400 nm femtosecond laser irradiation eradicates mature MRSA biofilms and inhibits their formation via quorum sensing dysregulation

Abstract

Biofilm infections caused by methicillin-resistant Staphylococcus aureus (MRSA) represent a critical global health challenge. This study comprehensively assessed the anti-biofilm efficacy of femtosecond laser treatment for both the prevention of biofilm formation and the destruction of pre-formed mature biofilms. The experimental setup utilized the INSPIRE HF100 laser system, which was pumped by the MAI TAI HP laser system, to provide femtosecond laser pulses at a wavelength of 400 nm, varying both the exposure duration and average power. We found that all the femtosecond laser treatments effectively impaired MRSA's ability to form biofilms, with the most potent effect observed at an average power of 50 mW for 45 min, with a significant 75.3% reduction in biofilm viability, 44.5% biomass reduction, and 60-85% reduction in metabolic activity achieved at 45 min (p < 0.0001 by ANOVA and Tukey test). Furthermore, the destruction of pre-formed biofilms was strongly dependent on the exposure duration, with 69% reduction in biofilm viability, 27.7% biomass reduction, and a 55.7% reduction in metabolic activity achieved at 45 min (p < 0.0001 by ANOVA and Tukey test). Gene expression analysis of 14 selected genes supports a model in which the femtosecond laser induced a significant transcriptional dysregulation across the quorum-sensing network, suggestive of a broad-scale attenuation of regulatory activity and leading to a functional failure in biofilm establishment, despite the transcriptional upregulation of adhesion genes. The integration of our quantitative (CFU), physical (CV), metabolic (MTT), morphological (SEM), and molecular (RT-qPCR) data provides a comprehensive "multi-dimensional" understanding of how 400 nm femtosecond laser irradiation mitigates MRSA biofilm formation and facilitates the eradication of mature biofilms. This work reports the optimization of femtosecond laser parameters for both anti-virulence and biofilm destruction, positioning it as a promising antibacterial modality against multidrug-resistant bacterial infections.