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Anders Backman, Karim Makdoumi; Riboflavin/UVA-mediated Elimination of Pathogenic Bacteria with and without Antibiotic Resistance Using an In Vitro Model of Keratitis Treatment.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6273.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the in vitro effect of riboflavin/UVA-mediated photo-oxidative stress on pathogenic bacterial species with and without antibiotic resistance properties.
Four different pairs (n=8) of pathogenic bacterial species that can be isolated in keratitis were studied. The experiments were done pairwise, in order to directly compare each pathogen with and without antibiotic resistance(R). Well-characterized reference strains were used: Staphylococcus epidermidis (ATCC12228) and (CCUG32257) (Methicillin/Oxacillin-R); Staphylococcus aureus (ATCC29213) and (ATCC43300) (Oxacillin-R); Pseudomonas aeruginosa ( ATCC27853) and CCUG37384 (Ciprofloxacin-R); Enterococcus faecalis ( ATCC29212) and CCUG34062 (Vancomycin-R). Bacteria cultured on blood agar plates were diluted in PBS to 4x 105/mL, dispersed in Riboflavin (R7649-25G, Sigma-Aldrich, Schnelldorf, Germany) and dissolved in RPMI (GIBCO No: 11835) resulting in a concentration 0.01% riboflavin. 15 µL was applied in a 7mm well on a diagnostic microscope slide (CEL-30-2325BLHTC, Thermo Scientific, Braunchweig, Germany), creating a thin layer of approximately 400 µm of bacterial solution. The fluid was exposed to UVA (365 nm) for 30 min, dose 5.4 J/cm2 (OPTO Xlink1.0, Brazil /PESCHKE Meditrade GmgH, Switzerland). Withdrawn samples were diluted and cultured on blood-agar plates and a colony forming unit value was determined for each solution. The results were compared to samples from two negative controls (-UVA, +Riboflavin) on a separate slide. Every strain was tested eight times and an average was caculated.
: In all strains the Riboflavin/UVA exposure resulted in a statistically significant elimination of bacteria (p<0.05). Similar degrees of eradication were seen in the antibiotic resistant strains as in the non-resistant pathogens. The degree of elimination varied between different bacterial species, ranging from 52 to 92 % in the different strains.
Riboflavin/UVA as is utilized in Collagen Crosslinking seems to be equally efficient in eradication of antibiotic resistant as in non-resistant strains, in vitro. Antibiotic resistance does not seem to be protective against the photo-oxidative stress generated by riboflavin/UVA and this study indicates that riboflavin photosensitization could be used in cases of keratitis caused by antibiotic resistant bacteria.
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