Antibacterial efficacy of Doxycycline as a Photosensitizer in Photodynamic Therapy against Subgingival Plaque Bacteria

Eka Fitria Augustina¹, Ernie Maduratna Setiawatie¹, Offia Melda Permata Hartamto²,

Yunita Marwah³

¹Department of Periodontics, Faculty of Dentistry, Airlangga University, Indonesia.

²Student of the Faculty of Dentistry, Airlangga University, Indonesia.

³Resident of a Periodontology Program, Faculty of Dentistry, Airlangga University, Indonesia.

*Corresponding Author E-mail: ekafitri91@gmail.com

ABSTRACT:

Background: Periodontitis is an inflammation that occurs in the periodontium and is caused by the host immune responses and subgingival plaque bacteria. Subgingival plaque bacteria can initiate periodontal disease because they have the potential to induce the release of proinflammatory cytokines. Mechanical debridement alone not always possible to completely remove pathogenic bacteria that cause periodontal infections due to accessibility and location. New treatments, such as photodynamic therapy, which uses lasers, can offer an alternative option. Photodynamic therapy is a non-invasive therapy that uses photons of light energy for medical purposes. The mechanism of photodynamic therapy is based on a triad consisting of a photosensitizer, a light source, and molecular oxygen. Microorganisms first absorb the photosensitizer, then are activated by light of a specific wavelength. Photosensitizer can transfer the received energy to molecular oxygen and convert oxygen into ROS, then cause the death of microorganisms by affecting their membranes, proteins, and nucleic acids. Doxycycline is widely used in periodontal therapy and is known to act as an exogenous photosensitizer. Objective: The aim of the research was to evaluate the potential of doxycycline as a photosensitizer in photodynamic therapy against subgingival plaque bacteria. Method: The research used the diffusion method to test the bacterial inhibition with 4 replications. This research was divided into 6 groups: (1) control, (2) photodynamic therapy, (3) 0,0125% doxycycline with photodynamic therapy, (4) 0,025% doxycycline with photodynamic therapy, (5) 0,05% doxycycline with photodynamic therapy, and (6) 0,1% doxycycline with photodynamic therapy. Subgingival plaque bacteria were collected from stock and put into a reaction tube that contained liquid BHIB. After that, the subgingival plaque bacteria culture was incubated for 48hours at 37ºC, and then the subgingival plaque bacteria culture was planted on Mueller Hinton agar with the spreading technique. Filter paper was inserted into each of the doxycycline concentrations and placed on the petridish that has been planted with subgingival plaque bacteria. Photodynamic therapy with a wavelength of 405nm for 30 seconds was exposed to the paper. Then, the agar plates were incubated for 48 hours at 37ºC. Result: The avarage inhibition zone of subgingival plaque bacteria was obtained in the following groups: (1) 0mm, (2) 13.375mm, (3) 14.6125mm, (4) 15.450mm, (5) 17.325mm, and (6) 19.2875mm. Conclusion: The doxycycline 0.1% concentration group that is combined with photodynamic therapy for 30 seconds has the biggest inhibition zone in the subgingival plaque bacteria.

KEYWORDS: Subgingival plaque bacteria, Photodynamic therapy, Antibacterial, Photosensitizer, Doxycycline.

INTRODUCTION: 

Periodontitis is an inflammation that occurs in the periodontium. Periodontitis consists of three words: perio, which means gingiva and other tissues around the teeth; dont, which means tooth; and itis, which means inflammation.¹ So periodontitis is the result of progressive and persistent inflammation that occurs in the tissues supporting the teeth, which can cause periodontal pocket formation, alveolar bone loss, and clinical attachment loss.^1,2 Periodontitis is caused by the host immune responses and subgingival plaque bacteria.³ Subgingival plaque bacteria can be found below the gingival margin, between the gingival pocket epithelium and the tooth. Subgingival plaque bacteria have an important role in causing periodontal tissue damage. Plaque bacteria can initiate periodontal disease, and bacterial antigens can cross the junctional epithelium, so plaque bacteria can promote an inflammatory process. Subgingival plaque bacteria have the potential to induce the release of proinflammatory cytokine.⁴

The goals of periodontal disease therapy are to improve the hygiene of the affected teeth, reduce the number of bacteria, and change the bacterial composition of the biofilm. A reduction in plaque biofilm volume can be achieved with mechanical instrumentation.⁵ Scaling and root planing (SRP) can remove soft and hardened microbial deposits from the root surface.⁶ However, in controlling periodontal disease with mechanical debridement alone, it is not always possible to completely remove pathogenic bacteria that cause periodontal infections due to accessibility and location.^5,7,8 The gold standard therapy for periodontal disease is SRP, which can reduce the number of pathogenic bacteria and is combined with additional therapy using antibiotics to increase its effectiveness. Antibiotics can be administered systemically or topically through the gingival sulcus.⁹ Adjuvant therapy with systemic antibiotics has been shown to increase the efficacy of nonsurgical periodontal therapy. The combination of SRP with systemic antibiotics can reduce pocket depth and increase clinical attachment levels, especially in pockets with a depth of more than 6 mm and in severe periodontitis. Additional systemic antibiotics usually do not need to be prescribed for mild-to-moderate chronic periodontitis because the side effects outweigh the clinical benefits.⁵

Doxycycline is seen to be widely used in periodontal therapy.¹⁰ Doxycycline is a semisynthetic tetracycline with a broad spectrum that has bacteriostatic properties. Doxycycline can inhibit bacterial protein biosynthesis by interfering with transfer RNA (tRNA) and messenger RNA (mRNA) in the ribosome. In aggressive periodontitis, doxycycline can be given at a dose of 100 – 200mg/day for 21 days. In chronic periodontitis, doxycycline can be given at a dose of 20 mg/day twice a day for 3 – 9 months, which is recommended as sub-antimicrobial-dose doxycycline (SDD) to inhibit collagenase. However, doxycycline has adverse effects, including reducing the efficacy of oral contraceptives, photosensitivity (sensitivity to sunlight), hypersensitivity reactions, nausea, vomiting, and esophaegal irritation. Tetracycline use in children aged 12 years or younger can cause tooth discoloration because tetracycline has the ability to chelate with calcium and therefore gets deposited in mineralized tissues such as bone teeth during the mineralization process, resulting in yellow to brown discoloration of teeth.⁴ In addition, long-term administration of doxycycline can also cause resistance to antibiotics and superimposed infections.^10,11 In the last decade, the government has expressed seriousness about antimicrobial drug resistance.¹² New treatments, such as photodynamic therapy, which uses lasers, can offer an alternative option.^13,14

Photodynamic therapy has been introduced as a periodontal treatment approach since the 1990s.¹⁵ Photodynamic therapy has been considered a promising new therapy to kill pathogenic bacteria in periodontal diseases.¹³ Photodynamic therapy is a non-invasive therapy that uses photons of light energy for medical purposes.¹⁴ The mechanism of photodynamic therapy is based on a triad consisting of a non-toxic molecule known as a photosensitizer, a light source (e.g., lasers, LEDs, and fluorescent lamps), and molecular oxygen.¹⁶ Photodynamic therapy can stimulate photosensitizers to produce singlet oxygen and other highly reactive free radicals, which oxidize and destroy bacterial cells.^7,17 The benefit of photodynamic therapy is that it has low toxicity to the host site. Furthermore, photodynamic therapy does not require systemic treatment because it has localized effects and has no side effects.¹⁶ In addition, photodynamic therapy can not only kill the pathogenic bacteria but can also cause endotoxin detoxification. It has been proven in vitro that lipopolysaccharide treated with photodynamic therapy can prevent mononuclear cells from producing proinflammatory cytokines.¹³

Photosensitizers are dyes that can be absorbed by microorganisms, cells, or tissues.⁹ Photosensitizers that bind to bacteria can cause electrostatistic interactions between the bacterial cell wall and increase the permeability of the wall bacterial cell.¹⁸ Furthermore, photosensitizers also have the ability to absorb specific lights so that they can cause the transfer of the received energy to molecular oxygen, then convert oxygen into reactive oxygen species (ROS). ROS can cause the death of microorganisms by affecting their membranes, proteins, and nucleic acids, so ROS can act as bactericidal antibiotics.^16,18,19 In general, bacteria produce porphyrin compounds that are sensitive to light, so porphyrins can become endogenous photosensitizers. Porphyrins produced by bacteria have very strong absorption in the visible light spectrum at a wavelength of 405nm. Doxycycline is also known to act as an exogenous photosensitizer at wavelengths of 375 – 780 nm.¹⁴ The purpose of this research was to evaluate the potential of doxycycline as a photosensitizer in photodynamic therapy against subgingival plaque bacteria.

MATERIALS AND METHODS:

The type of research used in this research is an experimental laboratory methodology with a post-test-only control group design. This research was conducted at the Research Center of the Faculty of Dentistry, Airlangga University. The ethical clearance certificate number is 291/HRECC.FDOM/V/2019.

The research used the diffusion method to see the inhibitory power of bacteria. The sample size in this study used the Federer formula; each sample required 4 replications. This research was divided into 6 groups: (1) control, (2) photodynamic therapy, (3) 0,0125% doxycycline with photodynamic therapy, (4) 0,025% doxycycline with photodynamic therapy, (5) 0,05% doxycycline with photodynamic therapy, and (6) 0,1% doxycycline with photodynamic therapy.

The materials used are a stock of subgingival plaque bacteria, BHIB (Brain Heart Infusion Broth), Mueller Hinton agar, Doxycycline, Diode laser, sterile loop, reaction tube, petridish, micropipette, filter paper, bunsen, methylated spirits, alcohol, an anaerobic jar, incubator, and refrigerator (for storage). First, subgingival plaque bacteria were collected from stock using a sterile loop and put into a reaction tube that contained liquid BHIB (Brain Heart Infusion Broth). After that, the subgingival plaque bacteria culture was inserted into the anaerobic jar, and then the anaerobic jar was incubated in an incubator for 48 hours at room temperature 37ºC. After that, observe the turbidity synchronized with the 0,5 McFarland standard (1,5 x 10⁸ CFU/ml). After that, a subgingival plaque bacteria culture was planted on Mueller Hinton agar with the spreading technique. Filter paper with a diameter of 5 mm was inserted into each of the doxycycline concentrations, and the paper was placed on the petridish that has been planted with subgingival plaque bacteria. Photodynamic therapy with a wavelength of 405 nm for 30 seconds was exposed to the paper at a distance as close as possible. After that, the agar plates were incubated for 48 hours at room temperature 37ºC.

Bacterial inhibition was determined by measuring the inhibition zone around the filter paper. In this study, two or more groups were compared using free samples, so the statistical analysis used the one-way ANOVA test.

RESULTS:

The results of the antibacterial efficacy of doxycycline as a photosensitizer on photodynamic therapy against subgingival plaque bacteria can be seen in Table 1. The clear area around the treated wells indicates antibacterial efficacy.

Figure 1. The result of inhibition zone. (A) control, (B) photodynamic therapy, (C) 0,0125% doxycycline with photodynamic therapy, (D) 0,025% doxycycline with photodynamic therapy, (E) 0,05% doxycycline with photodynamic therapy, and (F) 0,1% doxycycline with photodynamic therapy.

Table 1. Inhibition zone of subgingival plaque bacteria

Group	N	Mean	SD
Control	4	0	0
Photodynamic therapy	4	13,375	0,07217
Doxycycline 0,0125% + Photodynamic Therapy	4	14,6125	0,09465
Doxycycline 0,025% + Photodynamic Therapy	4	15,450	0,07071
Doxycycline 0,05% + Photodynamic Therapy	4	17,325	0,08660
Doxycycline 0,1% + Photodynamic Therapy	4	19,2875	0,10308

Table 2. The result of Tukey-HSD test

	Control	PDT	0,0125% + PDT	0,025% + PDT	0,05% + PDT	0,1% + PDT
Control	-----	0,00*	0,00*	0,00*	0,00*	0,00*
PDT		-----	0,00*	0,00*	0,00*	0,00*
0,0125% + PDT			-----	0,00*	0,00*	0,00*
0,025% + PDT				-----	0,00*	0,00*
0,05% + PDT					-----	0,00*
0,1% + PDT						-----

* = significant

PDT = photodynamic therapy

The research data was subjected to a normality test using the Shapiro-Wilk test because the number of samples was less than 50. The results of the normality test for all the data show a value of p > 0,05, which means the research data have a normal distribution. Then, the data were subjected to see the homogeneity of the data using the Levene test. The result of the Levene test shows that p = 0,124 (p>0,05), which means the data is homogeneous. Test differences between groups were analyzed using the one-way ANOVA test. The results of this test have a significant value of p = 0,000 (p<0,05), so we get a difference from the treatment group. The one-way ANOVA test results show that there is a significant difference in the inhibition power for subgingival plaque bacteria. The next analysis used the Turkey-HSD test because the data was homogeneous. The Tukey-HSD test is used to determine significance between research groups. The results of this test show that p = 0,000 ( p<0,05), which means that all groups have a significant difference. The term significant means that there is a meaningful difference or that the relationship being tested does not occur due to random error or chance.

DISCUSSION:

Photodynamic therapy is a therapy based on a triad consisting of a light source, a photosensitizer, and molecular oxygen, which can cause cell death (phototoxicity). A combination of a photosensitizer and a suitable visible light wavelength can form reactive oxygen species (ROS), which can cause the death of microorganisms by affecting their membranes, proteins, and nucleic acids.^16,18 ROS can damage membrane lipids so that it can increase cell membrane permeability and cause cell death. ROS can also damage DNA through deoxyribose oxidation, strand breaks, nucleotide removal, base modification, and DNA-protein cross-linking.²⁰ In this research, a light source used is a diode laser, and a photosensitizer used is doxycycline.

Diode lasers are currently being widely researched. Diode lasers in photodynamic therapy have an antibacterial effect, one of which is violet-blue light. Violet-blue light at a wavelength of 400 – 420nm has an antibacterial effect, with the highest antibacterial effect at a wavelength of 405nm.¹⁸ Therefore, in this research, we used a wavelength of 405nm.

Doxycycline is also known to act as an exogenous photosensitizer at wavelengths of 375 – 780nm.¹⁴ Photosensitizer can bind to pathogenic bacteria. Photosensitizers that bind to bacteria can cause electrostatistic interactions between the bacterial cell wall and the photosensitizer that ionize the release of Ca²⁺ and Mg²⁺ out of the cell, thereby increasing the permeability of the wall bacterial cell. Furthermore, photosensitizers also have the ability to absorb specific light. Photosensitizers that absorb light from lasers can produce reactive oxygen species, including singlet oxygen and H₂O_2, where ROS can cause oxidative damage and cell death.¹⁸ In this research, we use doxycycline as a photosensitizer in photodynamic therapy against subgingival plaque bacteria.

The research used the diffusion method to see the inhibitory power of bacteria. The sample size in this study was 4 replications. This research was divided into 6 groups: (1) control, (2) photodynamic therapy, (3) 0,0125% doxycycline with photodynamic therapy, (4) 0,025% doxycycline with photodynamic therapy, (5) 0,05% doxycycline with photodynamic therapy, and (6) 0,1% doxycycline with photodynamic therapy.

The research result showed that the lowest inhibition of subgingival plaque bacteria was group 1 (control), which was not treated with doxycycline or photodynamic therapy, which was 0mm. The second lowest inhibition of subgingival plaque bacteria was group 2, which was treated with photodynamic therapy with a wavelength of 405nm without being treated with doxycycline, which was 13,375mm. The group that was only treated with photodynamic therapy with a wavelength of 405nm could cause beacterial death because bacteria generally produce porphyrin compounds that are sensitive to light, where porphyrins can become endogenous photosensitizers. Porphyrins produced by bacteria can absorb the visible light spectrum at a wavelength of 405nm.

The group treated with doxycycline and exposed to photodynamic therapy with a wavelength of 405nm showed a higher bacterial inhibition result than the control group, and the group treated only exposed to photodynamic therapy with a wavelength of 405nm. This showed that doxycycline as a photosensitizer can bind to pathogenic bacteria and absorb light from lasers to produce reactive oxygen species, including singlet oxygen and other highly reactive free radicals, so that it can oxidize and destroy bacterial cells more than the group without an exogenous photosensitizer.

The research result showed that the greatest inhibition of subgingival plaque bacteria was in group 6, which was treated with 0,1% doxyxycline and exposed to photodynamic therapy with a wavelength of 405nm for 30seconds, which was 19,2875mm. This research shows that there is an increase in bacterial inhibitor power along with an increase in doxycycline concentration. This causes an increase in concentration, meaning it contains more doxycycline. Doxycycline with a concentration of 0,1% has a doxycycline content of 0,1 grams in 100ml, and doxycycline with a concentration of 0,0125% has a doxycycline content of only 0,0125 grams in 100ml, so group 3 contains less exogenous photosensitizer compared to group 6.

CONCLUSION:

The doxycycline 0.1% concentration group that is combined with photodynamic therapy at a wavelength of 405nm for 30 seconds has the biggest inhibition zone in the subgingival plaque bacteria. Further research needs to be done on differences in exposure time, power, and wavelength of photodynamic light for photoinactivation of subgingival plaque bacteria.

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Received on 30.11.2023            Modified on 09.03.2024

Accepted on 22.04.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(10):4929-4933.

DOI: 10.52711/0974-360X.2024.00758