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Year : 2019  |  Volume : 22  |  Issue : 12  |  Page : 1654-1661

Antimicrobial photodynamic therapy (light source; methylene blue; titanium dioxide): Bactericidal effects analysis on oral plaque bacteria: An in vitro study

1 Division of Periodontics, Department of Periodontics and Community Dental Sciences, College of Dentistry, Abha, Asir, KSA
2 Department of Medical Rehabilitation Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Asir, KSA
3 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Asir, KSA

Date of Submission05-Apr-2019
Date of Acceptance20-Jun-2019
Date of Web Publication3-Dec-2019

Correspondence Address:
Dr. M A Javali
Division of Periodontics, Department of Periodontics and Community Dental Sciences, College of Dentistry, King Khalid University, Abha, Asir
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njcp.njcp_189_19

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Background: Incomplete eradication of plaque bacteria from the plaque retentive sites and the emerging problem of antibiotic resistance led the scientific community to explore new antimicrobial strategies for improved results and shun antibiotic resistance. Objective: The purpose of this in-vitro study was to evaluate the antimicrobial effect of a novel light based therapy and to assess the susceptibility of oral plaque bacteria to light based technologies with and without photosensitizers. Materials and Methods: Four oral plaque bacterial strains were isolated from the dental plaque sample collected from the patients and exposed to various light based technologies and photodynamic therapy (PDT) with and without photosensitizers. The cultures were analysed for viable colony forming unit (CFU) counts. One-way analysis of variance was used to statistically analyse differences and the Student-Newman-Keuls method to perform multiple comparison procedures. Results: All groups showed remarkable reduction in the CFUs as compared to control group with use of light based technologies and PDT in this study. The difference of antimicrobial effect between all tested groups either with light based technologies and PDT with control group showed significant reduction in CFUs. Conclusions: From the results of this study, we concluded that light based technologies and PDT could be a valuable alternative therapy to mechanical debridement in the prevention of growth and recolonisation of oral plaque bacteria.

Keywords: Antimicrobial, blue light, laser, methylene blue, photodynamic therapy, titanium dioxide, ultraviolet

How to cite this article:
Javali M A, AlQahtani N A, Ahmad I, Ahmad I. Antimicrobial photodynamic therapy (light source; methylene blue; titanium dioxide): Bactericidal effects analysis on oral plaque bacteria: An in vitro study. Niger J Clin Pract 2019;22:1654-61

How to cite this URL:
Javali M A, AlQahtani N A, Ahmad I, Ahmad I. Antimicrobial photodynamic therapy (light source; methylene blue; titanium dioxide): Bactericidal effects analysis on oral plaque bacteria: An in vitro study. Niger J Clin Pract [serial online] 2019 [cited 2022 Nov 28];22:1654-61. Available from:

   Introduction Top

Dental plaque is a biofilm, consisting of a mixture of co-aggregated bacteria which is the main causative agent of periodontal disease.[1],[2] Due to that fact, the main goal of periodontal treatment is to reduce microbial agent, common treatment such as the mechanical debridement as well as the use of local or systemic antibiotics are considered as the main therapeutic measures in the treatment of chronic periodontitis.[3]

Although mechanical removal of plaque biofilm by scaling and root planing (SRP) is a crucial part of periodontal therapy, complete removal of plaque and plaque retentive factors is not always promising especially in less accessible sites such as deeper pockets and furcation areas.[4] Furthermore, bacteria that infiltrate the gingival tissue cannot be eliminated by mechanical instrumentation.[4]

The use of systemic or local antibiotics as an adjunct to conventional periodontal therapy has been needed. However, rising problem of antibiotic resistance has led to find alternative antimicrobial therapies which will kill resistant microorganisms and unlikely to produce resistance to develop to themselves.[5],[6] Therefore, it would be beneficial to develop an alternative application to eradicate bacteria from periodontal pockets. One such novel alternative is the application of photodynamic therapy (PDT) and other light-based therapies.[7] The striking advantages of these light-based antimicrobial therapies is their capacity to eliminate microbes irrespective of antibiotic resistance, and the essential unlikelihood of develop resistance to these light based therapies by the microorganisms due to nonspecific nature of the targets.[8]

PDT is an active and advanced antimicrobial method,[9] which includes the mixture of a non-toxic dye, photosensitizer and a source of light. It shows a great antimicrobial action in addition to better access to areas that are usually inaccessible to conventional therapy.[10] The use of PDT as an antimicrobial control technique has both local and definite effects.[10]

PDT as an alternative chemotherapy modality is widely used in cancer therapy.[11] Since PDT causes damage to different parts of microbial cells and affects different interaction pathways in the microorganisms, the development of antibiotic resistance against PDT can be excluded.[12] The photodynamic antimicrobial chemotherapy (PACT) has been indicated as an alternative to conventional antimicrobial therapy to kill oral bacteria.[13],[14],[15] A number of studies have shown to kill or inactivate microorganisms with the application of light source after sensitizing the microorganisms with a low concentration of some dye, such as methylene blue (MB), toluidine blue O (TBO) or titanium dioxide (TiO2).[16],[17],[18],[19]

The aim of this in-vitro study was to evaluate the antimicrobial effect of a novel therapy against oral plaque bacteria and to assess the susceptibility of oral plaque bacteria to various light-based technologies with and without photosensitizers.

   Materials and Methods Top

Study design

The present in-vitro cross sectional study was conducted in College of Dentistry, Division of Periodontics of King Khalid University, Saudi Arabia. The research methodology followed the international standards and ethical directives of the Helsinki. Information and confirmation principles for research purposes were strictly respected; the signed informed consent for study inclusion was obtained from each patient for collection of plaque samples. The study protocol was approved by Institutional review board and ethical approval was sought from scientific research committee, King Khalid University, College of Dentistry, Abha, Saudi Arabia (SRC/ETH/2018-19/002).

Patients visiting Out Patient Department, College of Dentistry of King Khalid University, Saudi Arabia were selected for the study for plaque sample collection. Patients taken periodontal treatment in the last 12 months or antibiotic therapy in the last 6 months, patients with infectious systemic diseases, patients taking various types of medications which can affect the periodontal status, pregnant and lactating mothers, and smokers, were excluded from the study.

Plaque sample collection

Plaque samples were collected under strict aseptic conditions. Prior to the collection of plaque sampling, patient was asked to rinse the mouth with water, and area of collection was isolated with cotton rolls. The food debris on the chewing surface was removed using a dental excavator. The dental plaque sample was collected from the patient using an excavator or curette and transferred into the 2ml broth of transport medium or brain heart infusion (BHI) in proper sterile screw cap bottles. The plaque samples were mixed well using a magnetic stirrer before incubation. The samples were inoculated using the streak plate technique on to the selective culture media.

Isolation of bacteria

The bacterial strains isolated from the sample collected in the study were Aggregatibacter actinomycetemcomitans (A.a), Fusobacterium nucleatum Scientific Name Search  (F.n), Porphromonas gingivalis (P.g) and Trepenoma denticola (T.d). All the bacteria were maintained by weekly subculture on  Brucella More Details blood agar plates (BBAP) (Oxoid, Lenexa, KS, USA) enriched with haemin and menadione, and were incubated in an anaerobic workstation (Electrotek, West Yorks, UK) at 3 degree.[13],[14] After an overnight culture in BHI broth (Hi Media, Mumbai, Maharashtra, India), each tested bacteria was quantified with a colorimeter (Amersham Biosciences, Amersham Buckinghamshire, UK). The bacteria were then diluted in solution to an optical density of McFarland standard at 405 nm (approximate numbers 1 × 102 cells). Sample of adjusted bacterial suspension was put into the wells of a flat bottom 96-well microtitre plate in triplicate. The plate was then placed on a microplate mixer (Tomy Seiko, Co. Tokyo, Japan) for even shaking, prior to the following test.

Study groups

Samples collected were distributed into five test groups; Group 1. Control – untreated; Group 2. Light alone – bacterial suspensions radiated with varying wavelengths; Group 3. MB alone – methylene blue was added to each sample to a final concentration of 0.01% weight/volume; Group 4. MB and light based technologies – bacterial samples were added with photosensitizer as in group 3 and then subsequently treated with light of varying wavelength as in group 2; Group 5. TiO2 and light-based technologies – bacterial samples were added with TiO2 and then subsequently treated with light of varying wavelength as in group 2.

The distance between the laser fiber end and sample was maintained at 5 mm approximately. After irradiation, 195 μl of pre-reduced dispersing media was added in each well, and 1 μl of each diluted suspension will be cultured by pour plate method on the BBAP. After 48-96 hours of anaerobic incubation at 37°C, bacterial colonies were counted and converted into CFU. Visual counting of CFUs was done under optical microscopy using pencil marks on dishes.

Photosensitizers and light source

The light sources used in this study were Ultraviolet light – 313 nm, Ultraviolet-A (315-400 nm wavelength), InfraRed (EnrafNonius) R1J001, 230 V, 3.26 amp, 750 w, Laser (Leve Laser) M1000 plus, max power 1w#CW, and Elipar™ Light emitting diode (LED) Curing light (430-480 nm, 100-127 V 50/60 Hz). We followed all the steps from the operatory protocol, according to manufacturer's recommendations.

The photosensitizing agent applied was methylene blue (MB) (PubChem, Rockville Pike, Bethesda, USA). MB was dissolved in 0.9% saline solution to obtain concentrations of 5, 10, 25 and 50 mg/ml 21. MB solutions were freshly prepared and stored in a dark place prior to use. In saline solution, MB has absorption maxima at 590 nm, 620 nm and 637 nm. The broadband light source used in this study allows optimal light absorption by MB.

Bacterial viability

An aliquot of bacterial suspension (100 μl) was serially diluted to 10-8 and plated on the selective media by pour plate method. The plates were incubated anaerobically at 37°C for 24 h, and then colonies that formed were counted as described.[20] These experiments were repeated thrice.

Statistical analysis

SPSS version 20 software served for statistical analysis. Statistical analysis was performed. One-way analysis of variance was used to statistically analyse differences and using the Student–Newman–Keuls method to perform multiple comparison procedures. Data was finally analyzed with descriptive statistics and two-way ANOVA (α = 0.05) was applied.

   Results Top

Counting of all CFUs in each of the test groups was done to determine the effectiveness and tabulated for four light based therapies used in this study. [Table 1] and [Figure 1] shows the susceptibility of the various bacteria to ultraviolet light following treatment with or without photosensitizer (MB or TiO2). The effects of the 180-230 nm infrared light are shown in [Table 2] and [Figure 2], laser light 30J in [Table 3] and [Figure 3] and the effects of the blue light (LED) of 430 nm are shown in [Table 4] and [Figure 4].
Table 1: Susceptibility of oral plaque bacteria to Ultraviolet light (UVL) following treatment with and without Photosensitizer

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Figure 1: Susceptibility of oral plaque bacteria to ultraviolet light

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Table 2: Susceptibility of oral plaque bacteria to Infrared light (IRL) following treatment with and without Photosensitizer

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Figure 2: Susceptibility of oral plaque bacteria to infrared light

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Table 3: Susceptibility of oral bacteria to Laser light following treatment with and without Photosensitizer

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Figure 3: Susceptibility of oral bacteria to laser light

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Table 4: Susceptibility of oral bacteria to blue light (LED) following treatment with and without Photosensitizer

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Figure 4: Susceptibility of oral bacteria to blue light (LED)

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For all bacterial strains used in this study, the control group (no treatment) showed a significantly higher bacterial growth, while the MB group and only light group presented no significant bacteria reduction showing that the dye alone and light alone appears not to reduce bacterial viability. This demonstrates that there is no direct toxicity with photosensitizers used at the concentration of 0.01% wt/vol. However, in the MB or TiO2 and light based therapies groups, all bacterial strains were sensitive to the PDT protocol, presenting a significantly less expressive bacterial growth.

Similarly, irradiation with either light based therapies alone used in this study for up to 60 s had no much significant effect on the viability of colony counts for any of the targeted bacteria. In contrast, exposure to either light based therapies in presence of photosensitizers (MB or TiO2) from ultraviolet light at an energy density of 400 nm, infrared light at 300 nm, laser light of 30J and blue light (LED) at 430 nm resulted in a significant decrease in viable colony counts. There was a significant decrease in counts when the exposure energy was increased but even then, about 45-50% of tested organisms survived by viable counts. For all tested microorganisms at any energy level used in this study points, there was a significant reduction in the CFUs when treated with both photosensitizer and light based therapies (group 4 and 5). Furthermore, there was a significant difference in reduction of CFUs between the different light based therapies used.

However, maximum reduction was seen in group 5 [Table 3], which was treated with laser and titanium dioxide. When compared [Table 1] with [Table 2], ultraviolet light group had more reduction in CFUs as compared to infrared light group. Laser light group [Table 3] also showed more decrease in colony forming bacteria as compared to [Table 4] which was irradiated with LED. The difference between all tested groups either with ultraviolet, infrared, laser or LED light, when treated with or without photosensitizers with untreated control group was significant. The antimicrobial effect of all tested groups is of great clinical significance as the elimination rate was more than 75%.

   Discussion Top

Oral plaque bacteria can be eliminated or eradicated by conventional therapy like SRP and local or systemic antimicrobials, but conventional therapy has its own limitations. So, recently the effectiveness of other treatment modalities like light-based therapy was studied for its efficacy against oral plaque bacteria in vitro as well as in vivo.[21]

Various research studies have been conducted directing to find alternative treatment modality to conventional therapy and avoid developing antibiotic resistance used for periodontal diseases. Light based therapies and PDT presents some advantages over conventional mechanical debridement and antibiotic therapy, such as fast elimination of target microorganisms and absence of maintaining a high concentration of photosensitizer on lesions during hours or days as observed in conventional therapy.

Among all the bacteria involved in the etiology of oral health problems, A.a, P.g, T.d and F.n has an important role in periodontitis,[22] and therefore has been paid much attention by many researchers. In this in vitro study, we aimed primarily to evaluate the antimicrobial effect of light-based technologies and photodynamic therapy as an alternative modality against oral plaque bacteria and to assess the susceptibility of oral plaque bacteria to various light-based technologies with and without photosensitizers.

In this study, oral plaque bacteria (i.e. A.a, P.g, T.d and F.n) wereexposed to ultraviolet, Infrared, Laser and LED light with and without photosensitizers (MB or TiO2). Zanin IC et al.[23] in 2006 used TBO and irradiation of 85.7 J cm to kill in vitro generated single-species biofilm of Streptococcus mutans and other oral streptococci. The elimination rates of the bacteria were found to vary between 95 and 99.9%. In our study, elimination rate was around 75%. Microbial biofilm, which mainly contains bacteria up to 500 times more resistant to antimicrobials than their planktonic counterparts, is a challenging target for the most modern antibacterial agents (Costerton JW et al. 1999).[24] The authors stated that the antibacterial effect of antimicrobial PDT on oral biofilm bacteria is stronger than that found after antibiotic treatment under similar conditions. Furthermore, Moslemi N et al.[25] in 2010 reported the elimination of different periopathogens and the photoinactivation of an in vitro A.a biofilm using antimicrobial PDT with TBO dye and a diode laser. Hence, an impact of antimicrobial PDT using infrared and TBO can be assumed, but has to be further studied.

Titanium dioxide was also used in his study which has studied to have photocatalytic effects on bacteria.[26] The photocatalytic properties of TiO2 have been utilized in many industries for example, in self-cleaning glass and antibacterial tiles.[27] Jesline A et al.[28] concluded from their study that TiO2 nanoparticles showed considerably good activity against the isolates, and it can act as promising, antibacterial agents. Similar results were presented by Shirai R et al.[29] when Ultraviolet-irradiated TiO2 significantly reduced the number of P. gingivalis when compared with non-irradiated controls.

Also, in our study, maximum reduction in the colony forming units for either strain of plaque bacteria was seen in group 5 [Table 3] which was exposed to laser light (30J) and TiO2. Similar results were presented by Chan Y, Lai CH[30] and Mattiello FD et al.[31] in there study with diode laser and TBO. Also, Ustun K et al.[32] from the results of their study concluded that Er, Cr: YSGG laser has bacteriocidal effects and can be used as an adjunct to SRP in periodontal treatment.

Al-Ahmad A et al.[33] from there study has shown that antimicrobial PDT in combination with TBO and infrared irradiation is a promising method for killing bacteria during initial oral colonization.

The bactericidal effect of blue light against bacteria has been proved both in vitro and in vivo, the results of our study in [Table 4] shows significant reduction in CFUs for all strains when compared to control. Sandulescu M[34] concluded from his study that the dental halogen curing light was effective in reducing oral plaque pathogens in planktonic state and that this method could be used for the treatment of periodontal disease. Maria AM et al.[35] studied photo activated disinfection therapy using LED and TBO and concluded, this as a viable adjunctive method to conventional mechanical plaque removal and the LED source is also less aggressive than the usual lasers, providing a safer and more accessible method in improving the periodontal clinical parameters.

PDT has been widely applied in periodontal infections where gram-negative bacteria are really common and are thought to be responsible for the most damaging infections. The viabilities of Aggregatibacter actinomycetemcomitans, P. gingivalis, Fusobacterium sp, Prevotella sp and Prevotella intermedia bacteria were reduced significantly after TBO mediated PDT was used.[13],[36],[37] Similar results were shown in our study [Figure 5] and [Figure 6]. Recently, a systematic review on use of photodynamic therapy as an adjunct therapy by Joseph B et al.[38] concluded that antimicrobial PDT is emerging as a useful therapeutic option in the treatment of periodontitis but long-term, multicenter studies with larger sample sizes are needed to accept it as an alternative therapy.
Figure 5: Comparison of P. gingivalis in the Control (a) and TiO2and Ultraviolet Light (b)

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Figure 6: Comparison of T. denticola growth in Control (a) and TiO2and Infrared Light (b), Control (c) and TiO2and LED (d). Comparison of Fusobacterium nucleatum growth in the Control (e) and TiO2and Laser Light (f)

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Therefore, the present study highlighted the effectiveness of various light sources with or without photosensitizers in eliminating the oral bacterial colonization, an impact on mature oral biofilm formed in-vitro is also a necessity if this procedure is to be applied clinically. Hence, the efficacy of various light sources within the oral cavity has to be established in order to conduct further in vivo clinical studies. Nevertheless, the limited ability of different antimicrobial agents to eradicate biofilm microbiota will encourage researchers to pay more attention to the application of light sources with the aim of developing more effective treatment.

   Conclusion Top

This study has shown that light based therapy and PDT with or without photosensitizer is an effective way of killing bacteria. Taking the colony forming count into consideration as well, this technique can prove to be helpful in the treatment of oral plaque induced infections. Furthermore, long term scientific studies are needed to prove its advantageous effect over conventional therapy. Also, new photosensitizers and efficient light delivery modality is to be explored.


The authors are thankful to the Deanship of Scientific Research and Dean, College of Dentistry, King Khalid University for all the support in the conduct of this study.

Financial support and sponsorship

This study was financially supported by Research Grant from the Deanship of Scientific Research of King Khalid University, Abha, Saudi Arabia (Research grant no. G.R.P-99-1439).

Conflicts of interest

There are no conflicts of interest.

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[PUBMED]  [Full text]  
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4]

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