Bactericidal effects of 310 nm ultraviolet light-emitting diode irradiation on oral bacteria

Small UVB (310 nm) and UVC (265 nm) LED irradiation devices were prepared (Fig. 1). The irradiation probe was shaped to a blue LED pen-type light. Power densities of the UVB device and UVC device were 1.75 mW/cm² and 1.77 mW/cm², respectively. Both of them were driven by DC power of the UV control system (10–200 mA and 12 V). The currents of the 310 nm LED and 265 nm LED at a voltage of 12 V were 70 mA and 150 mA (the property of each device being decided by a preliminary experiment by NIKKISO), respectively, for giving an approximate irradiance at the surface of each well in the examinations. The amount of exposure at each radiation time was calculated as E = P × t, where E is energy density (dose) in mJ/cm², P is power density (irradiance) in mW/cm², and t is time in seconds. The data are summarized in Table 1.

Brain-Heat Infusion (BHI) agar was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Modified GAM broth and modified GAM agar were purchased from Nissui Pharmaceutical Co., LTD (Tokyo, Japan). Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC 25586, Streptococcus sanguinis ATCC 10556, and Streptococcus mutans ATCC 25175 were purchased from the American Type Culture Collection (Manassas, VA). P. gingivalis and F. nucleatum were grown on GAM broth and GAM agar, and S. sanguinis and S. mutans were grown on BHI broth and BHI agar in an anaerobic chamber with Anaeropack Kenki (Mitsubishi Gas Chemical Company, Tokyo, Japan) at 37 °C.

A human oral squamous epithelial carcinoma cell line Ca9–22 was obtained from RIKEN Bioresource Center (Ibaraki, Japan). Ca9–22 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO₂ at 37 °C.

All of the bacteria were grown at 37 °C until the stationary growth phase. The bacterial cells were harvested by centrifugation (8000 × g for 10 min) and re-suspended in sterile phosphate-buffered saline (PBS) to yield a suspension at optical densities (ODs) of 0.4–0.5 at 600 nm with a photospectrometer. Aliquots each of 0.1 ml were used in the experiments. Bacterial suspensions each of 0.1 ml were poured into 96-well plates and were irradiated by the UVB (310 nm)-LED for different times (10, 30, 60, and 120 s). The distance from the light source of UV-LED to bacterial culture (bottom of a 96-wells plate) was 10 mm. The bactericidal effect of UVC (265 nm)-LED irradiation was used as a reference. After irradiation, each of the bacterial suspensions was diluted and seeded on an agar plate. The results were provided by the number of colonies on each agar plate and calculated as CFU/ml.

After incubation for 1–7 days, colonies on the plates were counted. The bactericidal effect was presented as % viability of bacteria calculated by the following formula: %Viability = colony forming unit (CFU)/ml (10, 30, 60, and 120-s irradiations)/ CFU/ml (0-s irradiation) × 100.

The toxicity of UV-LED irradiation to oral epithelial cells was measured using the Cell Counting Kit-8 (Dojindo, Tokyo, Japan) of WST-8 assay. Briefly, Ca9–22 cells were seeded in 96-well tissue culture plates at a density of 2 × 10⁴ cells/well. The cells were washed three times with phosphate-buffered saline (PBS) and 0.1 ml of PBS was added to each well. The cells were then irradiated by the UV-LED amount of 105, 210, 315, and 420 mJ/cm², that equal irradiation for 60, 120, 180, and 240 s. After UVB light exposure, the supernatants were removed, and fresh DMEM media were added to the wells and the cells were then incubated until desired time points. The activity of dehydrogenase in the supernatants was measured according to the manufacturer's instructions.

Monolayers of Ca9–22 cells on a chamber slide (Thermo Fisher Science, Waltham, NY) were irradiated by the 310 nm UVB-LED for 60 s. After 24 h, production of nitric oxide in Ca9–22 cells was examined using DAF2-DA (SEKISUI MEDICAL Co., Ltd., Tokyo, Japan).

A monolayer of Ca9–22 cells on a chamber slide was irradiated by the 310 nm UVB-LED for 60 s and then incubated for 24 h. The cells were washed with PBS three times and were fixed with 4% paraformaldehyde for 10 min. After washing with PBS three times, 0.2% TritonX-100 was added to the chamber for 5 min. After washing with PBS again, the cells were incubated with a blocking buffer (5% serum of goat /0.3% TritonX-100) for 1 h. After washing with PBS three times, the cells were incubated with an anti-iNOS rabbit monoclonal antibody (ab178945: Abcam, Cambridge, UK) at 4 °C overnight. After washing again with PBS three times, the cells were incubated with Alexa Fluor® 488 goat anti-rabbit IgG (H + L) antibody (Life Technologies Japan Inc., Tokyo, Japan) for 1 h. After washing with PBS five times, the nuclei of the cells were stained with DAPI (BioGenex, Fremont, CA). The cells were then observed by a confocal laser scanning microscope (Leica Microsystems, Welzlar, Germany).

Immunohistochemical studies were carried out using a polymer staining technique with Histofine Simple Stain MAX-PO(R) (NICHIREI, Tokyo, Japan). A monolayer of Ca9–22 cells on a chamber slide was irradiated by the 310 nm UVB-LED for 60 s and then incubated for 24 h. The cells were washed with PBS three times and fixed with 4% paraformaldehyde for 10 min. After washing with PBS three times, 0.2% TritonX-100 was added to the chamber for 5 min. After washing with PBS again, the cells were incubated with a blocking buffer (5% goat serum/0.3% TritonX-100) for 1 h. After washing with PBS three times, the cells were incubated with an anti-iNOS rabbit monoclonal antibody (ab178945: Abcam, Cambridge, UK) at 4 °C overnight. After washing again with PBS three times, the cells were incubated with a horseradish peroxidase-labeled anti-rabbit immunoglobulin- binding amino acid polymer [Nichirei Simple Stain MAX, PO (R) kit, Nichirei, Co., Tokyo, Japan] for 60 min at room temperature. After washing with PBS five times, sections were incubated in peroxidase substrate solution (ImmPACT DAB Substrate, VECTOR LABORATORIES Inc., CA, USA) until the desired stain was obtained. After washing with PBS five times, the nuclei of cells were stained with hematoxylin. INOS expression was analyzed by NIH-ImageJ macro. The colors of hematoxylin and DAB were digitally separated using Ruifrok and Johnston’s 2 color deconvolution method implemented as NIH-ImageJ macro. Then, iNOS expression was analyzed as the ratio of DAB color, which was calculated by dividing the degree of DAB staining by the number of nuclei.

Ca9-22 cells were irradiated by the 310 nm UVB-LED for 60 s and then incubated for 24 h. Total RNA was extracted from the cells with TRIzol reagent according to the instructions of the manufacturer (Life Technologies). Complementary DNA (cDNA) was synthesized from total RNA using ReverTra Ace -α- (TOYOBO, Osaka, Japan). Real-time PCR was performed using SYBR green Real-time PCR Master Mix (TOYOBO Osaka, Japan) to analyze expression of the iNOS gene (GAPDH being used as a housekeeping gene). The PCR protocol consisted of 40 cycles of denaturation at 95 °C. The set of primers for iNOS was 5′- TCT GCT GGC TTC CTG CTT TC-3′(forward) and 5′-CTG TCC TTC TTC GCC TCG TA-3′(reverse), and the set of primers for GAPDH were 5′-TTT GGT ATC GTG GAA GGA CTC A-3′(forward) and 5′- ATC TCG GGT GTG GTA GGT GA -3′(reverse). RNA expression levels were compared using the ΔΔCt method.

Hydrogen peroxide production in Ca9–22 cell cultures was measured using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, California, USA) according to the manufacturer's instructions. Briefly, a monolayer of Ca9–22 cells in 96-well plates was irradiated by the 310 nm UVB-LED for 60 s. The medium was changed to a reaction mixture in Krebs-Ringer phosphate buffer consisting of 145 mM NaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mM CaCl, 1.22 mM MgSO₄, 5.5 mM glucose, pH 7.35 and incubated at 37 °C for 0, 1, 3, and 6 h. Fluorescence in the wells was then measured by using a micro plate reader (excitation at 550 nm, emission at 590 nm).

DEA NONOate, a nitric oxide donor, was added to 0.1 ml of bacterial suspensions (ODs of 0.4–0.5 at 600 nm in PBS) in 96-well plates. One hour later, the suspensions were diluted and plated on agar plates, and incubated for 1–7 days at 37 °C under anaerobic condition. The colonies on the agar plates were then counted and % viabilities were calculated.

Hydrogen peroxide (1 μM and 1 mM) was added to 0.1 ml of bacterial suspensions (ODs of 0.4–0.5 at 600 nm in PBS) in 96-well plates. One hour later, the suspensions were diluted and plated on agar plates, and incubated for 1–7 days at 37 °C under anaerobic condition. The colonies on the agar plates were then counted and % viabilities were calculated.

The bactericidal effects of irradiation with the 310 nm and 265 nm UV-LEDs on oral bacteria are shown in Figs. 2 and 3. Irradiation with the 310 nm UVB-LED also had bactericidal effects on oral bacteria (Fig. 2), but the activity was lower than that of 265 nm UVC-LED irradiation (Fig. 3). With irradiation by the 310 nm UVB-LED for more than 30 s, viability of P. gingivalis was decreased to 47–58% (Fig. 2). The viability of F. nucleatuim was reduced to 53.5% by irradiation for 30 s. In addition, viabilities of S. sanguinis were decreased to 36–50% by irradiation for more than 10 s, and those of S. sanguinis were decreased to 52–58% by irradiation for more than 60 s. On the other hand, irradiation of 265 nm UVC for 10 s killed more that than 97% of these oral bacteria (Fig. 3). We investigated the effect of UVB-LED on biofilms of P. gingivalis and S. mutans. As shown in Additional file 1, irradiation with the 310 nm UVB-LED also had bactericidal effects on biofilms of P. gingivalis and S. mutans. With irradiation by the 310 nm UVB-LED for 10–120 s, viability of S. mutans was decreased to 69–74%. Viability of P. gingivalis was also decreased to 41–54% by the irradiation. A bactericidal effect was also observed by confocal laser scanning microscopy using a LIVE/DEAD BacLight Bacterial Viability Kit (Additional file 2). These results suggest that UVB-LED irradiation is also effective for microbial biofilms.

To examine the toxicity of 310 nm UVB irradiation to oral epithelial cells, Ca9–22 cells in culture were irradiated by 310 nm UVB. The results of a WST-8 assay showed that irradiation with 310 nm UVB-LED up to 60 s did not have any cytotoxicity to Ca9–22 cells, though irradiation with the 265 nm UVC-LED for 60 s showed strong cytotoxicity to the cells (Fig. 4). The LD₅₀ values of 310 nm and 265 nm were 229.6 s (401.8 mJ/cm²) and 32.2 s (57.0 mJ/cm², respectively. These results indicated that 310 nm UV-LED irradiation did not injure oral epithelial cells within 60 s.

UV irradiation induces production of reactive oxygen species (ROS) such as nitric oxide and hydrogen peroxide, and these ROS kill bacteria [18, 19]. Therefore, we examined whether UVB-LED irradiation induces the production of nitric oxide and hydrogen peroxide and whether oral bacteria are killed by these ROS. Production of nitric oxide was observed by 310 nm UVB-LED irradiation for 60 s in Ca9–22 cells (Fig. 5). In addition, production of iNOS was enhanced by irradiation for 60 s in the cells (Fig. 6). In addition, we examined iNOS mRNA expression in UVB-irradiated and non-irradiated cells by RT-PCR. We also observed a significant tendency that iNOS mRNA expression induced in UVB-irradiated Ca9–22 cells was stronger than that in non-irradiated cells (Additional file 3). These results suggest that UVB induced NO production in epithelial cells. Enhanced production of hydrogen oxide was also observed in Ca9–22 cell cultures after irradiation with the 310 nm UVB-LED for 60 s followed by incubation for 1–6 h (Fig. 7).

We next examined the cytocidal effects of these ROS on the tested oral bacteria. Addition of the nitric oxide donor DEA NONOate (10 μM) for 1 h in the cultures killed 56% of P. gingivalis, but F. nucleatum, S. sanguinis, and S. mutans were not killed by the treatment (Fig. 8). In addition, the viability of P. gingivalis and S. mutans was suppressed up to 50% by the addition of 1 μM hydrogen peroxide to the culture, but the viability of F. nucleatum and S. sanguinis was not suppressed, although 1 mM hydrogen peroxide partially suppressed the growth of S. sanguinis and S. mutans (Fig. 9).