 29213, from 50% at 5.4 J/cm(2)to 92% at 10.8 J/cm(2), to 100% at 16.2 J/cm(2)and above. Conclusions: Applying higher UV fluences substantially increases the bacterial killing rates. Safety limits for clinical application require further investigation.