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Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis
Accepted Manuscript Title: Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis Author: M Pourhajibagher N Chiniforush S Shahabi R Ghorbanzadeh A Bahador PII: DOI: Reference:
S1572-1000(16)30058-8 http://dx.doi.org/doi:10.1016/j.pdpdt.2016.06.003 PDPDT 789
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
12-5-2016 5-6-2016 13-6-2016
Please cite this article as: Pourhajibagher M, Chiniforush N, Shahabi S, Ghorbanzadeh R, Bahador A.Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2016.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis
Pourhajibagher M1a, Chiniforush N2a, Shahabi S3, Ghorbanzadeh R4, and Bahador A5,2,1* 1
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Laser Research Center of Dentistry, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 3 Dental biomaterials Department, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran. 4 Private practice, Tehran, Iran. 5 Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 2
*Correspondence Address: Abbas Bahador, Ph.D., Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Keshavarz Blvd, 100 Poursina Ave., Tehran, Iran. 14167-53955. Tel.: +9821 6405 3210; Fax: +98218895 5810. E-mail: [email protected]; alternate address: [email protected]. a
Equal contribution.
Running title: Antimetabolic and antibiofilm potential of sPDT against E. faecalis
Highlights: 1) Higher doses of sPDT reduced biofilm formation and metabolic activity of E. faecalis. 2) Lower doses of sPDT increased biofilm formation and metabolic activity of E. faecalis. 3) ICG-sPDT showed a higher antibiofilm and antimetabolic activity than MB- and TBO-sPDT.
Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis
Abstract: Background: During photodynamic therapy (PDT) in the treatment of a primary endodontic infection, it is extremely likely that microorganisms would be exposed to sub-lethal doses of PDT (sPDT). Although sPDT cannot kill microorganisms, it can considerably influence microbial virulence. This study was conducted to characterize the effect of sPDT using toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) on biofilm formation ability and metabolic activity of Enterococcus faecalis. Methods: The antimetabolic and antibiofilm potential of ICG-, TBO-, and MB-sPDT against E. faecalis was analyzed at sub-lethal doses (1/2–1/64 minimum inhibitory concentration) using the XTT reduction assay, crystal violet assay, and scanning electron microscopy. Results: Higher doses of sPDT adversely affected biofilm formation ability and metabolic activity. ICG-, TBO-, and MB-PDT at a maximum sub-lethal dose markedly reduced the formation of biofilm up to 42.8%, 22.6%, and 19.5%, respectively. ICG-, TBO-, and MBsPDT showed a marked reduction in bacterial metabolic activity by 98%, 94%, and 82%, respectively. ICG-PDT showed a stronger inhibitory effect on biofilm formation in E. faecalis than MB- and TBO-PDT at sub-lethal levels. Interestingly, a gradual increase in metabolic activity and biofilm formation upon exposure to a lower dose of test sPDT were observed. Conclusion: sPDT showed dual effect on biofilm formation ability and metabolic activity of E. faecalis. High doses revealed antimetabolic and antibiofilm potential activity, whereas lower doses had conflicting results. Hence, when PDT is prescribed in clinical settings, the dose of PDT used in vivo should be taken into consideration. Keywords: Enterococcus faecalis, biofilm, photodynamic therapy, indocyanine green, toluidine blue O, methylene blue, root canal 1. Introduction Enterococcus faecalis, a facultative anaerobic gram-positive coccus, has an important role in persistent/secondary infection of endodontically treated teeth due to biofilm-associated resistance to common endodontic irrigants [1-3]. Biofilm formation in the dentinal tubules and lateral canals of teeth is considered the major virulence factor of E. faecalis contributing to endodontic infections. Biofilm bacteria are up to 1,000-fold more resistant to antimicrobial agents than their planktonic counterparts [4]. Elimination of microbial biofilms and effective killing of microorganisms biofilm are the primary aims in the management of endodontic infections [5]. Although chemomechanical debridement plays a key role in the reduction of microbial biofilm in the infected root canal, because of the complex root canal anatomy, as well as sometimes coverage of the root dentinal walls with smear layer and blocking the lateral canals, some of the instrumented root canal area is left untouched [6]. Hence, microorganisms, endotoxins, and other microbial products result in treatment failure [7]. Antimicrobial chemical agents such as sodium hypochlorite, chlorhexidine, and MTAD, which is a mixture of doxycycline, citric acid, and Tween 80, are used as root canal irrigants [8, 9]. Although root canal irrigants with broad antimicrobial activity and the ability to prevent smear layer formation appear to be effective at reducing microorganisms load, most studies reported insufficient and unreliable evidence showing the superiority of any
irrigants [8-10]. In addition, microorganisms in mature biofilm can resist the action of antimicrobial irrigants [11]. Recently, antimicrobial photodynamic therapy (PDT) was introduced as an innovative and relatively new approach in endodontic treatment. PDT is mediated through the exposure of photosensitizers (PSs) with light at a specific wavelength to produce reactive oxygen species (ROS) that can directly damage sub-cellular components [8]. The successful outcome of PDT depends on the type and concentration of PS, exposure time of the microorganisms with PS, and dose of light irradiation [12]. If PDT was used in the treatment of primary endodontic infection, it is extremely likely that PS would reach the target site at sub-lethal concentrations and may subsequently be activated by light at sub-lethal doses. As a result, any microorganism, such as E. faecalis, not eradicated and remaining viable at the site of infection would be exposed to doses of PDT that would not result in cell death, i.e., sub-lethal doses of PDT (sPDT), exposing survivors to ROS stress. ROS has been recognized to play an active and important role in processes such as cell death and biofilm/colony development in bacteria [13]. Previous studies have been shown that the exposure of bacteria to sPDT could lead to the extension of resistance to antimicrobial agents and result in an increased risk of poor clinical outcomes, and more consumption of medical resources [14, 15]. On the other hand, sPDT treatment affected the expression of proteins involved in the metabolic activity of bacteria [16]. To the best of our knowledge, there is no report on the effects of PDT treatment on the biofilm formation ability and metabolic activity of E. faecalis. In the present study, we evaluated whether E. faecalis cells exposed to indocyanine green (ICG)-, toluidine blue (TBO)-, and methylene blue (MB)sPDT exhibited changes in metabolic activity and biofilm formation. 2. Materials and Methods 2.1. Bacterial strain and culture conditions E. faecalis strain ATCC 29212 was obtained from the Iranian Biological Resource Center (Tehran, Iran). The organism was aerobically grown in fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37°C until logarithmic growth phase (4–5 h old) to a final concentration of 1.0 × 106 colony forming units (CFU)/mL, which was verified by spectrophotometry (optical density [OD]600: 0.2) [17]. 2.2 PSs and light sources Stock solutions of TBO and MB (Sigma-Aldrich, Steinheim, Germany) at 0.2 mg/mL were prepared in sterile 0.9% (wt/vol) NaCl. ICG stock solution was prepared by dissolving one ICG tablet (Emundo®, A.R.C. laser GmbH, Nurn-berg, Germany) in 1.0 mL sterile distilled water (2.0 mg/mL) and kept under dark conditions before use. Diodes laser (DL) (Konftec, Taiwan) at wavelengths of 660 nm with output power of 150 mW and 635 nm with output power of 220 mW were used for MB and TBO, respectively. A DL at a wavelength of 810 nm (A.R.C.laser GmbH, Nurnberg, Germany) with an output of 200 mW was used for ICG. The output powers of all wavelengths were measured by a power meter (Laser Point s.r.l, Milano, Italy). 2.3. Determination of minimum inhibitory concentrations (MICs) of PSs The MICs of TBO, MB, and ICG against E. faecalis were determined by broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI) and International Organization for Standardization (ISO) [18-20]. The MIC was determined as the lowest concentration of PSs in which visible bacterial growth was significantly inhibited. In this method, a single 96-well round-bottomed sterile polystyrene microplates (TPP; Trasadingen, Switzerland) was used for a PS. Each PS was tested eight times (rows A–H). Susceptibility panel in the microplates were prepared by pipetting 100 μL of 2× Cation-Adjusted Mueller Hinton broth (CAMHB; Himedia, India) to each well; 100 μL
of PSs solutions (2 mg/mL ICG, 0.2 mg/mL TBO, and 0.2 mg/mL MB) were added to the wells in column 1 (far left of plate) and the PSs concentration was diluted to 1:2 (i.e., 1 mg/mL ICG, 0.1 mg/mL TBO, and 0.1 mg/mL MB. PSs were diluted two-fold by transferring 100 µL aliquots from column 1 to column 2. Therefore, column 2 is a two-fold dilution of column 1 (i.e., 0.5 mg/mL ICG, 0.05 mg/mL TBO, and 0.05 mg/mL MB). The procedure was repeated down across the microplate to column 10 and then 100 μL was discarded from column 10 rather than dispensing it into column 11. Starting from column 11 to column 1, the columns were inoculated with fresh CAMHB bacterial cultures (100 μL/well) and adjusted to a concentration of 1.0 × 106 CFU/mL using multi-channel pipet to prevent PSs carry-over. The final bacterial cell concentration in the wells was 5 × 105 CFU/mL. The concentrations of PSs were in range of 1 mg/mL to 1.9 μg/mL for ICG and 0.1 mg/mL to 0.19 μg/mL for both TBO and MB after inoculation from column 1 to column 10. Column 11 contained the positive (growth) control and column 12 was not inoculated and served as the sterility control. Purity was checked by transferring and spreading 10 µL from the positive growth control well to a non-selective, enriched nutrient agar (e.g., blood agar plates). To ensure that a proper dilution was made and that the inoculum contained 2–8 × 105 CFU/mL (the acceptable range), 10 µL from the positive growth control well was transferred to 10 mL saline (1:1,000 dilution). The suspension was mixed and 100 µL of suspension was transferred to a non-selective agar medium (e.g., trypticase soy agar) and spread over the entire agar surface with a sterile spreader (1:10 dilution). The microplates (MIC panels), colony count plates, and purity plates were incubated for 24 h at 37°C. The MICs of PSs were defined as that concentration of PS that significantly inhibited visible bacterial growth. Colony count was conducted using a colony counter (Model CC-1, Merk Boeco, Germany). Each colony was multiplied by the dilution factor (1:10,000). For the purity plates, the inoculum can be considered pure if all the colonies were similar to the colonies used in the original plate. If there were any other colony present, contamination may have occurred in the MIC panel, and thus the assay was repeated. 2.4. Determination of sub-lethal DL irradiation time To find out the sub-lethal DL (sDL) irradiation time against E. faecalis, wells containing 200 µL of bacterial suspensions at a final concentration of 2.5 × 105 CFU/mL were exposed to different DL irradiation times at wavelengths of 660 and 635 nm (1, 2, 3, 4, 5, and 6 min with 23.43, 46.87, 70.31, 93.75, 117.18, and 140.62 J/cm2 and 34.37, 68.75, 103.12, 137.5, 171.87 and 206.25 J/cm2, respectively and 810 nm (0.5, 1, and 2 min with 15.62, 31.25, and 62.5 J/cm2, respectively) at room temperature (25 ± 2°C). To prevent transmission of light to neighboring wells, 15 wells of each microplate, with 2-well distance between them, were selected for testing and the peripheral wells were filled with MB. The probes of DL were fixed 1 mm above the surface of the microplates by a microphone stand. To prevent beam reflection from the table top during DL irradiation, sheets of black paper that have zero and ten percent light absorption and reflection, respectively, were used under the microplates at wavelengths between 400–1,000 nm [21].The diameter of the irradiated area was the same as the well diameter at the bottom of the microplate (6.39 mm). sDLs were defined when the DL irradiation times in the last well showed growth. 2.5. Determination of sPDT To determine sPDT, 100 μL of PSs at 2 × MIC were serially diluted two-folds in flatbottom 96-well microtiter plates (TPP; Trasadingen, Switzerland) to 1/64 MIC in 2 × CAMHB according to the methods mentioned above in section 2.3. The wells were then inoculated with fresh CAMHB bacterial cultures (100 μL/well) that were adjusted to a
concentration of 1.0 × 106 CFU/mL. The final bacterial cell concentration in the wells was 5 × 105 CFU/mL. After inoculation, the concentrations of PSs were in range of 1/2–1/64 MIC from column 1 to column 6. The microplates were incubated for 5 min in the dark at room temperature. The treated bacterial suspensions in the wells were immediately exposed to DL with different exposure times as mentioned above. sPDT was defined as the lowest concentration of PS with the shortest irradiation time of DL in the last well showing growth. The control group did not have any treatment and all experiments were performed in triplicate. 2.6. Effects of sPDT treatments on biofilm formation ability of E. faecalis Using the crystal violet assay, sPDTs based on TBO, MB, and ICG were tested for their potential to prevent E. faecalis biofilm formation. The effects of the treatments were further evaluated by scanning electron microscopy (SEM). 2.7. Quantitation of biofilm formation ability of treated planktonic E. faecalis by crystal violet Quantitative analysis and interpretation criteria of the biofilm formation ability of E. faecalis were performed according to a previous study [22]. Briefly, 200 μL aliquots of freefloating bacteria in planktonic suspension at a final concentration of 5 × 105 CFU/mL were transferred to flat-bottomed sterile polystyrene microplates. Bacterial cells were treated with sPDT at sub-lethal doses (1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 of the MIC) as mentioned above and the plates were incubated for 24 h at 37°C to allow for biofilm formation. After incubation, the microplate contents were emptied out from each well and washed three times with phosphate-buffered saline (PBS) (10 mM Na2HPO4, 2 mM NaH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) to remove free-floating planktonic bacteria. The cells in the biofilm were stained with 200 μL of 0.1% (wt/vol) crystal violet solution at room temperature for 15 min. After washing twice with PBS, 100 μL of 95% ethanol was added to each well and the plates were incubated at room temperature for 10 min to fix the cells. Afterward, the wells were rinsed three times with PBS and air dried. To quantify the biofilms, 150 μL of 33% (v/v) acetic acid was poured in each wells and the absorbance was determined at 570 nm using a microplate reader (Thermo Fisher Scientific, US). Based on the OD of the treated biofilm and that of the negative control (ODc), the samples were classified as follows: strong (4× ODc < OD), moderate (2× ODc < OD ≤ 4xODc), weak (ODc < OD ≤ 2× ODc), or non-producer of biofilm (OD ≤ ODc). 2.8. Visualization of treated and untreated E. faecalis biofilm with SEM Biofilm formation was initiated on the MBEC™ high-throughput (HTP) plates (Innovotech, Alberta, Canada) as previously described [23]. Briefly, after each treatment, 200 μL of bacterial suspension was grown on the pegs of the HTP plates containing BHI broth supplemented with 0.1% glucose at 37°C for 48 h according to the manufacturer’s recommendations. SEM was performed to observe the morphology and the biofilm formation ability of treated E. faecalis as previously described [24]. Briefly, following incubation of HTP plates, the pegs were removed using a flamed plier and rinsed with PBS for 1 min to remove any loosely attached and floating cells. The pegs were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylic acid at 4°C for 24 h. After fixation, the pegs were rinsed with the same buffer for 15 min, immersed in 1% aqueous osmium tetroxide, and incubated for 60 min at room temperature (25 ± 2°C). The pegs were washed once with deionized water for 15 min, followed by gradual dehydration with ethanol, and 1.5 h of critical point drying (Bal-Tec CPD 030, the Netherlands). The pegs were then coated with gold–palladium using a sputter
coater (Bal-Tec SCD 005, the Netherlands) and examined with a scanning electron microscope (LEO, 1455 VP, Germany). 2.9. Quantitation of metabolic activity of treated planktonic E. faecalis by XTT assay We used the XTT reduction assay to assess the effect of sPDT on metabolic activity of planktonic cells, developing biofilm, and mature biofilms of E. faecalis. Metabolic activity of another parallel set of E. faecalis treated in a similar way was assessed using the XTT [2, 3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay, based on the principle that tetrazolium salt can be reduced into a colorimetric detectable water-soluble product formazan by metabolically active cells [25]. The XTT (Sigma Aldrich, Steinheim, Germany) solution (1 mg/mL) in PBS was filter sterilized with a 0.22-µm syringe filter and stored at −80°C before use. Filter-sterilized menadione (Sigma-Aldrich, Steinheim, Germany) solution (1 mM) was prepared in acetone immediately before each assay. Following each sPDT treatment under identical conditions as described in the determination of sPDT section, aliquots of XTT-menadione solution (12.5:1 v/v; 12 μL) were added to each well. The plate was incubated for 3 h in the dark at 37°C. Following incubation, the color intensity was determined by an automatic microplate reader at 490 nm. The absorbance values for the wells without the biofilms, which served as a blank, were then subtracted from the values of the tested wells to eliminate false results due to background interference. In the XTT assay, heat-killed bacteria were used as a negative control. The percentage of metabolic activity of cells was calculated using the equation [(OD cultures of non-treated E. faecalis as growth control -OD sample)/OD growth control] × 100100. 2.10. Statistical Analysis Data from the experiments were evaluated using the two-way analysis of variance (ANOVA) followed by the Tukey’s test. Significance was defined as P-values < 0.05.
3. Results 3.1. MICs of PSs The treatment of bacterial cells with up to 1,000 µg/mL ICG as well as 100 µg/mL of both TBO and MB in the dark did not completely inhibit E. faecalis growth. ICG (31.2–1,000 µg/mL), TBO, and MB (both at 6.2–100 µg/mL) significantly reduced E. faecalis growth when compared to untreated bacteria (control; P < 0.05) (Fig 1, a–c). Lower concentrations of ICG (15.6–1.9 µg/mL), TBO, and MB (both at 3.1–0.19 µg/mL) also affected E. faecalis growth, but this was not statistically significant (P > 0.05; Fig 1, a–c). The inhibitory concentrations (ICs) at which 50 and 90% of the number of primary inoculum were killed (IC50 and IC90, respectively) were as follows (μg/mL): ICG, 62.5 and 1,000, TBO, 25 and >100; and MB, 50 and >100. 3.2. sDL irradiation times Significant decreases in the cell viability after irradiation at 660, 635, and 810 nm with energy density of 93.75–140.62, 137.5–206.25, and 31.25–62.5 J/cm2, respectively were observed in a light dose-dependent manner (P < 0.05; Fig. 1, d–f). Lower dose exposure of bacterial cells to 660 nm (23.43–70.31 J/cm2), 635 nm (34.37–103.12 J/cm2), and 810 nm (15.62 J/cm2) did not exhibit significant toxicity against the bacterial cells compared to the survival rates of untreated controls (P > 0.05). The DL exposure dose required to kill 50% of the primary inoculum in 635 and 660 nm were 140.62 and 206.25 J/cm2, respectively.
3.3. sPDT ICG-mediated PDT using 62.5–1,000 µg/mL at a fluency of 15.62 J/cm2 and 31.2– 1,000 µg/mL at a fluency of 15.62 J/cm2 showed a significant dose-dependent reduction against E. faecalis growth when compared to the control group (P < 0.05; Fig 2). Treatment at 12.5–100 µg/mL of TBO and MB with irradiation at a fluency of 103.12 and 70.31 J/cm2, respectively as well as treatment at 6.2–100 µg/mL of TBO and MB with irradiation at a fluency of 137.5 and 93.75 J/cm2, respectively revealed a significant dose-dependent reduction against E. faecalis growth (P < 0.05; Fig 2). ICG- (7.8–31.2 µg/mL at a fluency of 15.62 J/cm2), TBO- and MB-sPDT (both at 1.5–6.2 µg/mL at a fluency of 103.12 and 70.31 J/cm2) did not exhibit a significant CFU/mL reduction (P > 0.05; Fig 2). Taken together, 31.2 µg/mL of ICG at a fluency of 15.62 J/cm2, 6.2 µg/mL of TBO at a fluency of 103.12 J/cm2 and 6.2 µg/mL of MB at a fluency of 70.31 J/cm2 were determined as the sPDT. 3.4. High dose of sPDT reduced E. faecalis biofilm formation ability E. faecalis ATCC 29212 produced strong biofilms. Table 1 shows the surviving count as a function of light dose of cells in suspension treated with different concentration of each PS. The effects of the PDT on the biofilm formation ability of E. faecalis varied as a function of the association between PSs and the fluence of light (Table 1). The biofilm formation ability of E. faecalis decreased gradually with increasing dose of test sPDT as shown in Table 1. The fluence of light that significantly reduced biofilm formation of E. faecalis was 15.62 J/cm2 associated with MIC (concentration) – 1/16 MIC (62.5–3.9 µg/mL) of ICG. Our results demonstrated that 31.2 μg/mL (1/2 MIC) ICG-sPDT prevented approximately 50% of biofilm formation of E. faecalis when used at a fluency of 15.62 J/cm2, suggesting that ICG-sPDT is a potent inhibitor of strong biofilm producers. When the fluence at 103.12 J/cm2 was assessed, the use of TBO at MIC to 1/8 MIC (12.5–1.56 µg/mL) resulted in OD values that were statistically lower than the values obtained from the untreated controls (P < 0.05). Biofilm formation of E. faecalis was not significantly reduced with 1/16 MIC (0.78 µg/mL) TBO at a fluence of 103.12 J/cm2. In contrast, when the bacterial cells were irradiated at 70.31 J/cm2, the concentrations of MB (MIC to 1/4 MIC; 12.5–3.12 µg/mL) tested significantly reduced OD values when compared to the untreated group (P < 0.05). At a maximum sub-lethal dose, ICG-, TBO- and MB-sPDT significantly reduced the formation of biofilm up to 42.8, 22.6, and 19.5%, respectively. Interestingly, the reduction in biofilm formation ability of E. faecalis treated with ICG-sPDT was significantly higher than treatment with TBO-sPDT or MB-sPDT (P < 0.05). The reduction induced by TBO-sPDT on biofilm formation was approximately similar to MB-sPDT (P > 0.05). 3.5. Low dose of sPDT induced E. faecalis biofilm formation ability The crystal violet assay showed that E. faecalis biofilm formation was induced by low sub-lethal ICG-, MB- and TBO- mediated PDT (Table 1). All sPDT treatments could reduce/inhibit E. faecalis biofilm formation up to 1/4 MIC. In contrast, at 1/8–1/64 MIC, there was a proportional increase in biofilm formation depending on the PS. As shown in Table 1, there was a gradual increase in biofilm formation upon exposure to lower concentrations of MB (1/8–1/64 MIC), TBO (1/16–1/64 MIC), and ICG (1/32–1/64 MIC). 3.6. High dose of sPDT reduced E. faecalis cell metabolic activity As shown in Figure 3, sPDT affected E. faecalis metabolic activity. Treatment of bacterial cells with ICG-sPDT (62.5 μg/mL with irradiation at 15.62 J/cm2), TBO-sPDT (12.5 µg/mL with irradiation at 103.12 J/cm2), and MB-sPDT (12.5 µg/mL with irradiation at 70.31 J/cm2) showed a significant reduction in bacterial metabolic activity (98%, 89%, and 77%,
respectively; P < 0.05), which was estimated using the XTT assay. It is worth noting that ICGsPDT (1/32 MIC; 1.9 μg/mL with irradiation at 9 J/cm2), TBO-sPDT (1/8 MIC; 1.56 μg/mL with irradiation at 103.12 J/cm2), and MB-sPDT (1/8 MIC; 1.56 μg/mL with irradiation at 70.31 J/cm2) reduced metabolic activity by 50%. The data suggest that the sPDT at relatively low doses does not inhibit biofilm formation, but metabolic activity is affected. 3.7. Low dose of sPDT induced E. faecalis cell metabolic activity The XTT assay showed a gradual decrease in metabolic activity with increasing concentrations of MB-, TBO- (both at1/2–1/16 MIC), and ICG-sPDT (1/2–1/32 MIC) (Fig 3). As shown in Figure 3, E. faecalis metabolic activity was induced by lower sub-lethal MB, TBO (both at 1/32 and 1/64 MIC), and ICG-mediated PDT (1/64 MIC) at the corresponding of fluency. 3.8. Biofilm architecture visualized by SEM In the present report, a preliminary study was carried out to visualize the effect of sPDT on overall morphology of the E. faecalis in the biofilm. For this purpose, the cell morphology of the PDT treated and untreated cells were studied using SEM and results are shown in Figure 4. The untreated bacterial cells of E. faecalis retained their original cocci shape with very smooth morphology in biofilm format i.e., clusters of cells (Fig. 4a). On the other hand, PDT treated cells showed reduction in the numbers of bacterial cells as well as significant changes in morphology, especially with irregular in shape and size (Fig. 4b, c, d). A more substantial decrease in the number of cells and irregular shaped cells were observed when the biofilm was treated with ICG-sPDT. 4. Discussion The tolerance of microorganisms to antimicrobial agents can emerge from the physical and physiological status of microbial cells in the biofilms, which allows for the development of resistance [26]. In E. faecalis, a common causative agent of secondary endodontic infection, the formation of biofilms is the major virulence factor that protects the bacteria from many environmental stresses including root canal irrigants. This could result in E. faecalis developing tolerance to these substances, which is of major clinical importance [27]. There are many reasons as to why microorganisms could be exposed to sub-inhibitory concentrations of PDT during treatment for an endodontic infection. These include poor compliance, lower dosage of light, PSs concentrations below MIC, and inaccessibility to infection site. The sPDT can attenuate microbial virulence factors and their ability to confer pathogenicity, such as flagellum of Vibrio vulnificus [28]; biological activities of the lipopolysaccharides and protease activity of Pseudomonas aeruginosa [29]; V8 protease, αhaemolysin, and sphingomyelinase of Staphylococcus aureus [30]; and germ tube formation of Candida albicans [31]. The present study evaluated the anti-virulence activity, anti-biofilm formation ability, and anti-metabolic activity of ICG-, TBO-, and MB-PDT against E. faecalis at sub-lethal levels because sPDT does not have bactericidal activity but has inhibitory effects on bacterial physiology. The results showed that ICG, TBO, and MB did not completely inhibit bacterial growth. In this study, ICG exhibited higher antibacterial activity than TBO and MB alone. These variations could be due to the difference in type and concentration of PS, uptake rates of ICG, laser energy fluence rate, and time of irradiation. In the present study, ICG-, TBO-, and MB-sPDT did not exert bactericidal activity (Figure 2), suggesting that all of the sPDT used did not influence the growth or survival of the bacterial cells. The parameters which used in DL groups for determination the sub-bactericidal activity was applied in PDT groups. DL groups with different wavelengths (635, 660, and
810 nm) showed reductions in the biofilm formation of E. faecalis compared with those of the control groups, which demonstrates a small bactericidal effect. This can be attributed to the phenomenon that some bacterial cells are known to synthesize endogenous porphyrins that act as PS [32]. Similar results were also observed using only laser application by Xhevdet et al. [33], who reported a reduction in E. faecalis biofilm formation. In addition, Misba et al. [22] showed divergent results in which irradiation in the absence of PS application did not result in any difference in the reduction of biofilm formation compared to the control group. To the best of our knowledge, this is the first report providing strong evidence that ICG-sPDT (42.8%) efficiently reduce the biofilm formation ability and metabolic activity of E. faecalis compared to TBO- and MB-sPDT. The latter two only reduced biofilm formation by 22.6% and 19.5%, respectively. The higher effectiveness of ICG-sPDT as compared to the TBO- and MB-sPDT may be more complex because sequestration of divalent cations, such as Ca+2 and Mg+2, by ICG (which can behave as an anion) may decrease biofilm formation and the integrity of biofilm [34]. A number of studies have shown that divalent cations can modulate the development of bacterial biofilm [35, 36]. Similar results have been reported for cation chelators, such as disodium and tetrasodium ethylenedinitrilotetraacetic acid (TEDTA), which sequestrates divalent cations [37]. In bacteria, biofilm formation is associated with the activation of quorum-sensing signals in response to environmental stress factors [38]. Our data suggest that E. faecalis biofilm formation can be induced by conditions that are potentially toxic for the bacterial cell (i.e., exposure to ROS from PDT). In addition, our data confirm previous observations on biofilm activation by oxidative stress [39]. Current studies imply that the biofilm development is a crucial mechanism for withstanding hostile environmental conditions [40]. In this respect, bacteria in biofilms, including E. faecalis, are characterized by an increased inherent antimicrobial resistance [41]. In this study, we analyzed the influence of sPDT on E. faecalis biofilm formation. Our results indicate that the biofilm formation can be strongly enhanced by lower sub-lethal ICG-, MB- and TBO-mediated PDT. Biofilm formation was inhibited when the PDT dosage was increased to higher levels (>1/4 of the sub-lethal). To the best of our knowledge, no data on the potential increase in E. faecalis biofilm formation as a result of using sPDT have been reported. Nevertheless, the results presented here emphasize that PDT should be used at the correct dosage because low sub-lethal dosage could increase biofilm formation. The XTT assay indicated that metabolic activity was reduced in bacterial cell (Fig. 3) with the greatest reduction being that for ICG-sPDT (98%). This may be due to the inhibitory action of ICG-sPDT conjugates against quorum sensing, extracellular polymeric substance production, and virulence traits [42], which can affect biofilm formation by reducing metabolic activity. This consequently inhibits the development of a secondary endodontic infection. However, reduction in biofilm formation did not decrease at the same rate as metabolic activity, suggesting that viable cells remaining in the biofilm under stressful conditions decreased their metabolic activity. Therefore, it is likely that the decrease in metabolic activity after sPDT may be due to a ROS stress response, which acts on the cell membrane and causes loss of membrane potential and reduction in intracellular Adenosine triphosphate [43]. The effect of ICG-sPDT, TBO-, and MB-sPDT on metabolic activity may indirectly interfere with the ability of the bacteria to adhere and form biofilms as shown in the present study and other studies [44]. The present study demonstrates an effect of sPDT on cellular metabolic activity of E. faecalis. However, further studies are required to see how these two
effects are related. It is important to study how the effects of sPDT on metabolic activity of E. faecalis biofilm affect the pathogenicity of the bacteria in secondary endodontic infections. This study shows that the metabolic activity can be induced by low doses of sPDT and confirms previous observations on metabolic activity by low reactive level laser therapy [45]. Our results indicate that the metabolic activity can be enhanced by the lower sub-lethal ICG-, MB- and TBO-mediated PDT. Currently, no data on the potential increase of metabolic activity in E. faecalis as a result of using sPDT have been reported. To visualize the effect of sPDT on E. faecalis biofilm formation, SEM studies were conducted. High cell density in large cellular aggregates was observed in the control group, whereas the cell density after sPDT was reduced. This indicates not only the loss of cells in the biofilm but also significant changes in morphology, including irregular biofilm shapes and sizes. This may be due to the loss of physical contact between the cells, which can lead to cell death within the biofilm [37]. These observations were concomitant with the study conducted by López-Jiménez et al. [13] which showed the susceptibility of E. faecalis to PDT. They suggested that the antibacterial activity is due to changes in membrane permeability, cell division machinery, and inhibition of bacterial cell wall synthesis. However, further investigations are required to identify the molecular mechanisms involved in the reduction of metabolic activity and biofilm formation by sPDT in E. faecalis. 5. Conclusion Our results demonstrate that exposure of E. faecalis to all three sPDT treatments (1/2– 1/4 MIC MB-PDT, 1/2–1/8 MIC TBO-PDT, and 1/2–1/16 MIC ICG-PDT) adversely affect biofilm formation and metabolic activity in a dose-dependent manner. Interestingly, ICGPDT showed a stronger inhibitory effect on biofilm formation and metabolic activity in E. faecalis than MB- and TBO-PDT at sub-lethal levels. The reduction in biofilm formation and metabolic activity following sPDT, specifically ICG-sPDT, suggests that therapies based on the optimal dosage of PDT could be a useful approach for the eradication E. faecalis infections and is crucial to prevent secondary endodontic infection. Lower sPDT treatment (≤1/8-, ≤1/16-, and ≤1/32-MIC for MB-, TBO- and ICG-PDT, respectively) may provide protect E. faecalis cells by inducing biofilm formation. Our in vitro results indicate that when PDT is prescribed for patients with primary endodontic infection, the concentration of PS and output power of light (fluency) used in vivo as well as the susceptibility of E. faecalis against PDT should be taken into consideration. Acknowledgement This research has been supported by Tehran University of Medical Sciences & health Services grant No. 94-04-30-27681. Disclosure Statement No competing financial interests exist. References [1] G.C. Rezende, L. Massunari, I.O. Queiroz, J.E. Gomes Filho, R.C. Jacinto, C.S. Lodi, et al., Antimicrobial action of calcium hydroxide-based endodontic sealers after setting, against E. faecalis biofilm, Braz Oral Res. 30 (2016) 30:e38. [2] M.E. Łysakowska, A. Ciebiada-Adamiec, M. Sienkiewicz, J. Sokołowski, K. Banaszek, The cultivable microbiota of primary and secondary infected root canals, their susceptibility to antibiotics and association with the signs and symptoms of infection. Int Endod J. (2015) 1-9.
[3] L.E. hávez de Paz, G. Bergenholtz, G. Svensäter, The effects of antimicrobials on endodontic biofilm bacteria, J Endod. 36 (2010) 70-77. [4] U. Furustrand Tafin, I. Majic, C. Zalila Belkhodja, B. Betrisey, S. Corvec, W. Zimmerli, et al., Gentamicin improves the activities of daptomycin and vancomycin against Enterococcus faecalis in vitro and in an experimental foreign-body infection model, Antimicrob Agents Chemother, 55 (2011) 4821-4827. [5] L. Giardino, E. Ambu, E. Savoldi, R. Rimondini, C. Cassanelli, E.A. Debbia, Comparative evaluation of antimicrobial efficacy of sodium hypochlorite, MTAD, and Tetraclean against Enterococcus faecalis biofilm, J Endod. 33 (2007) 852-855. [6] D.R. Violich, N.P. Chandler, The smear layer in endodontics - a review, Int Endod , J. 43 (2010) 2-15. [7] L.L. Narayanan, C. Vaishnavi, Endodontic microbiology, J Conserv Dent. 13 (2010) 233-239. [8] N. Chiniforush, M. Pourhajibagher, S. Shahabi, A. Bahador, Clinical Approach of High Technology Techniques for Control and Elimination of Endodontic Microbiota, J Lasers Med Sci. 6 (2015) 139-50. [9] Y. Liu, L. Guo, Y. Li, X. Guo, B. Wang, L. Wu, In vitro comparison of antimicrobial effectiveness of QMix and other final irrigants in human root canals, Sci Rep. 5 (2015) 1-6. [10] A.V. Keenan, No evidence favouring one irrigant versus another in root canal Treatments, Evid Based Dent. 13 (2012) 107.
[11] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biofilms. Lancet 358 (2001) 135-138. [12] A. Reinhard, W.J. Sandborn, H. Melhem, L. Bolotine, M. Chamaillard, L. Peyrin-Biroulet, Photodynamic therapy as a new treatment modality for inflammatory and infectious conditions, Expert Rev Clin Immunol. 11 (2015) 637-657. [13] L. López-Jiménez, E. Fusté, B. Martínez-Garriga, J. Arnabat-Domínguez, T. Vinuesa, M. Viñas. Effects of photodynamic therapy on Enterococcus faecalis biofilms, Lasers Med Sci, 30 (2015) 1519-1526. [14] C. Serrano, N. Torres, C. Valdivieso, C. Castano, M. Barrera, A. Ca-brales, Antibiotic resistance of periodontal pathogens obtained from frequent antibiotic users, Acta Odontol Latinoam. 22 (2009) 99-104. [15] M.R. Cole, J.A. Hobden, I.M. Warner, Recycling antibiotics into gumbos: A new combination strategy to combat multi-drug- resistant bacteria, Molecules 20 (2015) 6466-6487. [16] R. Dosselli, R. Millioni, L. Puricelli, P. Tessari, G. Arrigoni, C. Franchin, et al., Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach, J Proteomics. 77 (2012) 329-343. [17] Y.H. Li, M.N. Hanna, G. Svensäter, R.P. Ellen, D.G. Cvitkovitch, Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms, J Bacteriol. 183 (2001) 6875-6884. [18] Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically.: Approved Standard. Clinical and Laboratory Standards Institute Wayne, PA (2015). [19] Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. Clinical and Laboratory Standards Institute Wayne, PA (2015). [20] Clinical laboratory testing and in vitro diagnostic test systems- Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices- Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. ISO/FDIS. 20776-1, International Organization for Standardization (2006). [21] S. Butun, and K. Aydin, Structurally tunable absorption bands using ultrathin broadband plasmonic absorbers, Optics Express. 22 (2014) 19547-19568. [22] L. Misba, S. Kulshrestha, A.U. Khan, Antibiofilm action of a toluidine blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy, Biofouling. 32 (2016) 313-328. [23] D.C. Coraça-Hubér, M. Fille, J. Hausdorfer, K. Pfaller, M. Nogler, Evaluation of MBEC™-HTP biofilm model for studies of implant associated infections, J Orthop Res. 30 (2012) 1176-1180.
[24] G. Ramage, S.P. Saville, B.L. Wickes, J.L. López-Ribot, Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule, Appl Environ Microbiol. 68 (2002) 5459-5463. [25] M.A. Nordin, F. Abdul Razak, W.H. Himratul-Aznita, Assessment of Antifungal Activity of Bakuchiol on Oral-Associated Candida spp, Evid Based Complement Alternat Med. 2015 (2015) 1-7. [26] P.S. Stewart, Antimicrobial Tolerance in Biofilms, Microbiol Spectr. 3 (2015) 1-30. [27] Z. Mohammadi, M.K. Soltani, S. Shalavi, An update on the management of endodontic biofilms using root canal irrigants and medicaments, Iran Endod J. 9 (2014) 89-97. [28] T.W. Wong, Y.Y. Wang, H.M. Sheu, Y.C. Chuang, Bactericidal effects of toluidine blue-mediated photodynamic action on Vibrio vulnificus, Antimicrob Agents Chemother. 49 (2005) 895-902. [29] N. Kömerik, M. Wilson, S. Poole, The effect of photodynamic action on two virulence factors of gramnegative bacteria, Photochem Photobiol. 72 (2000) 676-680. [30] S. Tubby, M. Wilson, S.P. Nair, Inactivation of staphylococcal virulence factors using a light-activated antimicrobial agent, BMC Microbiol. 9 (2009) 1-10. [31] I.T. Kato, R.A. Prates, C.P. Sabino, B.B. Fuchs, G.P. Tegos, E. Mylonakis, et al., Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and Reduces In Vivo Pathogenicity, Antimicrob Agents Chemother. 57 (2013) 445-451. [32] F. Cieplik, L. Tabenski, W. Buchalla, T. Maisch, Antimicrobial photodynamic therapy for inactivation of biofilms formed by oral key pathogens, Front Microbiol. 5 (2014) 1-17.. [33] A. Xhevdet, D. Stubljar, I. Kriznar, T. Jukic, M. Skvarc, P. Veranic, et al., The disinfecting efficacy of root canals with laser photodynamic therapy, J Lasers Med Sci. 5 (2014) 19-26. [34] S. George, M.R. Hamblin, A. Kishen, Uptake pathways of anionic and cationic photosensitizers into bacteria, Photochem Photobiol Sci. 8 (2009) 788-795. [35] E. Banin, , K. M. Brady, E. P. Greenberg, Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm, Appl. Environ. Microbiol. 72 (2006) 2064-2069. [36] S. L.Percival, , P. Kite, K. Eastwood, R. Murga, J. Carr, M. J. Arduino, et al., Tetrasodium EDTA as a novel central venous catheter lock solution against biofilm, Infect. Control Hosp. Epidemiol. 26 (2005) 515-519. [37] M. Sharma, L. Visai, F. Bragheri, I. Cristiani, P.K. Gupta, P. Speziale, Toluidineblue-mediated photodynamic effects on staphylococcal biofilms, Antimicrob Agents Chemother. 52 (2008) 299-305. [38] O.E. Petrova, K. Sauer, Escaping the biofilm in more than one way: desorption, detachment or dispersion, Curr Opin Microbiol. 30 (2016) 67-78. [39] D. Vesić, C.J. Kristich, A Rex family transcriptional repressor influences H2O2 accumulation by Enterococcus faecalis, J Bacteriol. 195 (2013) 1815-1824. [40] J.A. Shapiro. Thinking about bacterial populations as multicellular organisms, Annu Rev Microbiol. 52 (1998) 81-104. [41] J.L. Dale, J. Cagnazzo, C.Q. Phan, A.M. Barnes, G.M. Dunny. Multiple roles for Enterococcus faecalis glycosyltransferases in biofilm-associated antibiotic resistance, cell envelope integrity, and conjugative transfer. Antimicrob Agents Chemother, 59 (2015) 4094-4105. [42] F. Barra, E. Roscetto, A.A. Soriano, A. Vollaro, I. Postiglione, G.M. Pierantoni, et al., Photodynamic and Antibiotic Therapy in Combination to Fight Biofilms and Resistant Surface Bacterial Infections, Int J Mol Sci. 16 (2015) 20417-20430. [43] A.P. Castano, T.N. Demidova, M.R. Hamblin, Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death, Photodiagnosis Photodyn Ther. 2 (2005) 1-23. [44] A.S. Garcez, S.C. Núñez, N. Jr. Azambuja, E.R. Fregnani, H.M. Rodriguez, M.R. Hamblin, et al., Effects of photodynamic therapy on Gram-positive and Gram-negative bacterial biofilms by bioluminescence imaging and scanning electron microscopic analysis, Photomed Laser Surg. 31 (2013) 519-525.
[45] E.L. Nussbaum, L. Lilge, T. Mazzulli, Effects of low-level laser therapy (LLLT) of 810nm upon in vitro growth of bacteria: relevance of irradiance and radiant exposure, J Clin Laser Med Surg. 21 (2003) 283-290.
Figure Captions
o PSs and sDL irradiaation time: (a) MB; (b b) TBO; (cc) ICG; (d) Energy Figure 1. MICs of w 660 nm ; (ee) Energy density d of DL D at wavellength 635 nm; n and density of DL at wavelength y of DL at wavelengthh 810 nm. *, P < 0.05; **, P < 0.01; signiificantly (f) Energy density c (no treatment). differennt from the control
Figure 2. Effect of o sPDT on E. faecaliss CFU/mL: (a) MB-sP PDT; (b) TB BO-sPDT; (c) ( ICGsPDT. **Significanttly differentt from the control, c P <00.05.
Figure 3. Metabolic activity of o treated pllanktonic E.. faecalis ceells by XTT T assay. *, P < 0.05; **, P < 0.01; signifficantly diffferent from the control (no treatmeent).
Figure 4. Effect of o sPDT onn E. faecallis biofilm architecturee. SEM imaages of (a) control biofilm; (b) MB with light; (c)) TBO withh light; and (d) ( ICG witth light.
Table 1. Biofilm formation of treated planktonic E. faecalis cells with TBO-, MB-, and ICGsPDT. *Significantly different from the control; P <0.05. Treatment
MIC
None (Control)
-
12.5 µg/mL at 3 min
TBO
of the MIC -
ICG
12.5 µg/mL at 3 min
Biofilm Inhibition (%) Induction (%)
P value
2.45 ± 0.21
-
‐
-
1/2 1/4 1/8 1/16 1/32 1/64
1.89 ± 0.23 1.99 ± 0.17 2.11 ± 0.26 2.66 ± 0.20 2.80 ± 0.21 3.01 ± 0.14
22.6 18.7 13.8 -
8.5 14.4 19.1
0.02 0.04 0.04 0.06 0.03 0.02
1.97 ± 0.21 2.05 ± 0.13 2.17 ± 0.18 2.31 ± 0.13 2.42 ± 0.18 2.44 ± 0.14
19.5 15.1
-
1/2 1/4 1/8 1/16 1/32 1/64
-
11.4 12.7 15.8 17.2
0.02 0.04 0.04 0.04 0.03 0.03
1/2 1/4 1/8 1/16 1/32 1/64
1.40 ± 0.23 1.66 ± 0.18 2.00 ± 0.15 2.15 ± 0.18 2.31 ± 0.20 2.40 ± 0.19
42.8 32.2 18.3 12.2 -
MB
OD570 ± SD
62.5 µg/mL at 0.5 min
-
0.01 0.02 0.04 0.04 0.29 0.04
5.7 10.4
S1572-1000(16)30058-8 http://dx.doi.org/doi:10.1016/j.pdpdt.2016.06.003 PDPDT 789
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Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
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Please cite this article as: Pourhajibagher M, Chiniforush N, Shahabi S, Ghorbanzadeh R, Bahador A.Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2016.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis
Pourhajibagher M1a, Chiniforush N2a, Shahabi S3, Ghorbanzadeh R4, and Bahador A5,2,1* 1
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Laser Research Center of Dentistry, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 3 Dental biomaterials Department, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran. 4 Private practice, Tehran, Iran. 5 Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 2
*Correspondence Address: Abbas Bahador, Ph.D., Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Keshavarz Blvd, 100 Poursina Ave., Tehran, Iran. 14167-53955. Tel.: +9821 6405 3210; Fax: +98218895 5810. E-mail: [email protected]; alternate address: [email protected]. a
Equal contribution.
Running title: Antimetabolic and antibiofilm potential of sPDT against E. faecalis
Highlights: 1) Higher doses of sPDT reduced biofilm formation and metabolic activity of E. faecalis. 2) Lower doses of sPDT increased biofilm formation and metabolic activity of E. faecalis. 3) ICG-sPDT showed a higher antibiofilm and antimetabolic activity than MB- and TBO-sPDT.
Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis
Abstract: Background: During photodynamic therapy (PDT) in the treatment of a primary endodontic infection, it is extremely likely that microorganisms would be exposed to sub-lethal doses of PDT (sPDT). Although sPDT cannot kill microorganisms, it can considerably influence microbial virulence. This study was conducted to characterize the effect of sPDT using toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) on biofilm formation ability and metabolic activity of Enterococcus faecalis. Methods: The antimetabolic and antibiofilm potential of ICG-, TBO-, and MB-sPDT against E. faecalis was analyzed at sub-lethal doses (1/2–1/64 minimum inhibitory concentration) using the XTT reduction assay, crystal violet assay, and scanning electron microscopy. Results: Higher doses of sPDT adversely affected biofilm formation ability and metabolic activity. ICG-, TBO-, and MB-PDT at a maximum sub-lethal dose markedly reduced the formation of biofilm up to 42.8%, 22.6%, and 19.5%, respectively. ICG-, TBO-, and MBsPDT showed a marked reduction in bacterial metabolic activity by 98%, 94%, and 82%, respectively. ICG-PDT showed a stronger inhibitory effect on biofilm formation in E. faecalis than MB- and TBO-PDT at sub-lethal levels. Interestingly, a gradual increase in metabolic activity and biofilm formation upon exposure to a lower dose of test sPDT were observed. Conclusion: sPDT showed dual effect on biofilm formation ability and metabolic activity of E. faecalis. High doses revealed antimetabolic and antibiofilm potential activity, whereas lower doses had conflicting results. Hence, when PDT is prescribed in clinical settings, the dose of PDT used in vivo should be taken into consideration. Keywords: Enterococcus faecalis, biofilm, photodynamic therapy, indocyanine green, toluidine blue O, methylene blue, root canal 1. Introduction Enterococcus faecalis, a facultative anaerobic gram-positive coccus, has an important role in persistent/secondary infection of endodontically treated teeth due to biofilm-associated resistance to common endodontic irrigants [1-3]. Biofilm formation in the dentinal tubules and lateral canals of teeth is considered the major virulence factor of E. faecalis contributing to endodontic infections. Biofilm bacteria are up to 1,000-fold more resistant to antimicrobial agents than their planktonic counterparts [4]. Elimination of microbial biofilms and effective killing of microorganisms biofilm are the primary aims in the management of endodontic infections [5]. Although chemomechanical debridement plays a key role in the reduction of microbial biofilm in the infected root canal, because of the complex root canal anatomy, as well as sometimes coverage of the root dentinal walls with smear layer and blocking the lateral canals, some of the instrumented root canal area is left untouched [6]. Hence, microorganisms, endotoxins, and other microbial products result in treatment failure [7]. Antimicrobial chemical agents such as sodium hypochlorite, chlorhexidine, and MTAD, which is a mixture of doxycycline, citric acid, and Tween 80, are used as root canal irrigants [8, 9]. Although root canal irrigants with broad antimicrobial activity and the ability to prevent smear layer formation appear to be effective at reducing microorganisms load, most studies reported insufficient and unreliable evidence showing the superiority of any
irrigants [8-10]. In addition, microorganisms in mature biofilm can resist the action of antimicrobial irrigants [11]. Recently, antimicrobial photodynamic therapy (PDT) was introduced as an innovative and relatively new approach in endodontic treatment. PDT is mediated through the exposure of photosensitizers (PSs) with light at a specific wavelength to produce reactive oxygen species (ROS) that can directly damage sub-cellular components [8]. The successful outcome of PDT depends on the type and concentration of PS, exposure time of the microorganisms with PS, and dose of light irradiation [12]. If PDT was used in the treatment of primary endodontic infection, it is extremely likely that PS would reach the target site at sub-lethal concentrations and may subsequently be activated by light at sub-lethal doses. As a result, any microorganism, such as E. faecalis, not eradicated and remaining viable at the site of infection would be exposed to doses of PDT that would not result in cell death, i.e., sub-lethal doses of PDT (sPDT), exposing survivors to ROS stress. ROS has been recognized to play an active and important role in processes such as cell death and biofilm/colony development in bacteria [13]. Previous studies have been shown that the exposure of bacteria to sPDT could lead to the extension of resistance to antimicrobial agents and result in an increased risk of poor clinical outcomes, and more consumption of medical resources [14, 15]. On the other hand, sPDT treatment affected the expression of proteins involved in the metabolic activity of bacteria [16]. To the best of our knowledge, there is no report on the effects of PDT treatment on the biofilm formation ability and metabolic activity of E. faecalis. In the present study, we evaluated whether E. faecalis cells exposed to indocyanine green (ICG)-, toluidine blue (TBO)-, and methylene blue (MB)sPDT exhibited changes in metabolic activity and biofilm formation. 2. Materials and Methods 2.1. Bacterial strain and culture conditions E. faecalis strain ATCC 29212 was obtained from the Iranian Biological Resource Center (Tehran, Iran). The organism was aerobically grown in fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37°C until logarithmic growth phase (4–5 h old) to a final concentration of 1.0 × 106 colony forming units (CFU)/mL, which was verified by spectrophotometry (optical density [OD]600: 0.2) [17]. 2.2 PSs and light sources Stock solutions of TBO and MB (Sigma-Aldrich, Steinheim, Germany) at 0.2 mg/mL were prepared in sterile 0.9% (wt/vol) NaCl. ICG stock solution was prepared by dissolving one ICG tablet (Emundo®, A.R.C. laser GmbH, Nurn-berg, Germany) in 1.0 mL sterile distilled water (2.0 mg/mL) and kept under dark conditions before use. Diodes laser (DL) (Konftec, Taiwan) at wavelengths of 660 nm with output power of 150 mW and 635 nm with output power of 220 mW were used for MB and TBO, respectively. A DL at a wavelength of 810 nm (A.R.C.laser GmbH, Nurnberg, Germany) with an output of 200 mW was used for ICG. The output powers of all wavelengths were measured by a power meter (Laser Point s.r.l, Milano, Italy). 2.3. Determination of minimum inhibitory concentrations (MICs) of PSs The MICs of TBO, MB, and ICG against E. faecalis were determined by broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI) and International Organization for Standardization (ISO) [18-20]. The MIC was determined as the lowest concentration of PSs in which visible bacterial growth was significantly inhibited. In this method, a single 96-well round-bottomed sterile polystyrene microplates (TPP; Trasadingen, Switzerland) was used for a PS. Each PS was tested eight times (rows A–H). Susceptibility panel in the microplates were prepared by pipetting 100 μL of 2× Cation-Adjusted Mueller Hinton broth (CAMHB; Himedia, India) to each well; 100 μL
of PSs solutions (2 mg/mL ICG, 0.2 mg/mL TBO, and 0.2 mg/mL MB) were added to the wells in column 1 (far left of plate) and the PSs concentration was diluted to 1:2 (i.e., 1 mg/mL ICG, 0.1 mg/mL TBO, and 0.1 mg/mL MB. PSs were diluted two-fold by transferring 100 µL aliquots from column 1 to column 2. Therefore, column 2 is a two-fold dilution of column 1 (i.e., 0.5 mg/mL ICG, 0.05 mg/mL TBO, and 0.05 mg/mL MB). The procedure was repeated down across the microplate to column 10 and then 100 μL was discarded from column 10 rather than dispensing it into column 11. Starting from column 11 to column 1, the columns were inoculated with fresh CAMHB bacterial cultures (100 μL/well) and adjusted to a concentration of 1.0 × 106 CFU/mL using multi-channel pipet to prevent PSs carry-over. The final bacterial cell concentration in the wells was 5 × 105 CFU/mL. The concentrations of PSs were in range of 1 mg/mL to 1.9 μg/mL for ICG and 0.1 mg/mL to 0.19 μg/mL for both TBO and MB after inoculation from column 1 to column 10. Column 11 contained the positive (growth) control and column 12 was not inoculated and served as the sterility control. Purity was checked by transferring and spreading 10 µL from the positive growth control well to a non-selective, enriched nutrient agar (e.g., blood agar plates). To ensure that a proper dilution was made and that the inoculum contained 2–8 × 105 CFU/mL (the acceptable range), 10 µL from the positive growth control well was transferred to 10 mL saline (1:1,000 dilution). The suspension was mixed and 100 µL of suspension was transferred to a non-selective agar medium (e.g., trypticase soy agar) and spread over the entire agar surface with a sterile spreader (1:10 dilution). The microplates (MIC panels), colony count plates, and purity plates were incubated for 24 h at 37°C. The MICs of PSs were defined as that concentration of PS that significantly inhibited visible bacterial growth. Colony count was conducted using a colony counter (Model CC-1, Merk Boeco, Germany). Each colony was multiplied by the dilution factor (1:10,000). For the purity plates, the inoculum can be considered pure if all the colonies were similar to the colonies used in the original plate. If there were any other colony present, contamination may have occurred in the MIC panel, and thus the assay was repeated. 2.4. Determination of sub-lethal DL irradiation time To find out the sub-lethal DL (sDL) irradiation time against E. faecalis, wells containing 200 µL of bacterial suspensions at a final concentration of 2.5 × 105 CFU/mL were exposed to different DL irradiation times at wavelengths of 660 and 635 nm (1, 2, 3, 4, 5, and 6 min with 23.43, 46.87, 70.31, 93.75, 117.18, and 140.62 J/cm2 and 34.37, 68.75, 103.12, 137.5, 171.87 and 206.25 J/cm2, respectively and 810 nm (0.5, 1, and 2 min with 15.62, 31.25, and 62.5 J/cm2, respectively) at room temperature (25 ± 2°C). To prevent transmission of light to neighboring wells, 15 wells of each microplate, with 2-well distance between them, were selected for testing and the peripheral wells were filled with MB. The probes of DL were fixed 1 mm above the surface of the microplates by a microphone stand. To prevent beam reflection from the table top during DL irradiation, sheets of black paper that have zero and ten percent light absorption and reflection, respectively, were used under the microplates at wavelengths between 400–1,000 nm [21].The diameter of the irradiated area was the same as the well diameter at the bottom of the microplate (6.39 mm). sDLs were defined when the DL irradiation times in the last well showed growth. 2.5. Determination of sPDT To determine sPDT, 100 μL of PSs at 2 × MIC were serially diluted two-folds in flatbottom 96-well microtiter plates (TPP; Trasadingen, Switzerland) to 1/64 MIC in 2 × CAMHB according to the methods mentioned above in section 2.3. The wells were then inoculated with fresh CAMHB bacterial cultures (100 μL/well) that were adjusted to a
concentration of 1.0 × 106 CFU/mL. The final bacterial cell concentration in the wells was 5 × 105 CFU/mL. After inoculation, the concentrations of PSs were in range of 1/2–1/64 MIC from column 1 to column 6. The microplates were incubated for 5 min in the dark at room temperature. The treated bacterial suspensions in the wells were immediately exposed to DL with different exposure times as mentioned above. sPDT was defined as the lowest concentration of PS with the shortest irradiation time of DL in the last well showing growth. The control group did not have any treatment and all experiments were performed in triplicate. 2.6. Effects of sPDT treatments on biofilm formation ability of E. faecalis Using the crystal violet assay, sPDTs based on TBO, MB, and ICG were tested for their potential to prevent E. faecalis biofilm formation. The effects of the treatments were further evaluated by scanning electron microscopy (SEM). 2.7. Quantitation of biofilm formation ability of treated planktonic E. faecalis by crystal violet Quantitative analysis and interpretation criteria of the biofilm formation ability of E. faecalis were performed according to a previous study [22]. Briefly, 200 μL aliquots of freefloating bacteria in planktonic suspension at a final concentration of 5 × 105 CFU/mL were transferred to flat-bottomed sterile polystyrene microplates. Bacterial cells were treated with sPDT at sub-lethal doses (1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 of the MIC) as mentioned above and the plates were incubated for 24 h at 37°C to allow for biofilm formation. After incubation, the microplate contents were emptied out from each well and washed three times with phosphate-buffered saline (PBS) (10 mM Na2HPO4, 2 mM NaH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) to remove free-floating planktonic bacteria. The cells in the biofilm were stained with 200 μL of 0.1% (wt/vol) crystal violet solution at room temperature for 15 min. After washing twice with PBS, 100 μL of 95% ethanol was added to each well and the plates were incubated at room temperature for 10 min to fix the cells. Afterward, the wells were rinsed three times with PBS and air dried. To quantify the biofilms, 150 μL of 33% (v/v) acetic acid was poured in each wells and the absorbance was determined at 570 nm using a microplate reader (Thermo Fisher Scientific, US). Based on the OD of the treated biofilm and that of the negative control (ODc), the samples were classified as follows: strong (4× ODc < OD), moderate (2× ODc < OD ≤ 4xODc), weak (ODc < OD ≤ 2× ODc), or non-producer of biofilm (OD ≤ ODc). 2.8. Visualization of treated and untreated E. faecalis biofilm with SEM Biofilm formation was initiated on the MBEC™ high-throughput (HTP) plates (Innovotech, Alberta, Canada) as previously described [23]. Briefly, after each treatment, 200 μL of bacterial suspension was grown on the pegs of the HTP plates containing BHI broth supplemented with 0.1% glucose at 37°C for 48 h according to the manufacturer’s recommendations. SEM was performed to observe the morphology and the biofilm formation ability of treated E. faecalis as previously described [24]. Briefly, following incubation of HTP plates, the pegs were removed using a flamed plier and rinsed with PBS for 1 min to remove any loosely attached and floating cells. The pegs were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylic acid at 4°C for 24 h. After fixation, the pegs were rinsed with the same buffer for 15 min, immersed in 1% aqueous osmium tetroxide, and incubated for 60 min at room temperature (25 ± 2°C). The pegs were washed once with deionized water for 15 min, followed by gradual dehydration with ethanol, and 1.5 h of critical point drying (Bal-Tec CPD 030, the Netherlands). The pegs were then coated with gold–palladium using a sputter
coater (Bal-Tec SCD 005, the Netherlands) and examined with a scanning electron microscope (LEO, 1455 VP, Germany). 2.9. Quantitation of metabolic activity of treated planktonic E. faecalis by XTT assay We used the XTT reduction assay to assess the effect of sPDT on metabolic activity of planktonic cells, developing biofilm, and mature biofilms of E. faecalis. Metabolic activity of another parallel set of E. faecalis treated in a similar way was assessed using the XTT [2, 3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay, based on the principle that tetrazolium salt can be reduced into a colorimetric detectable water-soluble product formazan by metabolically active cells [25]. The XTT (Sigma Aldrich, Steinheim, Germany) solution (1 mg/mL) in PBS was filter sterilized with a 0.22-µm syringe filter and stored at −80°C before use. Filter-sterilized menadione (Sigma-Aldrich, Steinheim, Germany) solution (1 mM) was prepared in acetone immediately before each assay. Following each sPDT treatment under identical conditions as described in the determination of sPDT section, aliquots of XTT-menadione solution (12.5:1 v/v; 12 μL) were added to each well. The plate was incubated for 3 h in the dark at 37°C. Following incubation, the color intensity was determined by an automatic microplate reader at 490 nm. The absorbance values for the wells without the biofilms, which served as a blank, were then subtracted from the values of the tested wells to eliminate false results due to background interference. In the XTT assay, heat-killed bacteria were used as a negative control. The percentage of metabolic activity of cells was calculated using the equation [(OD cultures of non-treated E. faecalis as growth control -OD sample)/OD growth control] × 100100. 2.10. Statistical Analysis Data from the experiments were evaluated using the two-way analysis of variance (ANOVA) followed by the Tukey’s test. Significance was defined as P-values < 0.05.
3. Results 3.1. MICs of PSs The treatment of bacterial cells with up to 1,000 µg/mL ICG as well as 100 µg/mL of both TBO and MB in the dark did not completely inhibit E. faecalis growth. ICG (31.2–1,000 µg/mL), TBO, and MB (both at 6.2–100 µg/mL) significantly reduced E. faecalis growth when compared to untreated bacteria (control; P < 0.05) (Fig 1, a–c). Lower concentrations of ICG (15.6–1.9 µg/mL), TBO, and MB (both at 3.1–0.19 µg/mL) also affected E. faecalis growth, but this was not statistically significant (P > 0.05; Fig 1, a–c). The inhibitory concentrations (ICs) at which 50 and 90% of the number of primary inoculum were killed (IC50 and IC90, respectively) were as follows (μg/mL): ICG, 62.5 and 1,000, TBO, 25 and >100; and MB, 50 and >100. 3.2. sDL irradiation times Significant decreases in the cell viability after irradiation at 660, 635, and 810 nm with energy density of 93.75–140.62, 137.5–206.25, and 31.25–62.5 J/cm2, respectively were observed in a light dose-dependent manner (P < 0.05; Fig. 1, d–f). Lower dose exposure of bacterial cells to 660 nm (23.43–70.31 J/cm2), 635 nm (34.37–103.12 J/cm2), and 810 nm (15.62 J/cm2) did not exhibit significant toxicity against the bacterial cells compared to the survival rates of untreated controls (P > 0.05). The DL exposure dose required to kill 50% of the primary inoculum in 635 and 660 nm were 140.62 and 206.25 J/cm2, respectively.
3.3. sPDT ICG-mediated PDT using 62.5–1,000 µg/mL at a fluency of 15.62 J/cm2 and 31.2– 1,000 µg/mL at a fluency of 15.62 J/cm2 showed a significant dose-dependent reduction against E. faecalis growth when compared to the control group (P < 0.05; Fig 2). Treatment at 12.5–100 µg/mL of TBO and MB with irradiation at a fluency of 103.12 and 70.31 J/cm2, respectively as well as treatment at 6.2–100 µg/mL of TBO and MB with irradiation at a fluency of 137.5 and 93.75 J/cm2, respectively revealed a significant dose-dependent reduction against E. faecalis growth (P < 0.05; Fig 2). ICG- (7.8–31.2 µg/mL at a fluency of 15.62 J/cm2), TBO- and MB-sPDT (both at 1.5–6.2 µg/mL at a fluency of 103.12 and 70.31 J/cm2) did not exhibit a significant CFU/mL reduction (P > 0.05; Fig 2). Taken together, 31.2 µg/mL of ICG at a fluency of 15.62 J/cm2, 6.2 µg/mL of TBO at a fluency of 103.12 J/cm2 and 6.2 µg/mL of MB at a fluency of 70.31 J/cm2 were determined as the sPDT. 3.4. High dose of sPDT reduced E. faecalis biofilm formation ability E. faecalis ATCC 29212 produced strong biofilms. Table 1 shows the surviving count as a function of light dose of cells in suspension treated with different concentration of each PS. The effects of the PDT on the biofilm formation ability of E. faecalis varied as a function of the association between PSs and the fluence of light (Table 1). The biofilm formation ability of E. faecalis decreased gradually with increasing dose of test sPDT as shown in Table 1. The fluence of light that significantly reduced biofilm formation of E. faecalis was 15.62 J/cm2 associated with MIC (concentration) – 1/16 MIC (62.5–3.9 µg/mL) of ICG. Our results demonstrated that 31.2 μg/mL (1/2 MIC) ICG-sPDT prevented approximately 50% of biofilm formation of E. faecalis when used at a fluency of 15.62 J/cm2, suggesting that ICG-sPDT is a potent inhibitor of strong biofilm producers. When the fluence at 103.12 J/cm2 was assessed, the use of TBO at MIC to 1/8 MIC (12.5–1.56 µg/mL) resulted in OD values that were statistically lower than the values obtained from the untreated controls (P < 0.05). Biofilm formation of E. faecalis was not significantly reduced with 1/16 MIC (0.78 µg/mL) TBO at a fluence of 103.12 J/cm2. In contrast, when the bacterial cells were irradiated at 70.31 J/cm2, the concentrations of MB (MIC to 1/4 MIC; 12.5–3.12 µg/mL) tested significantly reduced OD values when compared to the untreated group (P < 0.05). At a maximum sub-lethal dose, ICG-, TBO- and MB-sPDT significantly reduced the formation of biofilm up to 42.8, 22.6, and 19.5%, respectively. Interestingly, the reduction in biofilm formation ability of E. faecalis treated with ICG-sPDT was significantly higher than treatment with TBO-sPDT or MB-sPDT (P < 0.05). The reduction induced by TBO-sPDT on biofilm formation was approximately similar to MB-sPDT (P > 0.05). 3.5. Low dose of sPDT induced E. faecalis biofilm formation ability The crystal violet assay showed that E. faecalis biofilm formation was induced by low sub-lethal ICG-, MB- and TBO- mediated PDT (Table 1). All sPDT treatments could reduce/inhibit E. faecalis biofilm formation up to 1/4 MIC. In contrast, at 1/8–1/64 MIC, there was a proportional increase in biofilm formation depending on the PS. As shown in Table 1, there was a gradual increase in biofilm formation upon exposure to lower concentrations of MB (1/8–1/64 MIC), TBO (1/16–1/64 MIC), and ICG (1/32–1/64 MIC). 3.6. High dose of sPDT reduced E. faecalis cell metabolic activity As shown in Figure 3, sPDT affected E. faecalis metabolic activity. Treatment of bacterial cells with ICG-sPDT (62.5 μg/mL with irradiation at 15.62 J/cm2), TBO-sPDT (12.5 µg/mL with irradiation at 103.12 J/cm2), and MB-sPDT (12.5 µg/mL with irradiation at 70.31 J/cm2) showed a significant reduction in bacterial metabolic activity (98%, 89%, and 77%,
respectively; P < 0.05), which was estimated using the XTT assay. It is worth noting that ICGsPDT (1/32 MIC; 1.9 μg/mL with irradiation at 9 J/cm2), TBO-sPDT (1/8 MIC; 1.56 μg/mL with irradiation at 103.12 J/cm2), and MB-sPDT (1/8 MIC; 1.56 μg/mL with irradiation at 70.31 J/cm2) reduced metabolic activity by 50%. The data suggest that the sPDT at relatively low doses does not inhibit biofilm formation, but metabolic activity is affected. 3.7. Low dose of sPDT induced E. faecalis cell metabolic activity The XTT assay showed a gradual decrease in metabolic activity with increasing concentrations of MB-, TBO- (both at1/2–1/16 MIC), and ICG-sPDT (1/2–1/32 MIC) (Fig 3). As shown in Figure 3, E. faecalis metabolic activity was induced by lower sub-lethal MB, TBO (both at 1/32 and 1/64 MIC), and ICG-mediated PDT (1/64 MIC) at the corresponding of fluency. 3.8. Biofilm architecture visualized by SEM In the present report, a preliminary study was carried out to visualize the effect of sPDT on overall morphology of the E. faecalis in the biofilm. For this purpose, the cell morphology of the PDT treated and untreated cells were studied using SEM and results are shown in Figure 4. The untreated bacterial cells of E. faecalis retained their original cocci shape with very smooth morphology in biofilm format i.e., clusters of cells (Fig. 4a). On the other hand, PDT treated cells showed reduction in the numbers of bacterial cells as well as significant changes in morphology, especially with irregular in shape and size (Fig. 4b, c, d). A more substantial decrease in the number of cells and irregular shaped cells were observed when the biofilm was treated with ICG-sPDT. 4. Discussion The tolerance of microorganisms to antimicrobial agents can emerge from the physical and physiological status of microbial cells in the biofilms, which allows for the development of resistance [26]. In E. faecalis, a common causative agent of secondary endodontic infection, the formation of biofilms is the major virulence factor that protects the bacteria from many environmental stresses including root canal irrigants. This could result in E. faecalis developing tolerance to these substances, which is of major clinical importance [27]. There are many reasons as to why microorganisms could be exposed to sub-inhibitory concentrations of PDT during treatment for an endodontic infection. These include poor compliance, lower dosage of light, PSs concentrations below MIC, and inaccessibility to infection site. The sPDT can attenuate microbial virulence factors and their ability to confer pathogenicity, such as flagellum of Vibrio vulnificus [28]; biological activities of the lipopolysaccharides and protease activity of Pseudomonas aeruginosa [29]; V8 protease, αhaemolysin, and sphingomyelinase of Staphylococcus aureus [30]; and germ tube formation of Candida albicans [31]. The present study evaluated the anti-virulence activity, anti-biofilm formation ability, and anti-metabolic activity of ICG-, TBO-, and MB-PDT against E. faecalis at sub-lethal levels because sPDT does not have bactericidal activity but has inhibitory effects on bacterial physiology. The results showed that ICG, TBO, and MB did not completely inhibit bacterial growth. In this study, ICG exhibited higher antibacterial activity than TBO and MB alone. These variations could be due to the difference in type and concentration of PS, uptake rates of ICG, laser energy fluence rate, and time of irradiation. In the present study, ICG-, TBO-, and MB-sPDT did not exert bactericidal activity (Figure 2), suggesting that all of the sPDT used did not influence the growth or survival of the bacterial cells. The parameters which used in DL groups for determination the sub-bactericidal activity was applied in PDT groups. DL groups with different wavelengths (635, 660, and
810 nm) showed reductions in the biofilm formation of E. faecalis compared with those of the control groups, which demonstrates a small bactericidal effect. This can be attributed to the phenomenon that some bacterial cells are known to synthesize endogenous porphyrins that act as PS [32]. Similar results were also observed using only laser application by Xhevdet et al. [33], who reported a reduction in E. faecalis biofilm formation. In addition, Misba et al. [22] showed divergent results in which irradiation in the absence of PS application did not result in any difference in the reduction of biofilm formation compared to the control group. To the best of our knowledge, this is the first report providing strong evidence that ICG-sPDT (42.8%) efficiently reduce the biofilm formation ability and metabolic activity of E. faecalis compared to TBO- and MB-sPDT. The latter two only reduced biofilm formation by 22.6% and 19.5%, respectively. The higher effectiveness of ICG-sPDT as compared to the TBO- and MB-sPDT may be more complex because sequestration of divalent cations, such as Ca+2 and Mg+2, by ICG (which can behave as an anion) may decrease biofilm formation and the integrity of biofilm [34]. A number of studies have shown that divalent cations can modulate the development of bacterial biofilm [35, 36]. Similar results have been reported for cation chelators, such as disodium and tetrasodium ethylenedinitrilotetraacetic acid (TEDTA), which sequestrates divalent cations [37]. In bacteria, biofilm formation is associated with the activation of quorum-sensing signals in response to environmental stress factors [38]. Our data suggest that E. faecalis biofilm formation can be induced by conditions that are potentially toxic for the bacterial cell (i.e., exposure to ROS from PDT). In addition, our data confirm previous observations on biofilm activation by oxidative stress [39]. Current studies imply that the biofilm development is a crucial mechanism for withstanding hostile environmental conditions [40]. In this respect, bacteria in biofilms, including E. faecalis, are characterized by an increased inherent antimicrobial resistance [41]. In this study, we analyzed the influence of sPDT on E. faecalis biofilm formation. Our results indicate that the biofilm formation can be strongly enhanced by lower sub-lethal ICG-, MB- and TBO-mediated PDT. Biofilm formation was inhibited when the PDT dosage was increased to higher levels (>1/4 of the sub-lethal). To the best of our knowledge, no data on the potential increase in E. faecalis biofilm formation as a result of using sPDT have been reported. Nevertheless, the results presented here emphasize that PDT should be used at the correct dosage because low sub-lethal dosage could increase biofilm formation. The XTT assay indicated that metabolic activity was reduced in bacterial cell (Fig. 3) with the greatest reduction being that for ICG-sPDT (98%). This may be due to the inhibitory action of ICG-sPDT conjugates against quorum sensing, extracellular polymeric substance production, and virulence traits [42], which can affect biofilm formation by reducing metabolic activity. This consequently inhibits the development of a secondary endodontic infection. However, reduction in biofilm formation did not decrease at the same rate as metabolic activity, suggesting that viable cells remaining in the biofilm under stressful conditions decreased their metabolic activity. Therefore, it is likely that the decrease in metabolic activity after sPDT may be due to a ROS stress response, which acts on the cell membrane and causes loss of membrane potential and reduction in intracellular Adenosine triphosphate [43]. The effect of ICG-sPDT, TBO-, and MB-sPDT on metabolic activity may indirectly interfere with the ability of the bacteria to adhere and form biofilms as shown in the present study and other studies [44]. The present study demonstrates an effect of sPDT on cellular metabolic activity of E. faecalis. However, further studies are required to see how these two
effects are related. It is important to study how the effects of sPDT on metabolic activity of E. faecalis biofilm affect the pathogenicity of the bacteria in secondary endodontic infections. This study shows that the metabolic activity can be induced by low doses of sPDT and confirms previous observations on metabolic activity by low reactive level laser therapy [45]. Our results indicate that the metabolic activity can be enhanced by the lower sub-lethal ICG-, MB- and TBO-mediated PDT. Currently, no data on the potential increase of metabolic activity in E. faecalis as a result of using sPDT have been reported. To visualize the effect of sPDT on E. faecalis biofilm formation, SEM studies were conducted. High cell density in large cellular aggregates was observed in the control group, whereas the cell density after sPDT was reduced. This indicates not only the loss of cells in the biofilm but also significant changes in morphology, including irregular biofilm shapes and sizes. This may be due to the loss of physical contact between the cells, which can lead to cell death within the biofilm [37]. These observations were concomitant with the study conducted by López-Jiménez et al. [13] which showed the susceptibility of E. faecalis to PDT. They suggested that the antibacterial activity is due to changes in membrane permeability, cell division machinery, and inhibition of bacterial cell wall synthesis. However, further investigations are required to identify the molecular mechanisms involved in the reduction of metabolic activity and biofilm formation by sPDT in E. faecalis. 5. Conclusion Our results demonstrate that exposure of E. faecalis to all three sPDT treatments (1/2– 1/4 MIC MB-PDT, 1/2–1/8 MIC TBO-PDT, and 1/2–1/16 MIC ICG-PDT) adversely affect biofilm formation and metabolic activity in a dose-dependent manner. Interestingly, ICGPDT showed a stronger inhibitory effect on biofilm formation and metabolic activity in E. faecalis than MB- and TBO-PDT at sub-lethal levels. The reduction in biofilm formation and metabolic activity following sPDT, specifically ICG-sPDT, suggests that therapies based on the optimal dosage of PDT could be a useful approach for the eradication E. faecalis infections and is crucial to prevent secondary endodontic infection. Lower sPDT treatment (≤1/8-, ≤1/16-, and ≤1/32-MIC for MB-, TBO- and ICG-PDT, respectively) may provide protect E. faecalis cells by inducing biofilm formation. Our in vitro results indicate that when PDT is prescribed for patients with primary endodontic infection, the concentration of PS and output power of light (fluency) used in vivo as well as the susceptibility of E. faecalis against PDT should be taken into consideration. Acknowledgement This research has been supported by Tehran University of Medical Sciences & health Services grant No. 94-04-30-27681. Disclosure Statement No competing financial interests exist. References [1] G.C. Rezende, L. Massunari, I.O. Queiroz, J.E. Gomes Filho, R.C. Jacinto, C.S. Lodi, et al., Antimicrobial action of calcium hydroxide-based endodontic sealers after setting, against E. faecalis biofilm, Braz Oral Res. 30 (2016) 30:e38. [2] M.E. Łysakowska, A. Ciebiada-Adamiec, M. Sienkiewicz, J. Sokołowski, K. Banaszek, The cultivable microbiota of primary and secondary infected root canals, their susceptibility to antibiotics and association with the signs and symptoms of infection. Int Endod J. (2015) 1-9.
[3] L.E. hávez de Paz, G. Bergenholtz, G. Svensäter, The effects of antimicrobials on endodontic biofilm bacteria, J Endod. 36 (2010) 70-77. [4] U. Furustrand Tafin, I. Majic, C. Zalila Belkhodja, B. Betrisey, S. Corvec, W. Zimmerli, et al., Gentamicin improves the activities of daptomycin and vancomycin against Enterococcus faecalis in vitro and in an experimental foreign-body infection model, Antimicrob Agents Chemother, 55 (2011) 4821-4827. [5] L. Giardino, E. Ambu, E. Savoldi, R. Rimondini, C. Cassanelli, E.A. Debbia, Comparative evaluation of antimicrobial efficacy of sodium hypochlorite, MTAD, and Tetraclean against Enterococcus faecalis biofilm, J Endod. 33 (2007) 852-855. [6] D.R. Violich, N.P. Chandler, The smear layer in endodontics - a review, Int Endod , J. 43 (2010) 2-15. [7] L.L. Narayanan, C. Vaishnavi, Endodontic microbiology, J Conserv Dent. 13 (2010) 233-239. [8] N. Chiniforush, M. Pourhajibagher, S. Shahabi, A. Bahador, Clinical Approach of High Technology Techniques for Control and Elimination of Endodontic Microbiota, J Lasers Med Sci. 6 (2015) 139-50. [9] Y. Liu, L. Guo, Y. Li, X. Guo, B. Wang, L. Wu, In vitro comparison of antimicrobial effectiveness of QMix and other final irrigants in human root canals, Sci Rep. 5 (2015) 1-6. [10] A.V. Keenan, No evidence favouring one irrigant versus another in root canal Treatments, Evid Based Dent. 13 (2012) 107.
[11] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biofilms. Lancet 358 (2001) 135-138. [12] A. Reinhard, W.J. Sandborn, H. Melhem, L. Bolotine, M. Chamaillard, L. Peyrin-Biroulet, Photodynamic therapy as a new treatment modality for inflammatory and infectious conditions, Expert Rev Clin Immunol. 11 (2015) 637-657. [13] L. López-Jiménez, E. Fusté, B. Martínez-Garriga, J. Arnabat-Domínguez, T. Vinuesa, M. Viñas. Effects of photodynamic therapy on Enterococcus faecalis biofilms, Lasers Med Sci, 30 (2015) 1519-1526. [14] C. Serrano, N. Torres, C. Valdivieso, C. Castano, M. Barrera, A. Ca-brales, Antibiotic resistance of periodontal pathogens obtained from frequent antibiotic users, Acta Odontol Latinoam. 22 (2009) 99-104. [15] M.R. Cole, J.A. Hobden, I.M. Warner, Recycling antibiotics into gumbos: A new combination strategy to combat multi-drug- resistant bacteria, Molecules 20 (2015) 6466-6487. [16] R. Dosselli, R. Millioni, L. Puricelli, P. Tessari, G. Arrigoni, C. Franchin, et al., Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach, J Proteomics. 77 (2012) 329-343. [17] Y.H. Li, M.N. Hanna, G. Svensäter, R.P. Ellen, D.G. Cvitkovitch, Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms, J Bacteriol. 183 (2001) 6875-6884. [18] Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically.: Approved Standard. Clinical and Laboratory Standards Institute Wayne, PA (2015). [19] Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. Clinical and Laboratory Standards Institute Wayne, PA (2015). [20] Clinical laboratory testing and in vitro diagnostic test systems- Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices- Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. ISO/FDIS. 20776-1, International Organization for Standardization (2006). [21] S. Butun, and K. Aydin, Structurally tunable absorption bands using ultrathin broadband plasmonic absorbers, Optics Express. 22 (2014) 19547-19568. [22] L. Misba, S. Kulshrestha, A.U. Khan, Antibiofilm action of a toluidine blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy, Biofouling. 32 (2016) 313-328. [23] D.C. Coraça-Hubér, M. Fille, J. Hausdorfer, K. Pfaller, M. Nogler, Evaluation of MBEC™-HTP biofilm model for studies of implant associated infections, J Orthop Res. 30 (2012) 1176-1180.
[24] G. Ramage, S.P. Saville, B.L. Wickes, J.L. López-Ribot, Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule, Appl Environ Microbiol. 68 (2002) 5459-5463. [25] M.A. Nordin, F. Abdul Razak, W.H. Himratul-Aznita, Assessment of Antifungal Activity of Bakuchiol on Oral-Associated Candida spp, Evid Based Complement Alternat Med. 2015 (2015) 1-7. [26] P.S. Stewart, Antimicrobial Tolerance in Biofilms, Microbiol Spectr. 3 (2015) 1-30. [27] Z. Mohammadi, M.K. Soltani, S. Shalavi, An update on the management of endodontic biofilms using root canal irrigants and medicaments, Iran Endod J. 9 (2014) 89-97. [28] T.W. Wong, Y.Y. Wang, H.M. Sheu, Y.C. Chuang, Bactericidal effects of toluidine blue-mediated photodynamic action on Vibrio vulnificus, Antimicrob Agents Chemother. 49 (2005) 895-902. [29] N. Kömerik, M. Wilson, S. Poole, The effect of photodynamic action on two virulence factors of gramnegative bacteria, Photochem Photobiol. 72 (2000) 676-680. [30] S. Tubby, M. Wilson, S.P. Nair, Inactivation of staphylococcal virulence factors using a light-activated antimicrobial agent, BMC Microbiol. 9 (2009) 1-10. [31] I.T. Kato, R.A. Prates, C.P. Sabino, B.B. Fuchs, G.P. Tegos, E. Mylonakis, et al., Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and Reduces In Vivo Pathogenicity, Antimicrob Agents Chemother. 57 (2013) 445-451. [32] F. Cieplik, L. Tabenski, W. Buchalla, T. Maisch, Antimicrobial photodynamic therapy for inactivation of biofilms formed by oral key pathogens, Front Microbiol. 5 (2014) 1-17.. [33] A. Xhevdet, D. Stubljar, I. Kriznar, T. Jukic, M. Skvarc, P. Veranic, et al., The disinfecting efficacy of root canals with laser photodynamic therapy, J Lasers Med Sci. 5 (2014) 19-26. [34] S. George, M.R. Hamblin, A. Kishen, Uptake pathways of anionic and cationic photosensitizers into bacteria, Photochem Photobiol Sci. 8 (2009) 788-795. [35] E. Banin, , K. M. Brady, E. P. Greenberg, Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm, Appl. Environ. Microbiol. 72 (2006) 2064-2069. [36] S. L.Percival, , P. Kite, K. Eastwood, R. Murga, J. Carr, M. J. Arduino, et al., Tetrasodium EDTA as a novel central venous catheter lock solution against biofilm, Infect. Control Hosp. Epidemiol. 26 (2005) 515-519. [37] M. Sharma, L. Visai, F. Bragheri, I. Cristiani, P.K. Gupta, P. Speziale, Toluidineblue-mediated photodynamic effects on staphylococcal biofilms, Antimicrob Agents Chemother. 52 (2008) 299-305. [38] O.E. Petrova, K. Sauer, Escaping the biofilm in more than one way: desorption, detachment or dispersion, Curr Opin Microbiol. 30 (2016) 67-78. [39] D. Vesić, C.J. Kristich, A Rex family transcriptional repressor influences H2O2 accumulation by Enterococcus faecalis, J Bacteriol. 195 (2013) 1815-1824. [40] J.A. Shapiro. Thinking about bacterial populations as multicellular organisms, Annu Rev Microbiol. 52 (1998) 81-104. [41] J.L. Dale, J. Cagnazzo, C.Q. Phan, A.M. Barnes, G.M. Dunny. Multiple roles for Enterococcus faecalis glycosyltransferases in biofilm-associated antibiotic resistance, cell envelope integrity, and conjugative transfer. Antimicrob Agents Chemother, 59 (2015) 4094-4105. [42] F. Barra, E. Roscetto, A.A. Soriano, A. Vollaro, I. Postiglione, G.M. Pierantoni, et al., Photodynamic and Antibiotic Therapy in Combination to Fight Biofilms and Resistant Surface Bacterial Infections, Int J Mol Sci. 16 (2015) 20417-20430. [43] A.P. Castano, T.N. Demidova, M.R. Hamblin, Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death, Photodiagnosis Photodyn Ther. 2 (2005) 1-23. [44] A.S. Garcez, S.C. Núñez, N. Jr. Azambuja, E.R. Fregnani, H.M. Rodriguez, M.R. Hamblin, et al., Effects of photodynamic therapy on Gram-positive and Gram-negative bacterial biofilms by bioluminescence imaging and scanning electron microscopic analysis, Photomed Laser Surg. 31 (2013) 519-525.
[45] E.L. Nussbaum, L. Lilge, T. Mazzulli, Effects of low-level laser therapy (LLLT) of 810nm upon in vitro growth of bacteria: relevance of irradiance and radiant exposure, J Clin Laser Med Surg. 21 (2003) 283-290.
Figure Captions
o PSs and sDL irradiaation time: (a) MB; (b b) TBO; (cc) ICG; (d) Energy Figure 1. MICs of w 660 nm ; (ee) Energy density d of DL D at wavellength 635 nm; n and density of DL at wavelength y of DL at wavelengthh 810 nm. *, P < 0.05; **, P < 0.01; signiificantly (f) Energy density c (no treatment). differennt from the control
Figure 2. Effect of o sPDT on E. faecaliss CFU/mL: (a) MB-sP PDT; (b) TB BO-sPDT; (c) ( ICGsPDT. **Significanttly differentt from the control, c P <00.05.
Figure 3. Metabolic activity of o treated pllanktonic E.. faecalis ceells by XTT T assay. *, P < 0.05; **, P < 0.01; signifficantly diffferent from the control (no treatmeent).
Figure 4. Effect of o sPDT onn E. faecallis biofilm architecturee. SEM imaages of (a) control biofilm; (b) MB with light; (c)) TBO withh light; and (d) ( ICG witth light.
Table 1. Biofilm formation of treated planktonic E. faecalis cells with TBO-, MB-, and ICGsPDT. *Significantly different from the control; P <0.05. Treatment
MIC
None (Control)
-
12.5 µg/mL at 3 min
TBO
of the MIC -
ICG
12.5 µg/mL at 3 min
Biofilm Inhibition (%) Induction (%)
P value
2.45 ± 0.21
-
‐
-
1/2 1/4 1/8 1/16 1/32 1/64
1.89 ± 0.23 1.99 ± 0.17 2.11 ± 0.26 2.66 ± 0.20 2.80 ± 0.21 3.01 ± 0.14
22.6 18.7 13.8 -
8.5 14.4 19.1
0.02 0.04 0.04 0.06 0.03 0.02
1.97 ± 0.21 2.05 ± 0.13 2.17 ± 0.18 2.31 ± 0.13 2.42 ± 0.18 2.44 ± 0.14
19.5 15.1
-
1/2 1/4 1/8 1/16 1/32 1/64
-
11.4 12.7 15.8 17.2
0.02 0.04 0.04 0.04 0.03 0.03
1/2 1/4 1/8 1/16 1/32 1/64
1.40 ± 0.23 1.66 ± 0.18 2.00 ± 0.15 2.15 ± 0.18 2.31 ± 0.20 2.40 ± 0.19
42.8 32.2 18.3 12.2 -
MB
OD570 ± SD
62.5 µg/mL at 0.5 min
-
0.01 0.02 0.04 0.04 0.29 0.04
5.7 10.4