Synergistic inactivation of Micrococcus luteus and its biofilms by 5-aminolevulinic acid-mediated photodynamic...

Micrococcus luteus is an opportunistic nosocomial bacterial species with the ability to form biofilms on medical devices, and the emergence of antibiotic tolerance in M. luteus necessitates the development of alternative therapeutic strategies. 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) represents a promising alternative, leveraging the bacterial endogenous heme biosynthetic pathway to generate cytotoxic reactive oxygen species upon light activation.

The intrinsic heme biosynthesis pathway of M. luteus was utilized for photosensitizer production. Bacterial cells and 4-day mature biofilms formed on titanium surfaces were incubated with varying concentrations of 5-ALA (0-0.5%) and subsequently irradiated using a 405 nm LED light source at fluences of up to 27 J/cm². Porphyrin accumulation was quantified by fluorescence spectroscopy. The antibacterial and anti-biofilm efficacies of ALA-PDT were systematically evaluated using colony-forming unit (CFU) counts, live/dead staining, fluorescence microscopy, and scanning electron microscopy (SEM).

Fluorescence spectroscopy revealed that treatment with 0.1% (w/v) 5-ALA for 2 h resulted in maximum porphyrin accumulation. When combined with light irradiation at 18 J/cm², this protocol achieved significant, concentration- and time-dependent killing of planktonic M. luteus, with no observed dark toxicity. CFU counts indicated that this optimized ALA-PDT protocol also effectively eradicated mature biofilms on titanium, leading to a substantial reduction in viable bacterial counts. Notably, the combination of 5-ALA and light exhibited superior bactericidal activity compared to either treatment alone, confirming the synergistic nature of this therapeutic approach. Fluorescence microscopy and SEM consistently revealed that ALA-PDT not only killed embedded bacteria but also disrupted the extracellular matrix and reduced biofilm thickness, whereas 5-ALA alone or light alone only diminished intercellular connections without causing cell death.

This study demonstrates that ALA-PDT is a highly effective approach for eliminating both planktonic and biofilm-associated M. luteus. The synergistic combination of 5-ALA and light irradiation achieves both bacterial killing and biofilm disruption. Our findings underscore the potential of ALA-PDT as a non-antibiotic strategy for managing infections caused by this resilient pathogen, especially those related to medical implants.