Photoinactivation Using Visible Light Plus Water-Filtered Infrared-A (vis+wIRA) and Chlorine e6 (Ce6) Eradicates Planktonic Periodontal Pathogens and Subgingival Biofilms

Abstract

Alternative treatment methods for pathogens and microbial biofilms are required due to the widespread rise in antibiotic resistance. Antimicrobial photodynamic therapy (aPDT) has recently gained attention as a novel method to eradicate pathogens. The aim of this study was to evaluate the antimicrobial effects of a novel aPDT method using visible light (vis) and water infiltrated infrared A (wIRA) in combination with chlorine e6 (Ce6) against different periodontal pathogens in planktonic form and within in situ subgingival oral biofilms. Eight different periodontal pathogens were exposed to aPDT using vis+wIRA and 100 μg/ml Ce6 in planktonic culture. Additionally, pooled subgingival dental biofilm was also treated by aPDT and the number of viable cells determined as colony forming units (CFU). Live/dead staining was used in combination with confocal laser scanning microscopy to visualize and quantify antimicrobial effects within the biofilm samples. Untreated negative controls as well as 0.2% chlorhexidine-treated positive controls were used. All eight tested periodontal pathogens including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Actinomyces odontolyticus, Fusobacterium nucleatum, Parvimonas micra, Slackia exigua, and Atopobium rimae and the aPDT-treated subgingival biofilm were eliminated over the ranges of 3.43–8.34 and 3.91–4.28 log10 CFU in the log10 scale, respectively. Thus, aPDT showed bactericidal effects on the representative pathogens as well as on the in situ subgingival biofilm. The live/dead staining also revealed a significant reduction (33.45%) of active cells within the aPDT-treated subgingival biofilm. Taking the favorable tissue healing effects of vis+wIRA into consideration, the significant antimicrobial effects revealed in this study highlight the potential of aPDT using this light source in combination with Ce6 as an adjunctive method to treat periodontitis as well as periimplantitis. The present results encourage also the evaluation of this method for the treatment of caries and apical periodontitis.

Antimicrobial photodynamic therapy alone or in combination with antibiotic local administration against biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis

“…Our data revealed that 75 mg/L and 100 mg/L PDZ-mediated aPDT promoted 0.97 and 1.12 log 10 reductions in CFU/mL, respectively. In contrast to our outcomes, a previous study demonstrated a significant effect against F. nucleatum ATCC 25586, with more than a 3 log 10 reduction when aPDT with visible light (vis) and water infiltrated infrared A (wIRA) were combined with Ce6[50]. The strong antimicrobial effect of aPDT against F. nucleatum…”

“…Despite the efficacy of aPDT against biofilm viability, an antimicrobial effect was not observed to ensure antibacterial properties since we obtained less than a 3 log 10 CFU reduction [57]. In contrast, an antimicrobial effect of aPDT using vis+wIRA combined with Ce6 was recently reported against different periodontal pathogens in a pool of subgingival oral biofilms with disruption of their tridimensional structures before the application of aPDT [50]. This disruption in the architecture of a biofilm can potentiate the effects of antimicrobial strategies.…”

“…Remarkably, the authors indicated a slight reduction in F. nucleatum cells even after light irradiation alone [56], which is consistent with our fluorescence information. However, in both reported studies [50,56], the treatment was performed on planktonic bacterial species; this approach was inconsistent with our experimental approach in which we developed biofilms that presented a complex tridimensional architecture. Thus, the difference in microbial culture type can explain why the effect of aPDT was lower in our studies than in previous studies [50,56].…”

“…Thus, the substantial differences in bacterial CFU reduction between this study and the previous study [38[50] can be explained by the different methodologies applied. In the previous study [50], subgingival oral biofilms were developed on in situ devices, but the biofilm samples were pooled, centrifuged and resuspended in saline solution [38]. Therefore, in that study, the treatment was applied to planktonic bacteria cells arising from biofilms; in contrast, in our study, aPDT was directly applied to a complex, organized pathogenic structure.…”

“…aPDT has previously been demonstrated to kill planktonic periodontal pathogens [41,[50][51][52]. However, the susceptibility of microorganisms to aPDT is considerably reduced when they are organized into biofilms.…”