Impact of hydrogen peroxide photolysis on viable bacterial count and composition of in vivo dental biofilm—an ex vivo study

A bactericidal technique utilizing hydrogen peroxide (H₂O₂) photolysis, which generates hydroxyl radicals, was developed. Since it has demonstrated the potential to kill in vitro Streptococcus mutans biofilms, this technique has the possibility to be applied to dental caries. However, its efficacy on in vivo cariogenic dental biofilms remains unclear. This study aimed to evaluate the bactericidal effect of H₂O₂ photolysis on in vivo biofilms obtained from volunteers, focusing on its potential application in dental caries treatment. Sixteen participants, prescreened for the presence of S. mutans and/or lactobacilli in their saliva, wore custom-made splints with hydroxyapatite (HA) discs for 5 days. The discs with 5-day-old biofilms were subjected to H₂O₂ photolysis or control treatments such as H₂O₂, light-emitting diode (LED) irradiation, or water for 90 s, followed by bacterial culturing and quantitative real-time polymerase chain reaction (qPCR). Statistical significances were assessed using the Steel–Dwass test. Scanning electron microscopy (SEM) and 16S rRNA gene sequencing were also used for biofilm evaluation. Discs with treated-biofilms were reinserted for an additional 5 days to evaluate biofilm regrowth and community shifts. SEM confirmed biofilm formation on HA surfaces. Bactericidal assays showed that the 90-s treatment with H₂O₂ photolysis significantly reduced viable bacterial counts, achieving a 3.5 log colony-forming unit (CFU)/specimen for total bacteria and a 2.6 log CFU/specimen for total streptococci, compared with 5.9- and 5.4 log CFU/specimen in the untreated controls, respectively (p < 0.05). qPCR confirmed that the bacterial proportion was initially equivalent across the groups and remained unchanged after regrowth. The 16 S sequencing revealed a diverse microbial community dominated by Streptococcus spp. No significant differences in alpha or beta diversity were observed between the treatment groups even after regrowth. These findings suggest that the H₂O₂ photolysis technique can kill bacteria within in vivo biofilms. The observed bactericidal effect supports the potentiality of H₂O₂ photolysis as a promising adjunctive approach for dental caries.