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.

Keywords: antibiotic resistance; antimicrobial photodynamic therapy; periodontitis; photosensitizer; subgingival biofilm.

FIGURE 1

Eradication rates of Gram-positive periodontitis-related oral microorganisms (Actinomyces odontolyticus, Parvimonas micra, Atopobium rimae, Slackia exigua) after the application of aPDT using vis+wIRA at a Ce6 concentration of 100 μg/ml. An untreated negative control and a 0.2% CHX-treated positive control were also tested, along with Ce6-treated bacteria in the absence of vis+wIRA and vis+wIRA-treated bacteria in the absence of Ce6. The colony forming units (CFU) are presented on a Log₁₀ scale per milliliter (Log₁₀/ml). Data shown are means ± SD (n = 6).

FIGURE 2

Eradication rates of Gram-negative periodontitis-related oral microorganisms (Eikenella corrodens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum) after the application of aPDT using vis+wIRA at a Ce6 concentration of 100 μg/ml. An untreated negative control and a 0.2% CHX-treated (CHX) positive control were also tested, along with Ce6-treated bacteria in the absence of vis+wIRA and vis+wIRA-treated bacteria in the absence of Ce6. The CFUs are presented on a Log₁₀ scale per milliliter (Log₁₀/ml). Data shown are means ± SD (n = 6).

FIGURE 3

Diagrams of the CFUs depicting the photodynamic efficiency against aerobic and anaerobic oral bacteria within the subgingival biofilm, respectively. Ce6 at a concentration of 100 μg/ml served as the photosensitizer. An untreated negative control and a 0.2% CHX-treated positive control were also tested, along with Ce6-treated biofilms in the absence of vis+wIRA and vis+wIRA-treated biofilms in the absence of Ce6. The CFUs are presented on a Log₁₀ scale per milliliter (Log₁₀/ml). Data shown are means ± SD (n = 6).

FIGURE 4

Boxplots illustrating percentages of the live bacteria as detected by live/dead staining after the application of photodynamic therapy against the subgingival biofilm. Ce6 at a concentration of 100 μg/ml served as the photosensitizer. An untreated negative control and a 0.2% CHX-treated positive control were also tested, along with Ce6-treated biofilms in the absence of vis+wIRA and vis+wIRA-treated biofilms in the absence of Ce6. The central line represents the median; whiskers indicate minimum and maximum.

FIGURE 5

Confocal Laser Scanning Microscopy (CLSM) images demonstrating the photodynamic effect on subgingival biofilms after live/dead staining. The panels illustrate the live (green) and dead (red) microbial populations of the untreated negative control (A), positive 0.2% CHX-treated control (B), Ce6-treated biofilm in the absence of vis+wIRA (C), vis+wIRA -treated biofilms in the absence of Ce6 (D), Ce6-treated (E) groups in the presence of vis+wIRA. Scale bar for all images 20 μm.