Comparison of the antimicrobial reduction effect of photodynamic inactivation with the addition of chlorophyll and curcumin photosensitizer in Aggregatibacter actinomycetemcomitans and Enterococcus faecalis [version 1; peer review: awaiting peer review]

Background: Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are pathogenic bacteria of the oral cavity that cause various diseases such as periodontitis and endodontics. These bacteria are easily resistant to antibiotics. Photodynamic inactivation (PDI) is a method of inactivating microorganisms that utilizes light to activate a photosensitizer agent (PS) that produces reactive oxygen species causing cell lysis. Methods: This study used the PDI method with a 405 nm diode laser at various energy density with the addition PS curcumin


Introduction
The oral cavity is one of the most important parts of the body that must be maintained. Infectious diseases of the teeth and mouth that are often found are periodontitis and endodontics. Periodontitis is a bacterial infection of the teeth that causes inflammation of the supporting tissues of the teeth, which include the gingiva, ligaments, cement, and alveolar bone. 1 Periodontitis is caused by pathogenic bacteria, predominantly gram-negative, anaerobic, or microaerophilic in the subgingival area. 2 Aggregatibacter actinomycetemcomitans bacteria are found in dental plaque, periodontal pockets, and buccal mucosa in up to 36% of the normal population. 3 Aggregatibacter actinomycetemcomitans bacteria can infect patients when the human immune system decreases and inhibits other organisms' growth in the oral mucosa, teeth, and nasopharynx.
In general, gram-positive bacteria, Enterococcus faecalis (E. faecalis), are found in the root canals of teeth. The bacterium Enterococcus faecalis is ovoid, with a diameter between 0.5 and 1 μm. 4 These bacteria are facultative anaerobes and can survive in extreme environments such as highly alkaline pH and high salt concentration conditions. The number of these bacteria in the human body can be minimized by paying attention to the food consumed and environmental conditions such as humidity. Furthermore, E. faecalis bacteria resist calcium hydroxide and antibiotics such as tetracycline. 5 Systemic treatment in the form of antibiotics has been widely used to treat periodontitis. However, several studies have reported cases of antimicrobial resistance to certain types of antibiotics. So alternative therapy is needed that is effective and does not cause antibiotic resistance. 6 Therefore, the recommended alternative therapy in this study is photodynamic inactivation (PDI).
Photodynamic inactivation (PDI) is a method of inactivating microorganisms by utilizing light to activate a photosensitizer (PS) agent that produces reactive oxygen species (ROS), causing cell lysis. 7,8 The suitability of the light spectrum with the PS absorption spectrum is the key to photophysical reactions, namely the absorption of light energy by PS agents, which will trigger photochemical and photobiological reactions to produce antimicrobial effects 9,10 and biomodulation. 11 PS is a light-sensitive molecule that plays a role in absorbing light energy. PS is divided into two types, namely endogenous and exogenous photosensitizers. The addition of exogenous PS aims to increase the effectiveness of light energy absorption. 12 Some natural ingredients that are exogenous PS include chlorophyll and curcumin. Chlorophyll is a green substance found in green plants that photosynthesizes. 13 In photosynthesis, chlorophyll acts as a light catcher, energy transfer, and light conversion and can absorb a maximum wavelength between 400-700 nm. 14 Curcumin is a curcuminoid compound with yellow pigment in turmeric rhizome, which is antitumor, antioxidant, anticarcinogenic, anti-inflammatory, antiviral, antifungal, antispasmodic, and hepatoprotective. 15 The absorption spectrum of curcumin is in the wavelength range of 375-475 nm. 16 Previous studies have reported the effectiveness of using PS chlorophyll in alfalfa leaves with a blue LED activator of 20.48 J/cm2 for the inactivation of A. actinomycetemcomitans bacteria by 81%. 17 The results of another study with the addition of PS curcumin and diode laser activator 403 nm 15.83 J/cm2 in Staphylococcus aureus resulted in a mortality rate of 85.48%. 16 Then, another study using curcumin and blue LEDs on S. aureus bacteria resulted in a mortality rate of 91.49%. 18 Continuing previous studies, 15,16,18 this study aims to compare the effectiveness of antimicrobial reduction from photodynamic inactivation (PDI) on bacteria A. actinomycetemcomitans and E. faecalis with PS curcumin and chlorophyll Medicago sativa L. using a 405 nm diode laser. Diode laser irradiation was carried out at various lengths of irradiation time, namely (30, 60, 90, 120, 150, and 180) seconds.

Methods
Bacterial culture Bacteria A. actinomycetemcomitans ATCC 43718 and E. faecalis ATCC 29212 were cultured in Tryptone Soy Broth (TSB). Then, they were incubated for 24 hours at 37 o C until the colonies reached~108 CFU/mL or 1.0 McFarland standard. The culture was placed 100 L on 96-well microplates and incubated for 48 hours.

Photosensitizer (PS)
Chlorophyll was extracted from Medicago sativa L (K-Link liquid, Indonesia) and Curcuma standard (Sigma Aldrich) with a concentration of 1.6 mg/ml diluted with sterile normal saline. The absorption spectrum of chlorophyll was measured using Shimadzu UV-VIS 1800 spectrometer.

Light source
The light source of a laser diode is 405 nm, and characterization was carried out using Jasco CT-10 monochromators to determine the peak wavelength. The power output was 2.49 mW, measured with power meter OMM-6810B-220V. The spot beam area size was 0.28 cm 2 . Diode laser irradiation was carried out with variations in the length of the irradiation time of 30, 60, 90, 120, 150, and 180 seconds. The energy density value can be calculated using equation 1 9 :

PDI treatment
The treatment samples consisted of two types of bacteria, A. actinomycetemcomitans and E. faecalis. The bacterial PDI treatment consisted of a negative control group without treatment (T0), a positive control group with the addition of chlorophyll and curcumin (T1), a 405 nm diode laser treatment group at various energy densities of 0.26; 0.53; 0.79; 1.06; 1.32; 1.59 J/cm 2 (S1), a 405 nm diode laser treatment group with the addition of chlorophyll Medicago sativa L 1.6 mg/ml (S2), and a 405 nm diode laser treatment group with the addition of curcumin PS 1.6 mg/ml (S3). In groups S2 and S3, samples were given chlorophyll or curcumin and they were incubated for 10 minutes, then irradiated with 405 nm diode laser with an exposure time (30, 60, 90, 120, 150, and 180 seconds). The treated samples were grown on TSA media, incubated for 24 hours at 37C, and the number of bacterial colonies grown was counted by the Total Plate Count method.

Statistical analysis
Each treatment was calculated as CFU/ml using equation 2. Next, the percentage of bacterial reduction was calculated using equation 3 based on the control group. The results of bacterial reduction were statistically analyzed by ANOVA test using IBM SPSS Statistics Version 21.
CFU ml ¼ number of colonies Â dilution factor volume of culture plate (2) %Viability reduction of bacteria =

Results
The chlorophyll and curcumin extracts were tested using UV-Vis at a wavelength range of 325 nm to 705 nm to determine the absorption spectrum of light. Then, the results of the characterization of the absorption spectrum of chlorophyll and curcumin to light are obtained, as shown in Figures 1 and 2 After that, antibacterial tests were carried out on Aggregatibacter actinomycetemcomitans and Enterococcus faecalis bacteria, which were exposed to a diode laser with and without a photosensitizer.

Discussion
This research was conducted using the PDI technique using a blue diode laser as a light source, chlorophyll Medicago sativa L and curcumin as PS to reduce bacteria E. faecalis and A. actinomycetemcomitans. The wavelength of light is an important factor in the photoinactivation process. The laser diode used in this study has a wavelength of 405 nm and output power of 2.49 mW. The results of the characterization of power against time and temperature show the stability of power and temperature so that the temperature factor does not cause the death of bacteria.
PS is a light-sensitive molecule. Exogenous PS is PS that is added to assist the photoinactivation process. This study used exogenous PS chlorophyll Medicago sativa L and curcumin. Medicago sativa L chlorophyll absorbance used for a laser wavelength of 405 nm was 85.1% and for curcumin was 80.64%. 18 The photoinactivation process occurs due to a  photophysical mechanism initiated by the absorption of light by PS. The energy of the absorbed photon will cause the excitation of the electron to increase to a higher energy level. If the energy excitation state overlaps with the triplet excitation state, an intersystem crossing occurs, a spin reversal that places the electron in a triplet excited state and triggers a photochemical reaction.
Photochemical reactions are divided into two types. The first type is the transfer of electrons to a biological substrate in the form of a redox reaction and produces singlet oxygen. The second type is the transfer of energy to the triplet electrons to produce singlet oxygen. Singlet oxygen is radical. If attached to lipids and membrane proteins, it will cause peroxidation and damage cell membranes, causing leakage and cell lysis. 18 The photochemical reactions in PDI are generally of the second type.
The study's results on E. faecalis bacteria showed a significant difference between treatments. Diode laser irradiation with an energy density of 1.59 J/cm 2 gave the percentage of bacterial death of E. faecalis 36.7% without the addition of PS, 69.30% with the addition of PS chlorophyll Medicago sativa L and 89.42% with the addition of PS curcumin. Meanwhile, in A. actinomycetemcomitans bacteria with energy density diode laser irradiation 1.59 J/cm 2 , the percentage of bacterial death was 35.81% with the addition of PS, 64.39% with the addition of PS chlorophyll Medicago sativa L and 89.82% with the addition of PS curcumin.
The results showed that adding PS curcumin increased the effectiveness of reducing E. faecalis and A. actinomycetemcomitans bacteria. PDI with PS curcumin was effectively used to reduce bacteria because its absorption followed endogenous porphyrins. 19 The addition of energy density will increase the reduction effect without and with the addition of PS Medicago sativa L 17 and curcumin. 18