Microbial Composition of Oral Biofilms after Visible Light and Water-Filtered Infrared a Radiation (VIS+wIRA) in Combination with Indocyanine Green (ICG) as Photosensitizer

Abstract

In view of increasing antibiotic resistance, antimicrobial photodynamic therapy (aPDT) is an alternative treatment method used to eradicate the microbial community of oral biofilms that can be responsible for different oral infections. In order to investigate changes in the microbial composition after application of aPDT with visible light and water-filtered infrared A (VIS+wIRA) in combination with indocyanine green (ICG), oral microorganisms of the initial and mature biofilm were evaluated by mass spectrometry (MALDI-TOF-MS). To determine surviving microorganisms using MALDI-TOF-MS, an in situ biofilm was irradiated with VIS+wIRA for five minutes in the presence of ICG (300 and 450 µg/mL, respectively). Treatment with chlorhexidine (0.2%) served as positive control. Identified microorganisms of the initial biofilm treated with ICG showed a clear reduction in diversity. The microbial composition of the mature oral biofilm also showed changes after the implementation of aPDT, which mainly resulted in a shift in the percentage of bacterial species. The resulting destruction of the microbial balance within the oral biofilm by aPDT using VIS+wIRA and ICG can be seen as an advantageous supplementary approach in the adjunctive treatment of periodontitis and peri-implantitis.

Keywords: antimicrobial photodynamic therapy (aPDT); indocyanine green (ICG); oral biofilm; visible light and water-filtered infrared A (VIS+wIRA).

Figure 1

Column diagrams of the different microorganisms of the initial biofilm in the untreated control. The composition is shown in strain (a), genus (b) and bacterial species within the highest genus (c). All percentages given refer to all examined subjects. The bars depict the average values in percentage of three discs (one from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses.

Figure 2

Graph of the surviving microorganisms after treatment of the initial biofilm with CHX. The subdivision is made into strain (a), genus (b) and bacterial species (c), whereby the occurrence is given in percentage, based on the total number of all detected bacteria in all subjects. The bars depict the average values in percentage of three discs (one from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses. The high standard deviations are caused by the fact that bacteria were detected only in the initial adhesion of two volunteers after the treatment with CHX. All of these bacteria are members of the genus Streptococcus, of which Streptococcus mitis was detected only in the initial biofilm of one volunteer and Streptococcus sanguinis survived in the initial oral biofilm of two participants.

Figure 3

Presentation of the reduced diversity after application of aPDT in combination with 300 µg/mL ICG on the initial biofilm. The graph is divided into strain (a), genus (b) and bacterial species of the dominant genus of all examined subjects (c). The proportion of bacteria is given in percentages. The bars depict the average values in percentage of six discs (two from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses.

Figure 4

Diagrams illustrating the diversity of in situ generated biofilm samples after a formation time of 3 days. The depiction of the negative control shown is divided into strain (a), genus (b) and bacterial species of the most representative genus (c). All percentages shown refer to the total of the determined microorganisms in all subjects. The bars depict the average values in percentage of three discs (one from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses.

Figure 5

Illustration of the changed composition of the mature oral biofilm after treatment with 0.2% CHX. Division of diversity into bacterial strain (a), genus (b) and bacterial species of the proportionately largest genus (c). All determined percentages take into account the results of all examined subjects. The bars depict the average values in percentage of three discs (one from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses.

Figure 6

Graph of the microbial composition of the mature oral biofilm after application of aPDT in combination with 300 µg/mL ICG. The percentages shown are related to total samples divided into strain (a), genus (b) and bacterial species of the dominant genus (c). The bars depict the average values in percentage of six discs (two from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses.

Figure 7

Visualization of surviving microorganisms within the mature biofilm treated with 450 µg/mL ICG after aPDT. The percentages of the bacteria in the total microbial composition of all examined subjects are given. The graph shows a division into strain (a), genus (b) and dominant bacterial species (c). The bars depict the average values in percentage of six discs (two from each volunteer). In addition to the average values, the standard deviations are also presented in parentheses.