Boosting porphyrin synthesis and ALA-mediated photoinactivation through near-infrared therapy

Photodynamic inactivation (PDI) combines the use of photosensitizers with visible light to produce reactive oxygen species that effectively eliminate pathogens. To investigate the impact of near- infrared therapy (NIRT) on heme biosynthesis and permeability of the pro-photosensitizers 5-aminolevulinic acid (ALA) and Hexyl-ALA (H-ALA) through biofilms, we applied sub-lethal conditions for both NIRT and PDI to maintain intact bacterial viability. During NIRT, the temperature remained below 37°C, permitting rapid heating (ΔT = 11°C) without causing thermal damage. NIRT potentiated the bactericidal effects of ALA-PDI by 3 logs in Staphylococcus aureus RN6390 biofilms and 4 logs in ST5-SCCmecI biofilms. With H-ALA-PDI, reductions of 4 and 6 logs, respectively, were observed. In the case of ALA, this enhancement was partly due to increased porphyrin synthesis, a result not replicated by simple heating. For H-ALA, the enhanced effect of PDI was likely due to biofilm or bacterial membrane destabilization caused by NIRT and H-ALA. Unlike biofilms, applying the same ALA-PDI conditions, the treatment was effective in planktonic S. aureus RN6390 cultures, reducing the bacteria by 3 logs, with no additional benefit from NIRT, suggesting that NIRT primarily aids in penetrating the biofilm matrix rather than the bacterial cells. Confocal fluorescence microscopy confirmed that NIRT-treated biofilms exposed to ALA exhibited higher porphyrin fluorescence than untreated biofilms. These findings support the use of NIRT in enhancing the effectiveness of PDI against bacterial biofilms.