Photodynamic Inactivation of Uropathogenic Biofilm-Forming Microorganisms: A Pilot Study

BACKGROUND: Methods for microbial inactivation, including physical approaches aimed at the destruction of biofilms formed by uropathogenic microorganisms for the prevention of infectious and inflammatory diseases in urology, remain insufficiently studied. The development of new strategies in this field remains relevant.

AIM: To evaluate the feasibility of photodynamic inactivation of biofilms formed by typical representatives of uropathogenic microorganisms using an antiseptic agent with a bacteriostatic effect—methylene blue—possessing photochemical properties.

METHODS: Cultures of Staphylococcus aureus and Escherichia coli isolated from renal calculi of patients from a urology department were used. In vitro experiments on photodynamic inactivation of microorganisms were conducted on mature preformed biofilms. Irradiation was performed using a diode laser emitting at a wavelength of 662 nm through a sterile 0.1% methylene blue solution in continuous mode across five setups (three control, two experimental). After irradiation, biofilms on the cover glasses were fixed on microscope slides using colorless varnish. The prepared specimens were stained with acridine orange solution, dried in the dark, examined under a fluorescence microscope at ×100 magnification using an immersion system, and photographed with a digital camera. Images were digitally processed using 3D modeling technologies with ImageJ software version 1.52a.

RESULTS: The impact of the photoactive agent and laser irradiation was assessed at two power settings—450 mW and 1100 mW. In the first case, partial destruction of the biofilms was noted (41.9% of the original biofilm structure for S. aureus and 82.4% for E. coli), whereas in the second case, exposure at 1100 mW resulted in complete degradation of the mature multilayer biofilm into single cells without extracellular matrix, corresponding to 97.7% destruction of the original biofilm structure for S. aureus and 96.5% for E. coli.

CONCLUSION: This study is the first to demonstrate the feasibility of photodynamic inactivation of uropathogenic biofilm-forming microorganisms using a photochemically active agent—methylene blue. The promising results suggest that combined laser irradiation and methylene blue application may serve as an alternative or adjunct to systemic antibiotic therapy in urological practice.