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

Abstract

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.