Collagen Cross-linking for Eradication Of Bacteria Using UVA And Riboflavin Is Effected By The Used Irradiance And The Exposure Time

Abstract

Purpose: : To investigate the influence of UVA-irradiance, dose and exposure time in eradication of bacteria by using collagen cross-linking method model.

Methods: : Dilutions of a Staphylococcus epidermidis strain (ATCC12228)( 2.5 x 10⁴/ml) was used in the experiments with riboflavin(SIGMA, R7649-25G)dilution (0.01%) in RPMI (GIBCO, Invitrogen). These mixtures (40 uL, approx.1 mm thin) were exposed to UVA (365 nm) using Collagen Cross-Linking lamps. The examined UVA-irradiance were 3.00, 6.29 mW/cm² using (8 mm spot, 4.5 cm distance)(OPTO Xlink, Pechke Meditrade GmbH, Switzerland) and 30 mW/ cm² (9mm spot, 9 cm distance)(KXL, Avedro inc. MA, USA) for generating the exposure doses of, 5.4 J/cm² and 10.8 J/cm². The corresponding exposure times were 30 min/60 min(3.00 mW/cm²), 14min 20 sec/ 28 min 40sec (6.29 mW/cm²), and 3 min/ 6 min (30 mW/cm²). Diluted samples from exposed mixtures were used for CFU-counts on blood agar-plates. Comparisons was made to the negative controls without UVA/riboflavin.

Results: : The experiments showed that eradication of S.epidermidis was almost complete (98 %) for the low irradiance (3.00 mW/cm²), over 60 min, compared to the unexposed negative control. The medium irradiance (6.29 mW/cm²) and (28 min 40 sec) eradicated around 64 % of the bacteria. The high irradiance (30 mW/cm²), over 6 min, only eradicated around 50 % of bacteria in the same comparison. The clinical radiant exposure dose (5.4 J/cm²), and exposure times of 3, 14 min 20 sec, and 30 min gave no eradication of bacteria in this model.

Conclusions: : The lower irradiance and long exposure-time of UVA and riboflavin (0.01%) would probably be more useful, than high irradiance and riboflavin, in treatment of bacterial keratitis in the clinical setting, as seen by our results. This means that the UVA-exposure dose is not the only factor influencing bacterial eradiacation. The single UVA-dose used in clinical treatments is probably sufficient for eradication of almost all S. epidermidis as the treated layer in the cornea is only around 200 µm, in contrast to this model, that uses the lowest possible, 1 mm layer of liquid for testing the eradication.

Keywords: cornea: clinical science • microbial pathogenesis: experimental studies • keratitis