Synergistic Effect of Combination of a Temoporfin-Based Photodynamic Therapy with Potassium Iodide or Antibacterial Agents on Oral Disease Pathogens In Vitro

5, 10, 15, 20-Tetrakis(3-hydroxyphenyl)chlorin (temoporfin) is a photosensitizer used in photodynamic therapy for oral cancer and periodontal disease treatment. This study determined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of temoporfin. Additionally, the combination of potassium iodide (KI) or antimicrobial agents in oral pathogens under hypoxic or normoxic conditions were determined. We also evaluated the biofilm removal effect and detected the expressions of the antibiotic resistance-related genes and biofilm formation-related genes of methicillin-resistant staphylococcus aureus (MRSA). The results provided reveal that the combination of the temoporfin and KI had a synergistic effect of reducing the MICs and MBCs of Lactobacillus acidophilus and Lactobacillus paracasei under normoxic and hypoxic conditions due to increasing H2O2 production. Temoporfin increased the biofilm removal of Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, and Staphylococcus aureus under normoxic condition, and it reduced the antibiotic resistance-related genes expression of MRSA. The combination of temoporfin with ampicillin or chlorhexidine significantly enhanced the bactericidal effect on MRSA. This study provides a potential application of temoporfin on the clinical side against oral pathogens and the prevention of oral diseases.


Introduction
The oral cavity provides an optimal environment for the growth and survival of various microbes, which exist primarily as biofilm [1][2][3]. Oral pathogens form a biofilm on the surfaces of teeth, commonly known as dental plaque. Dental plaque is colonized by complex, relatively specific, and strongly interdependent microorganisms. They include aerobic and anaerobic Gram-positive bacteria, Gram-negative bacteria, and fungus. Some microbes have been implicated in oral diseases, such as caries, periodontitis, and oral candidiasis [3][4][5]. Therefore, antibiotics and antiseptic agents, such as ampicillin and chlorhexidine (CHX), are often used to treat oral diseases. However, there are side effects,  However, it remains unclear whether this effect enhances bacterial growth or improves temoporfin's bactericidal ability. The kinetic microplate method was used to analyze bacterial growth inhibition over 24 h, as shown in Figure 2. The clear bacterial suspensions were spread on an agar plate to double-check. No colony was defined as being completely inhibited. Kinetic analysis showed that A. actinomycetemcomitans and S. mutans growth were completely inhibited after temoporfin treatment at 4 µg/mL ( Figure 2a) and 2 µg/mL (Figure 2b), respectively. A log phase delay or stationary phase delay in the growth curve after 24 h incubation implies that temoporfin inhibited bacterial growth and killed the bacteria. When treated with 0.25-4 mg/mL of KI, the growth curves of A. actinomycetemcomitans ( Figure 2c) and S. mutans (Figure 2d) showed no obvious effect compared with control.
net equation: 2H2O2 → 2H2O + O2 However, it remains unclear whether this effect enhances bacterial growth or improves temoporfin's bactericidal ability. The kinetic microplate method was used to analyze bacterial growth inhibition over 24 h, as shown in Figure 2. The clear bacterial suspensions were spread on an agar plate to double-check. No colony was defined as being completely inhibited. Kinetic analysis showed that A. actinomycetemcomitans and S. mutans growth were completely inhibited after temoporfin treatment at 4 μg/mL ( Figure 2a) and 2 μg/mL (Figure 2b), respectively. A log phase delay or stationary phase delay in the growth curve after 24 h incubation implies that temoporfin inhibited bacterial growth and killed the bacteria. When treated with 0.25-4 mg/mL of KI, the growth curves of A. actinomycetemcomitans ( Figure 2c) and S. mutans (Figure 2d) showed no obvious effect compared with control. Figure 2. The kinetic growth curve of A. actinomycetemcomitans and S. mutans were inhibited by temoporfin in a dose-dependent manner instead of KI. A. actinomycetemcomitans were treated with temoporfin (a) and KI (c); similarly, S. mutans were treated with temoporfin (b) and KI (d). The symbols on the right side of the graphs indicate various temoporfin (μg/mL) and KI (mg/mL) doses. The blue line indicates vehicle treatment (control) in each graph.

MIC/MBC of the Temoporfin and KI Co-Treatment for Oral Bacteria under Normoxic and Hypoxic Conditions
Different bacteria have different drug absorption abilities, tolerances, and intracellular oxygen content, which may affect the efficacy of aPDT. The effect of aPDT may be Figure 2. The kinetic growth curve of A. actinomycetemcomitans and S. mutans were inhibited by temoporfin in a dose-dependent manner instead of KI. A. actinomycetemcomitans were treated with temoporfin (a) and KI (c); similarly, S. mutans were treated with temoporfin (b) and KI (d). The symbols on the right side of the graphs indicate various temoporfin (µg/mL) and KI (mg/mL) doses. The blue line indicates vehicle treatment (control) in each graph.

MIC/MBC of the Temoporfin and KI Co-Treatment for Oral Bacteria under Normoxic and Hypoxic Conditions
Different bacteria have different drug absorption abilities, tolerances, and intracellular oxygen content, which may affect the efficacy of aPDT. The effect of aPDT may be affected under hypoxic conditions. In addition, we considered whether the synergistic activity of temoporfin and KI co-treatment could improve the killing effect on oral bacteria over temoporfin alone treatment. The MIC and MBC values of temoporfin and KI for the seven oral microbes are shown in Table 1. In the normoxic environment, the MIC and MBC values for L. acidophilus, L. paracasei, MRSA, and S. mutans were reduced by adding 1 mg/mL KI. Similarly, in the hypoxic environment, the MIC and MBC values for A. actinomycetemcomitans, L. acidophilus, L. paracasei, S. aureus, and MRSA were reduced by adding 1 mg/mL KI. In both normoxic and hypoxic environments, KI addition significantly enhanced the aPDT effect of temoporfin. KI and temoporfin co-treatment affected more bacterial species under hypoxic conditions than normoxic conditions. The co-treatment prominently reduced the MIC and MBC of L. acidophilus and L. paracasei under both normoxic and hypoxic conditions. The temoporfin and KI co-treatment showed the most significant antibacterial activity against L. acidophilus and L. paracasei. Therefore, it is important to verify these results rigorously. The dosage of 0.5-8 µg/mL temoporfin did not inhibit L. acidophilus and L. paracasei growth (Figure 3a), but the dosage of 0.25-4 mg/mL KI showed dose-dependent inhibition ( Figure 3b) under hypoxic conditions. The turbidity or optical density at a wavelength of 600 nm (OD600, mean ± S. E.) of L. acidophilus and L. paracasei decreased from 1.085 ± 0.1154 to 0.9322 ± 0.1607 and from 1.22 ± 0.1378 to 0.8463 ± 0.01027, respectively. Different doses of temoporfin (2, 4, and 8 µg/mL) and KI (0.25, 0.5, and 1 mg/mL) were used for 24 h under a hypoxic environment for further analysis. The OD600 of L. acidophilus treated with 8 µg/mL temoporfin combined with 0.5-1 mg/mL KI was significantly decreased (Figure 3c). Similarly, the OD600 of L. paracasei treated with 0.5, 1, and 2 µg/mL temoporfin combined with 0.25, 0.5, and 1 mg/mL KI was significantly decreased (Figure 3d). Thus, treatment with temoporfin combined with KI, even at low doses, significantly reduced the OD600 values of L. acidophilus and L. paracasei. CompuSyn software was used to analyze whether the effect of drug combinations was synergistic or antagonistic. A combination index (CI) < 0.3 is defined as strong synergism in the CompuSyn software. The combinations of 2 µg/mL temoporfin and 1 mg/mL KI, 8 µg/mL temoporfin and 0.5 mg/mL KI, and 8 µg/mL temoporfin and 1 mg/mL KI in L. acidophilus ( Figure 3e) showed a strong synergistic antibacterial effect. In addition, combinations of 0.5-2 µg/mL temoporfin and 0.25-1 mg/mL KI in L. paracasei ( Figure 3f) showed a strong synergistic antibacterial effect. Temoporfin and KI co-treatment showed a synergistic antibacterial effect on L. acidophilus and L. paracasei but not on MRSA (data not shown).

Combination of Temoporfin with Either Ampicillin or CHX Inhibited MRSA Growth
Although temoporfin and KI co-treatment showed no synergistic effect in MRSA, temoporfin and ampicillin co-treatment ( Figure 4a) and temoporfin and CHX co-treatment ( Figure 4b) clearly restricted MRSA growth. The 0.5-1 μg/mL temoporfin and 25-50 μg/mL ampicillin co-treatment slightly reduced MRSA growth, whereas the co-treatment with CHX could not reduce the growth when concentrations of CHX were 0.125-0.25

The Biofilm Removal Effect of Temoporfin in a Normoxic Environment
The biofilm removal assay revealed that the concentration of 2 μg/mL temoporfin had noticeably high biofilm removal efficacies against biofilms of A. actinomycetemcomitans, E. faecalis, and S. aureus. However, it had no apparent effect on biofilms of L. acidophilus, L. paracasei, S. mutans, and MRSA biofilms under normoxic conditions (Figure 5a). When the concentration of temoporfin was 8 μg/mL, there was only a slight removal effect The MRSA was treated with vehicle, temoporfin alone, CHX alone, and temoporfin and CHX co−treatment. CompuSyn report for ampicillin (c) and CHX (d). (e) The antibiotic-resistant genes mecI, mecR1, and mecA expression fold change in MRSA after temoporfin treatment for 24 h. * p < 0.05, ** p < 0.01, and *** p < 0.001 compare with vehicle (e). * CI < 0.05 (c,d). The biofilm removal assay revealed that the concentration of 2 µg/mL temoporfin had noticeably high biofilm removal efficacies against biofilms of A. actinomycetemcomitans, E. faecalis, and S. aureus. However, it had no apparent effect on biofilms of L. acidophilus, L. paracasei, S. mutans, and MRSA biofilms under normoxic conditions (Figure 5a). When the concentration of temoporfin was 8 µg/mL, there was only a slight removal effect on biofilms of L. acidophilus, L. paracasei, and MRSA.
1 μg/mL temoporfin treatment. The expression of sarA was not affected by temoporfin treatment. However, the temoporfin treatment and temoporfin and KI co-treatment did not show the MRSA-biofilm removal activity in normoxia ( Figure 5a) and hypoxia ( Figure  5b), respectively.
2.4.4. Endogenous Hydrogen Peroxide Production in the Species That Were Sensitive to Temoporfin and KI Co-Treatment KI and H2O2 reactions produce oxygen. It was unclear whether temoporfin utilized KI-produced oxygen to enhance antibacterial activity. To clarify why the synergistic effect of temoporfin and KI was limited in L. acidophilus and L. paracasei, we tested the amounts of H2O2 produced by MRSA, L. acidophilus, and L. paracasei under hypoxic conditions (Figure 5f). The amount of H2O2 produced was 12.36 ± 0.7248, 29.34 ± 1.446, and 35.64 ± 3.888 μM for MRSA, L. acidophilus, and L. paracasei, respectively. L. acidophilus and L. paracasei produced more H2O2 than MRSA (p < 0.05), and this was consistent with the synergistic effect observed only in L. acidophilus and L. paracasei.

Discussion
The effect of PDT depends on the absorption of photosensitizers, the light energy, and the intracellular oxygen content. At temoporfin MIC, the bacteria absorbed the photosensitizer for 3 h, and the best effect was obtained after 15 min of illumination.

The Biofilm Removal Effect of Temoporfin and KI Co-Treatment in a Hypoxic Environment
Hypoxic conditions enhanced the resistance to most antibiotics in the pathogens [30]. In the hypoxic environment, at concentrations of 1, 2, 4, and 8 µg/mL, temoporfin combined with 1 mg/mL KI did not demonstrate substantial removal efficacy against preformed MRSA biofilms (Figure 5b). Notably, 1 µg/mL temoporfin combined with 1 mg/mL KI had high biofilm removal efficacies against L. acidophilus and L. paracasei biofilms in the hypoxic environment (Figure 5c,d). This confirmed that the synergistic activity of KI and temoporfin can enhance the biofilm removal effect compared with temoporfin alone in vitro.

The Effect of Temoporfin on the Expression of Genes Regulating Biofilm Formation in MRSA
The transcription levels of genes (agrA, icaA, sarA, and srtA) associated with MRSA biofilms formation were determined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) (Figure 5e). The expressions of agrA, icaA, and sarA were downregulated by 0.125 µg/mL temoporfin treatment. However, srtA was upregulated by 1 µg/mL temoporfin treatment. The expression of sarA was not affected by temoporfin treatment. However, the temoporfin treatment and temoporfin and KI co-treatment did not show the MRSA-biofilm removal activity in normoxia ( Figure 5a) and hypoxia (Figure 5b), respectively.

Endogenous Hydrogen Peroxide Production in the Species That Were Sensitive to Temoporfin and KI Co-Treatment
KI and H 2 O 2 reactions produce oxygen. It was unclear whether temoporfin utilized KI-produced oxygen to enhance antibacterial activity. To clarify why the synergistic effect of temoporfin and KI was limited in L. acidophilus and L. paracasei, we tested the amounts of H 2 O 2 produced by MRSA, L. acidophilus, and L. paracasei under hypoxic conditions (Figure 5f). The amount of H 2 O 2 produced was 12.36 ± 0.7248, 29.34 ± 1.446, and 35.64 ± 3.888 µM for MRSA, L. acidophilus, and L. paracasei, respectively. L. acidophilus and L. paracasei produced more H 2 O 2 than MRSA (p < 0.05), and this was consistent with the synergistic effect observed only in L. acidophilus and L. paracasei.

Discussion
The effect of PDT depends on the absorption of photosensitizers, the light energy, and the intracellular oxygen content. At temoporfin MIC, the bacteria absorbed the photosensitizer for 3 h, and the best effect was obtained after 15 min of illumination. Additionally, the effect was weakened before this time-point (Figure 1c). In addition, the experimental results confirmed that temoporfin irradiation could be conducted multiple times. When 0.5 µg/mL temoporfin was administered for 5 min, the bacteria were not inhibited in the experiment based on method II but were inhibited in the experiment based on method III (Figure 1d). Method III involved exposure for 5 min a day for three consecutive days, and more temoporfin accumulated in the bacteria to suppress bacterial growth. The antibacterial effect of temoporfin was more pronounced. However, the long waiting time for absorption and the long irradiation time may limit the clinical application of aPDT.
KI is a biocompatible compound that is readily available, safe, and effective as an inorganic salt. A high dosage of KI still reduced S. mutans (Figure 2d), L. acidophilus, and L. paracasei growth (Figure 3b) instead of A. actinomycetemcomitans (Figure 2c). In the study, A. actinomycetemcomitans is a Gram-negative bacterium, and the others are Grampositive bacteria. The Gram-positive species have a thick and porous peptidoglycan cell wall surrounding a cytoplasmic membrane. The Gram-negative species have double lipid bilayers sandwiched between the peptidoglycan layer. Small molecules' penetration into Gram-positive species are easier than Gram-negative species [31]. The impermeable external membrane of the Gram-negative bacteria cell wall limits the anionic or neutral-charge molecule entrance [32]. Therefore, we supposed that the stress resistance of A. actinomycetemcomitans to KI is higher than that of S. mutans due to diverse cell wall structures. The effect of KI on Gram-positive bacteria is more obvious than that on Gram-negative bacteria.
In this study, A. actinomycetemcomitans was the only Gram-negative bacterium but did not show the worst response to temoporfin. The Gram-positive strains in this study-E. faecium and S. aureus-showed high sensitivity to temoporfin, but MRSA did not ( Table 1). The biofilm removal effect of temoporfin was weak under the normoxic environment ( Figure 5a). As temoporfin does not completely inhibit the genes associated with the formation of MRSA biofilms, there was no significant effect observed for biofilm removal under hypoxic conditions (Figure 5b,e). Ampicillin and CHX were antibiotic and antiseptic, respectively. Both ampicillin and CHX were commonly used in clinical dentistry. They interfered with the synthesis of the cell wall by different mechanisms. A high dose of CHX can disrupt the cell membrane and cause cell death. However, long-term usage of ampicillin and CHX will induce several side effects, such as antibiotic resistance, rash, nausea, diarrhea, skin irritation, teeth discoloration, and allergic reactions. Lactobacilli have been associated with dental caries [33]. Temoporfin and ampicillin combination and temoporfin and CHX combination show the synergistic effect of antibacterial activity in MRSA (Figure 4). The temoporfin suppressed the expression of the drug resistance gene mecA and helped reduce the working dosage of ampicillin and CHX. Therefore, a low dose Pharmaceuticals 2022, 15, 488 9 of 14 of temoporfin, ampicillin, CHX, and KI co-treatment can be used to reduce the Lactobacilli cell numbers and biofilms for caries prevention and therapy.
The present study results show that in normoxic and hypoxic environments, A. actinomycetemcomitans, S. mutans, E. faecalis, S. aureus, and MRSA can be completely inhibited by 1-8 µg/mL temoporfin but that L. acidophilus and L. paracasei cannot. However, the addition of KI enhanced the effect of temoporfin, especially against Lactobacillus acidophilus and Lactobacillus paracasei (Table 1, Figure 3c,d and Figure 5c,d). In normoxic and hypoxic environments, the MIC and MBC of L. acidophilus and L. paracasei were reduced by adding 1 mg/mL KI (Table 1). In synergistic analysis, we observed that the growth of L. acidophilus treated with 8 µg/mL temoporfin combined with 1 mg/mL KI significantly decreased. In addition, the growth of L. paracasei treated with 0.5 µg/mL temoporfin combined with 0.25 mg/mL KI significantly decreased (Figure 3). The reaction appears to be the addition of iodide and singlet oxygen to produce reactive peroxy iodide and hydrogen peroxide, producing a stable antimicrobial substance-iodine or tri-iodide. The chemical reaction is represented as follows [24,34]: These bactericidal components are probably responsible for the long-term bactericidal effect that persists after ceasing illumination [27]. Although L. paracasei and L. acidophilus were sensitive to the above active substances, KI did not distinctly enhance the photobactericidal effect of temoporfin on all test bacteria in our study. Thus, there are still other bacterial endogenous factors affecting the photobactericidal effect.
The Fenton reaction is a process of advanced oxidation during which the ferrous ion (Fe 2+ ) is oxidized by hydrogen peroxide (H 2 O 2 ) to the ferric ion (Fe 3+ ), forming a hydroxyl radical (OH • ) and a hydroxide ion (OH − ) in the process [35]. Only L. acidophilus and L. paracasei were cultured in de Man, Rogosa, and Sharpe (MRS) broth. In contrast, the other bacteria were cultured in brain heart infusion (BHI) and tryptic soy broth (TSB). We reconfirmed the composition of the MRS broth and TSB. There was no iron (Fe) component in either broth; therefore, we excluded the Fenton reaction. It was thus confirmed that in TSB, the H 2 O 2 produced by MRSA was not utilized by the Fenton reaction.
Under aerobic conditions, bacterial pyruvate metabolized carbon via pyruvate oxidase to form H 2 O 2 [36]. H 2 O 2 production decreased by two-to three-fold when certain bacteria were grown in a hypoxic environment. Lactobacilli produced bacteriocins, lactic acid, and H 2 O 2 to suppress the pathogenic growth of certain bacteria [37]. Regarding H 2 O 2 production in this study, we observed that L. acidophilus and L. paracasei produced more H 2 O 2 than MRSA did under a hypoxic environment (Figure 5f). Illuminated photofrin in the presence of KI produced hydrogen peroxide but not superoxide [29]. This evidence supports that KI enhances oxygen generation to promote the photobactericidal activity of temoporfin through endogenous and chemical productions reaction of H 2 O 2 . However, further research is needed to determine the pyruvate oxidase activity of L. acidophilus, L. paracasei, and MRSA strains. In addition to KI, manganese peroxide (MnO 2 ) and copper oxide (CuO) can also generate oxygen by reacting with H 2 O 2 . MnO 2 or CuO reaction with H 2 O 2 is relatively slower than KI, and it is unclear whether they can promote the effect of temoporfin. The effects of MnO 2 and CuO on bacteria or cells need to be evaluated. MRSA (ATCC 43300) were used in this study. A. actinomycetemcomitans was cultured in BHI broth. S. mutans, E. faecalis, S. aureus, and MRSA were cultured in TSB. L. acidophilus and L. paracasei were cultured in MRS broth. The bacteria were inoculated by loop transfer from frozen tubes into 3 mL of nutrient broth slant, and they were incubated at 37 • C for 24 h with constant shaking at 200 rpm. Bacteria from these cultures were transferred to the appropriate agar plates and incubated overnight. The selected single colony was transferred to a suitable liquid medium and incubated for 4-6 h to achieve logarithmic growth. The OD600 of each culture was adjusted to 1.0 using fresh broth to achieve a standard inoculum of 10 6 CFU/mL. Stock cultures were maintained at -80 • C in a growing broth containing 25% sterile glycerol [38,39].

Determination of Temoporfin Conditions, MIC, and MBC
Cell suspensions were prepared by inoculating 2 mL of 10 6 CFU/mL microbes from each logarithmic phase stock into 2 mL of broth containing various concentrations of the test compounds in 15 mL culture tubes. Temoporfin (ChemScene, Monmouth Junction, NJ, USA) was dissolved in dimethyl sulfoxide (DMSO) as 100 mg/mL stock solutions and stored at −20 • C. The bacterial suspensions were treated with various doses of temoporfin and incubated at 37 • C for 24 h with constant shaking at 200 rpm. Temoporfin treatments were used in conjunction with a diode laser (TI-818-1, Transverse Industries Co., Ltd., New Taipei City, Taiwan), a red light source with emission at 635 ± 5 nm. The device is designed with four independent light sources for laboratory use only [40,41]. The distance from the light to the sample was 15 cm, and the spot size diameter was 5.5 cm. The exposure times were 3, 5, 10, and 15 min (2-10 J/cm 2 ) or daily exposure for 5 min for 3 days. The concentration at which no visible turbidity was observed represented the MIC. It was subsequently inoculated on sterile 10 cm nutrient agar plates with no test compound and incubated for 24 h. The lowest concentration of the test compound with no growth was considered the MBC [38].

Growth Curve Assay
Growth curve analysis was performed in a 96-well format adapted from a previously described method [42]. Bacterial suspensions were prepared by inoculating 1 µL of 10 6 CFU/mL microbes from each logarithmic phase stock in 1 mL of the liquid medium containing various concentrations of temoporfin and potassium iodide (KI) (Sigma-Aldrich ® , St. Louis, MO, USA) in 15 mL culture tubes. After 3 h of incubation, the bacterial suspension was transferred to a 3 cm culture dish and exposed to 635 nm red light for 15 min (2-10 J/cm 2 ). The bacterial suspension (200 µL) was transferred to 96-well plates for testing, and 200 µL of sterile liquid broth was used as a blank. The 24-h growth curve analyses were performed for A. actinomycetemcomitans and S. mutans at 37 • C. The kinetic analysis included a 5 s shaking step before each of the OD600 time point measurements, which were recorded at 30 min intervals. The concentration was analyzed using a Ver-saMax™ ELISA microplate reader (Molecular Device, San Jose, CA, USA) and Softmax ® Pro (version 5.4.1) software.

Reverse Transcription (RT)-qPCR
Bacteria (10 6 CFU/mL) were inoculated into medium containing temoporfin concentrations of 0.125, 0.25, 0.5, and 1 µg/mL and incubated for 24 h. The microbes were collected for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The total ribonucleic acid (RNA) of cells treated with the drug was extracted using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). RT of total RNA was conducted using a random primer, and complementary deoxyribonucleic acid (cDNA) was used as the PCR template. The expression of antibiotic resistance-related genes (mecI, mecR1, and mecA) and biofilm formation-related genes (agrA, icaA, sarA, and srtA) was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression [38].

Biofilm Removal Assay
Bacteria (10 6 CFU/mL) were inoculated in a 96-well plate and incubated at 37 • C for 24 h to produce mature bacterial biofilms. After removing the planktonic bacteria, the biofilms were washed once with sterile phosphate-buffered saline (PBS). Then 100 µL of medium containing various concentrations of temoporfin and KI was added to each well. All treatment groups were exposed to a diode laser for 15 min. After 4 h of incubation at 37 • C, the medium was aspirated, and then the wells were washed with PBS twice and air-dried for 1 h. Crystal violet (150 µL of 0.1% w/v) was added to each well and left to stand at room temperature for 10-15 min. The crystal violet was aspirated, and the plate was rinsed four times with water. After aspirating water, 150 µL of 33% acetic acid was added to each well. Absorbance was determined at 550 nm on the VersaMax™ ELISA microplate reader using 30% acetic acid in water as the blank [40,44,45].

Hydrogen Peroxide Production Assay
Endogenous peroxide production was analyzed by a spectrophotometric assay, with reference to the experiment of Pericon et al. [36]. Under anaerobic conditions, selected colonies were transferred to a suitable liquid medium and incubated for 4-6 h to achieve logarithmic growth. The OD600 was adjusted to 0.6 using fresh broth to obtain bacterial suspensions, which were incubated for 2 h; then, the OD600 was adjusted to 1.0 using fresh broth. Cells were centrifuged at 4 • C for 20 min at 4000× g, washed twice in ice-cold PBS (pH = 7.4), and resuspended in PBS with 0.5 mM glucose to attain twice the original culture volume. H 2 O 2 production was measured in PBS to minimize the Fenton reaction. After 1 h of incubation at 37 • C under anaerobic conditions, the cultures were collected by centrifugation for 10 min at 10,000× g and filtered through a 0.2 µm (pore size) membrane. Before measuring H 2 O 2 production, phenol red (Ishizu Pharmaceutical Co., Ltd., Osaka, Japan) and horseradish peroxidase (Sigma-Aldrich ® , St. Louis, MO, USA) were added to the peroxide assay buffer at final concentrations of 0.46 mM and 0.046 U/mL, respectively. An aliquot of the filtered supernatant was added to the assay mixture at a ratio of 1 to 4 and incubated for 30 min at 37 • C. NaOH (final concentration of 0.004 N) was added to stop the reaction, and the absorbance was recorded at 610 nm. Concentrations were computed using a standard curve with known amounts of H 2 O 2 .

Statistical Analysis
Statistical data were obtained from three independent experiments. Data are shown as the mean ± standard error. Statistically, significant differences were determined using oneway ANOVA and paired t-test using Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between variants were considered significant at p < 0.05. CompuSyn software (Version 1.0, ComboSyn Inc., Paramus, NJ, USA) was used to quantify the synergism and antagonism of the drug combinations.

Conclusions
The current study investigated the MIC and MBC values of temoporfin against common oral microbes under normoxic and hypoxic conditions. The antibacterial activity of temoporfin was more effective under normoxic conditions than under hypoxic conditions. The combination of temoporfin with KI had synergistic effects of suppressing bacterial growth and enhancing biofilm removal activity in L. acidophilus and L. paracasei. They produce more H 2 O 2 than MRSA under hypoxic conditions. The combination of temoporfin with ampicillin or CHX also showed synergistic effects for reducing the antibiotic-resistant ability of MRSA. Since PDT treatment is expensive, reducing PDT dose and improving photosensitivity can help reduce medical costs. The combination of temoporfin and KI, as well as temoporfin and antibacterial agents, could be an effective remedy for treating oral and systemic diseases.

Data Availability Statement:
The data presented in this study are available in article.