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Comparison of the antimicrobial efficacy of photodynamic therapy with two mediators against Lactobacillus acidophilus in vitro
Accepted Manuscript Title: Comparison of the antimicrobial efficacy of photodynamic therapy with two mediators against Lactobacillus acidophilus in vitro Authors: Arash Azizi, Shiva Mousavian, Soudabeh Taheri, Shirin Lawaf, Elnaz Gonoudi, Arash Rahimi PII: DOI: Reference:
S1572-1000(17)30488-X https://doi.org/10.1016/j.pdpdt.2018.01.014 PDPDT 1105
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
27-10-2017 3-1-2018 22-1-2018
Please cite this article as: Azizi Arash, Mousavian Shiva, Taheri Soudabeh, Lawaf Shirin, Gonoudi Elnaz, Rahimi Arash.Comparison of the antimicrobial efficacy of photodynamic therapy with two mediators against Lactobacillus acidophilus in vitro.Photodiagnosis and Photodynamic Therapy https://doi.org/10.1016/j.pdpdt.2018.01.014 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.
Comparison of the antimicrobial efficacy of photodynamic therapy with two mediators against Lactobacillus acidophilus in vitro
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1-professor/first name:Arash/last name:Azizi/email: [email protected]./ oral medicine department. Islamic Azad University, Dental Branch, Tehran, Iran
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2-dr/first name:Shiva /last name: Mousavian/ email: [email protected]/ oral medicine specialist
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3-mrs/ first name: Soudabeh /last name: Taheri / email: [email protected] / Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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4-dr/ first name: Shirin/ last name: Lawaf/ email: [email protected] / prosthodontics department. Islamic Azad University, Dental Branch, Tehran, Iran 5 -⃰ (Corresponding)
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dr/ firstname :Elnaz / last name: Gonoudi/ email: [email protected]/ . oral medicine department. Islamic Azad University, Dental Branch, Tehran, Iran
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6-dr/ first name:Arash/ last name: Rahimi/ email: [email protected] / Biophysics Department, Islamic Azad University, Science and Research Branch, Tehran, Iran
Highlights
The separate use of MB and combined use of 660-nm laser and MB have a significant inhibitory effect on L. acidophilus growth.
Photodynamic therapy is the good way to eliminate microorganism of oral cavity. Photodynamic therapy is the conservative method to eliminate microorganism of oral cavity. Photodynamic therapy can be replace instead of antibiotic therapy.
Abstract Background and objectives: Lactobacillus is a cariogenic microorganism. Different therapeutic approaches including photodynamic therapy (PDT) have been suggested for treatment of bacterial infection. The purpose of the current study was to compare the effects of PDT with Indocyanine green (ICG) and Methylene blue (MB) photosensitizers (PSs) on Lactobacillus acidophilus (L. acidophilus).
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Materials and methods: In this in-vitro experimental study, 84 samples of L. acidophilus (1 McFarland standard) were compared in 14 experimental groups including: MB, ICG, 660-nm laser, 808-nm laser (pulsed, 74s/continuous-wave, 37s), different combinations of lasers and PSs, Chlorhexidine (CHX) 0.2%, sodium hypochlorite (NaOCl) 2.5%, penicillin 6.3.3 and control group. The samples were cultured in microplates containing blood agar culture medium. After incubation at 37°C for 48 hours, colony forming units (CFUs) of L. acidophilus were counted and compared before and after therapeutic interventions. Data were analyzed using SPSS19 software program according to one-way ANOVA and Kruskal-Wallis test.
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Results: This study showed that the separate use of ICG, 660- and 808-nm lasers (pulsed, 74s/continuous-wave, 37s), and the combined use of 808-nm laser (pulsed, 74s/continuouswave, 37s) and ICG have no significant inhibitory effect on L. acidophilus colonies (P>0.05), whereas the separate use of MB and the combined use of 660-nm laser (continuous-wave, 37s/pulsed, 74s) and MB significantly inhibited the growth of L. acidophilus in comparison with the control group (p<0.05). Likewise, CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 significantly inhibited the bacterial growth (p<0.05).
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Conclusion: The results showed that separate use of MB and combined use of 660-nm laser and MB have a significant inhibitory effect on L. acidophilus growth.
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Introduction
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Keywords: Photodynamic Therapy; Indocyanine Green; Methylene Blue; Chlorhexidine; Sodium Hypochlorite; Penicillin; Lactobacillus Acidophilus
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Lactobacilli, of the lactobacillaceae family, are gram-positive, rod-shaped, none-sporeforming and immobile bacteria. They produce energy through fermentation of sugar, which at least half of its products is comprised of lactic acid. In the oral cavity, lactic acid causes dental damage and induces dental caries [1-3]. Sodium hypochlorite (NaOCl) 2.5% and Chlorhexidine (CHX) 0.2% are antibacterial agents used regularly against different bacterial species; although they have some disadvantages, and in some cases, contradictory results have been reported after their implementation [4]. When chemical antibacterial agents are included in different preventive and therapeutic procedures, microorganisms may neutralize their action through developing resistance towards these agents; therefore, it is necessary to find alternative therapeutic approaches to substitute these chemicals [5].
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Photodynamic therapy (PDT) is a relatively novel therapeutic method based on the interaction between a safe light source and a light-sensitive substance or photosensitizer (PS) such as methylene blue (MB), toluidine blue, aluminum disulfonated phthalocyanine, chlorine derivatives and nontoxic Indocyanine green (ICG). This interaction, in the presence of oxygen, develops a special sequence of biologic events through producing different species of active oxygen, and as a result, microorganism apoptosis and death will occur [6]. Azizi et al in 2016 evaluated the effect of PDT with MB and ICG PSs on Candida albicans (C. albicans), and concluded that PDT with ICG resulted in the lowest amount of Candida albicans colonies [7]. Fekrazad and colleagues in 2017 assessed the antimicrobial effects of PDT with MB on the amount of salivary Streptococcus mutans (S. mutans) in children with severe early caries, and concluded that PDT decreases the amount of salivary S. mutans immediately after the treatment [8].
Materials and Methods
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Lactobacillus acidophilus (L. acidophilus) standard strain
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Considering the favorable effects of PDT established by previous studies, the aim of the present experiment was to determine the antimicrobial effect of PDT with 808- and 660-nm lasers mediated with MB and ICG PSs.
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Standard strain of L. acidophilus PTCC 1643 was procured from Iranian Research Organization for Science and Technology. L. acidophilus bacteria were cultured in brain heart infusion (BHI) liquid medium, and were incubated at 37°C with 10% CO2 for 48 hours. The solution holding L. acidophilus bacteria in the BHI liquid medium was diluted to 1 McFarland standard (approximately 3×108 bacteria/ml). For precise colony counting, the bacterial suspensions in the liquid medium were cultured on the surface of blood agar medium, and the number of colonies was counted after 48 hours (pre-intervention colony count) [4]. Lasers
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Diode lasers (Polaris Co., Poland) with the wavelengths of 808 nm (energy density of 9.48 J/cm2), and 660 nm (energy density of 1.88 J/cm2) were used in the present study. The diameter of the probe was 1cm for both lasers. The irradiation durations in pulsed and continuous-wave modes were 74 and 37 seconds, respectively. Duty cycle (DC) for continuous-wave mode was considered to be 50% with 100Hz frequency [9]. PSs and other antimicrobial agents
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MB 1% (Merck, Germany), which is activated at 660 nm wavelength and causes photooxidation, was used as a PS in the current study. It was prepared at the microbiology laboratory of School of Dentistry of Shahid Beheshti University of Medical Sciences. The second PS used in the current study was ICG (Arcos, New Jersey, USA), which was prepared in dimethyl sulfoxide (DMSO) solvent at the concentration of 0.01%. It triggers photooxidation at 808 nm wavelength. Other antimicrobial agents used in the present study include: CHX 0.2% mouthwash (Behsa Pharmaceuticals Co., Tehran, Iran), NaOCl 2.5% (Bojne Co., Tehran, Iran) and Penicillin 6.3.3.
The therapeutic groups (Table 1) 1- Control group 10µl of bacterial suspension was placed inside a 96-well microplate. The microplate was incubated at 37°C for 24 hours [4]. Afterwards, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted after 48 hours according to colony forming unit (CFU). 2- MB group (2 minutes)
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10µl of bacterial suspension was mixed with 100µl of MB (final concentration of 1%), and the mixture was placed inside a 96-well microplate (at the bottom of flat wells). The microplate was kept in a dark room for 2 minutes to allow adequate exposure of MB to the bacteria. Afterwards, the samples were cultured on blood agar medium, and the number of L. acidophilus bacterial colonies was counted after 48 hours (post-intervention colony count) [10]. 3- 660-nm continuous-wave laser (37 seconds)
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10µl of bacterial suspension was placed inside a 96-well microplate. Afterwards, continuouswave diode laser was irradiated with 40mW output power at 660 nm wavelength and energy density of 1.88 J/cm2 for 37 seconds. After 48 hours, the total number of L. acidophilus bacterial colonies was counted according to CFU [9].
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4- 660-nm pulsed laser (74 seconds)
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10µl of bacterial suspension was placed in a 96-well microplate. Afterwards, pulsed diode laser irradiation was performed with 40mW output power at 660 nm wavelength and energy density of 1.88 J/cm2 for 74 seconds. After 48 hours, the total number of L. acidophilus bacterial colonies was counted according to CFU [9]. 5- 660-nm continuous-wave laser (37 seconds) and MB
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10µl of bacterial suspension was mixed with 100µl of MB at the concentration of 1%. With the use of a sampler, the mixture was placed at the bottom of 6 wells of a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of MB to the bacteria. Afterwards, continuous-wave diode laser was irradiated in each well with 40mW output power at 660 nm wavelength and energy density of 1.88 J/cm2 for 37 seconds. Next, using an inoculating needle or a standard probe, the contents of each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar containing 3 to 5% CO2, and after 48 hours, the total number of L. acidophilus colonies was counted (CFU) [9]. 6- 660-nm pulsed laser (74 seconds) and MB 10µl of bacterial suspension was mixed with 100µl of MB at the concentration of 1%. With the use of a sampler, the mixture was placed at the bottom of 6 wells in a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of MB to the bacteria. Afterwards, 660-nm laser in pulsed mode was irradiated with 40mW output power and energy density of 1.88 J/cm2 for 74 seconds in each well. Next, using an inoculating needle or a standard probe, the contents of
each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar containing 3 to 5% CO2, and after 48 hours, the total number of L. acidophilus bacterial colonies (CFU) was counted [9]. 7- ICG group (2 minutes)
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10 µl of bacterial suspension was mixed with 100µl of ICG (final concentration of 0.01%), and the mixture was placed inside a 96-well microplate (at the bottom of flat wells). The microplate was kept in a dark room for 2 minutes to allow adequate exposure of ICG to the bacteria. Afterwards, the samples were cultured on blood agar medium, and the number of L. acidophilus bacteria was counted after 48 hours (post-intervention colony count) [10]. 8- 808-nm continuous-wave laser (37 seconds)
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10µl of bacterial suspension was placed in a 96-well microplate. Afterwards, continuouswave diode laser was irradiated with 200mW output power at 808 nm wavelength and energy density of 9.48 J/cm2 for 37 seconds. After 48 hours, the total number of L. acidophilus bacterial colonies (CFU) was counted [9]. 9- 808-nm pulsed laser (74 seconds)
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10µl of bacterial suspension was placed in a 96-well microplate. Afterwards, diode laser in pulsed mode was irradiated with 200mW output power at 808 nm wavelength and energy density of 9.48 J/cm2. After 48 hours, the total number of L. acidophilus colonies was counted [9].
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10- 808-nm continuous-wave laser (37 seconds) and ICG
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10µl of bacterial suspension was mixed with 100µl of ICG in DMSO at the final concentration of 0.01%. Using a sampler, the mixture was placed at the bottom of 6 wells in a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of ICG to the bacteria. Afterwards, 808nm continuous-wave diode laser was irradiated with 200mW output power and energy density of 9.48 J/cm2 for 74 seconds. Next, using an inoculating needle or a standard probe, the contents of each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar with 3 to 5% CO2, and after 48 hours, the total number of L. acidophilus colonies was counted [9]. 11- 808-nm pulsed laser (74 seconds) and ICG
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10µl of bacterial suspension was mixed with 100µl of ICG in DMSO at the final concentration of 0.01%. With the use of a sampler, the mixture was placed at the bottom of 6 wells in a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of ICG to the bacteria. Afterwards, pulsed diode laser was irradiated with 200mW output power at 808 nm wavelength and energy density of 9.48 J/cm2 for 74 seconds in each well. Next, using an inoculating needle or a standard probe, the contents of each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar containing 3 to 5% CO2. After 48 hours, the total number of L. acidophilus colonies (CFU) was counted [9]. 12- CHX 0.2%
10µl of bacterial suspension was mixed with 100µl of CHX 0.2%, and the mixture was placed in a 96-well microplate. The microplate was incubated at 37°C for 24 hours [4]. Next, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted 48 hours later. 13- NaOCl 2.5%
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10µl of bacterial suspension was mixed with 100µl of NaOCl 2.5%, and the mixture was placed in a 96-well microplate. The microplate was incubated at 37°C for 24 hours [4]. Afterwards, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted after 48 hours. 14- Penicillin 6.3.3
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10µl of bacterial suspension was mixed with 100µl of penicillin 6.3.3, and the mixture was placed in a 96-well microplate (at the bottom of flat wells). The microplate was incubated at 37°C for 24 hours. Before and after the intervention, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted 48 hours later [11].
Control
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MB
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Red laser
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Laser therapy mode and duration
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Table 1: Experimental groups
Number of strains 6 6 6
Red laser
Pulsed 74s
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Red laser and MB
Continuous-wave 37s
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Red laser and MB
Pulsed 74s
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ICG
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Continuous-wave 37s
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IR laser
Continuous-wave 37s
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IR laser
Pulsed 74s
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IR laser and ICG
Continuous-wave 37s
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11
IR laser and ICG
Pulsed 74s
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12
CHX 0.2%
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NaOCl 2.5%
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14
Penicillin
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Data collection method
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Data were statistically analyzed with SPSS19 software program. Central dispersion indices of L. acidophilus colonies after different therapeutic procedures were determined and reported accompanied by logarithmic values of colony numbers. The differences of CFUs of L. acidophilus among different groups were statistically analyzed using one-way analysis of variance (ANOVA) and Kruskal-Wallis test. Pairwise comparisons between the groups with regards to the number of CFUs of L. acidophilus were performed according to Dunn's test. The type1 error rate was considered to be 0.05 in the current experiment.
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Results
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The mean amounts of L. acidophilus bacteria in the 14 experimental groups are presented in Table 2. According to the results, the separate use of ICG, 660- and 808-nm lasers (continuous-wave, 37 seconds), 660- and 808-nm lasers (pulsed, 74 seconds), the combined use of 808-nm laser (continuous-wave, 37 seconds) and ICG and the combined application of 808-nm laser (pulsed, 74 seconds) and ICG had no significant inhibitory effect on L. acidophilus colonies (P>0.05).
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In contrast, MB 1% dye significantly decreased the number of bacterial colonies (p<0.001). Likewise, the combined use of 660-nm laser (continuous-wave, 37seconds/pulsed, 74 seconds) and MB significantly decreased the number of L. acidophilus in comparison with the control group (p<0.001). CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 also significantly inhibited the growth of L. acidophilus in comparison with the control group (p<0.001).
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Table 2: Mean amount of L. acidophilus colonies in 14 experimental groups after intervention* Number of strains
CFU
Control
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100×103>
MB
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49800±5303
660-nm continuous-wave laser (37s)
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100×103>
660-nm pulsed laser (74s)
6
100×103>
660-nm continuous-wave laser (37s) and MB
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11167±9280
660-nm pulsed laser (74s) and MB
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7167±3710
ICG
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100×103>
808-nm continuous-wave laser (37s)
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100×103>
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100×103>
808-nm continuous-wave laser (37s) and ICG
6
100×103>
808-nm pulsed laser (74s) and ICG
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100×103>
CHX 0.2%
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NaOCl 2.5%
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Penicillin
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*The average number of lactobacilli was equal to 106 before intervention.
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808-nm pulsed laser (74s)
As shown in Figure 1, the combined use of 660-nm laser (continuous-wave, 37 seconds/pulsed, 74 seconds) and MB dye significantly inhibited the growth of L. acidophilus in comparison with the control group (p<0.001).
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Figure 2 shows that different modes of 808-nm laser (continuous-wave, 37 seconds/pulsed, 74 seconds) accompanied by ICG dye had no significant inhibitory effect on L. acidophilus growth rate in comparison with the control group (p>0.05).
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According to Figure 3, MB showed a significant inhibitory effect on L. acidophilus growth rate in comparison with the control group and ICG (p<0.05).
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According to Figure 4, CHX, NaOCl and penicillin significantly decreased the growth rate of L. acidophilus in comparison with the control group (p<0.05).
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Discussion
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As demonstrated by the results, irradiation with 660-nm laser (pulsed, 74 seconds/ continuous-wave, 37 seconds) in combination with MB dye significantly decreased the number of L. acidophilus. In contrast, the separate use of ICG and 808-nm laser (pulsed, 74 seconds/continuous-wave, 37 seconds) and the combined use of 808-nm laser (pulsed, 74 seconds/ continuous-wave 37 seconds) and ICG dye did not show any significant inhibitory effects, whereas CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 significantly decreased the growth of L. acidophilus. Likewise, PDT with MB dye was effective against L. acidophilus. In a study by Usacheva et al in 2001, the clinical antibacterial characteristics of toluidine blue and MB against different microbial species were evaluated in light and darkness. The results showed that all the bacterial species were eliminated after laser irradiation; however, complete photo-degradation of the microorganisms exposed to MB dye was 2 to 7 times higher than that after solitary laser application. Also, bacterial death rate increased with prolonged incubation time. The results indicated that laser therapy has a positive inhibitory impact on bacterial growth [12]. In a study by Souza et al (2010) the impact of laser irradiation at 685 nm wavelength in combination with PSs was assessed on the viability of different C. albicans species. The authors concluded that laser and MB dye decreased the
number of fungal colonies by 88.6, 84.8 and 91.6%. Solitary use of laser decreased the number of all candida species expect for C. albicans. Therefore, photo activation of MB dye with red laser at the wavelength of 685 nm exhibited antifungal effects [13].
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Park et al (2010) assessed the antibacterial efficacy of PDT with pure chlorine against staphylococcus aureus, pseudomonas aeruginosa, Escherichia coli (E. coli) and salmonella enterica. They evaluated the inhibition zone and bacterial viability with the use of red and green fluorescence method. The results of bacterial inhibition zone assessment after PDT with chlorine indicated growth inhibition of staphylococcus aureus and pseudomonas aeruginosa; however, the inhibition was limited with respect to E. coli and salmonella, which shows the dependency of the results on laser energy density and chlorine concentration [14]. Rajesh and colleagues (2011) evaluated the influence of the proprietary parameters of visible low-power laser on biofilm S. mutans and C. albicans, and demonstrated that low-power laser irradiation leads to decreased cell viability and biofilm growth. The reaction of S. mutans species against laser irradiation was similar in all laser doses, and the biofilm growth rate was also dose-dependent. Albeit, when accompanied by C. albicans species, S. mutans bacteria showed higher resistance against low-power laser. The morphology of S. mutans species after low-power laser irradiation was not changed, but the association of microbial species leaded to decreased formation of C. albicans hyphae [15].
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In 2011, Guglielmi et al studied the effects of PDT mediated with MB on deep carious lesions, and showed that PDT significantly decreased S. mutans, lactobacillus and all other viable bacteria. They concluded that this treatment can be a suitable minimally-invasive therapy for deep carious lesions [16].
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In 2014, Ricatto and colleagues compared the effect of PDT with laser and MB dye on cariogenic bacteria (S. mutans and lactobacillus) in bovine dentin in vitro, and concluded that in S. mutans groups, the use of CCLED (dye/LED at 94 J/cm2) and CCLASER (dye/LASER at 94 J/cm2) showed significant reduction of CFUs, which was statistically higher than that in the control, SCLED (no dye/ LED at 94 J/cm2) and SCLASER (no dye/ LASER at 94 J/cm2) groups. Regarding lactobacillus, CCLASER and CCLED groups showed significant reduction of CFUs in comparison with SCLASER which showed moderate results. SCLED and control groups showed limited CFU reduction. Overall, they concluded that PDT with laser or LED mediated with MB dye has significant antimicrobial impacts on cariogenic bacteria in dentin [17]. In a study by Melo et al in 2015 on the antimicrobial effect of PDT mediated with toluidine blue on dentinal carious lesions, it was demonstrated that PDT significantly decreases S. mutans, lactobacillus and all other viable bacteria in comparison with control group [18].
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In a study by Neves et al in 2016 on the clinical impacts of PDT with MB dye on disinfection of deep dentinal layers of deciduous molars after relative caries removal, it was shown that PDT at 120 J/cm2 with 0.01% MB was unable to reduce bacterial contamination in deep dentinal layers. The difference between the conditions of clinical studies and those of the studies performed under completely controlled conditions can be an explanation for the controversy among the results [19].
In 2016, Azizi et al studied PDT with MB and ICG dyes against S. mutans, and concluded that this treatment and also the use of CHX mouthwash can completely eliminate S. mutans colonies [20]. In 2017, Araújo et al assessed the growth capacity of S. mutans and lactobacillus against PDT mediated with Curcumin in dentinal carious lesions. The findings indicated that both light intensities of 19 mW/cm2 and 47.5 mW/cm2 needed 5g/l of Curcumin to significantly reduce bacterial growth, and also high concentrations of Curcumin is toxic for microorganisms [21].
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According to our results, separate use of ICG dye and its combination with laser have no inhibitory effect against L. acidophilus. To date, no other study has evaluated the inhibitory effect of laser mediated with photoactive substances on L. acidophilus growth. Therefore, we found no study to compare our results with. It is recommended to further investigate the exact mechanism of laser irradiation mediated with ICG against L. acidophilus growth.
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According to previous studies, various mechanisms have been reported for the impact of laser irradiation mediated with ICG and MB dyes on inhibiting the growth of bacteria and fungi. The membranes of bacteria and fungi are cellular structures which separate the cell from its surroundings, and due to their specific structure, they are different among various species. Variances have been detected not only between gram-positive and gram-negative bacteria, but also among fungi, bacteria and viruses. Studies have shown that laser irradiation destroys the membrane of bacteria and leads to release of bacterial intracellular contents [22]. Also, it is possible that PDT mediated with PSs inhibits bacterial growth by changing the activity of ionic channels, surface receptors and even bacterial enzymes [10]. It has been stated that PDT acts through inhibition of dihydrofolate reductase. With the use of this enzyme, bacteria synthesize purine and pyrimidine to thicken their cell walls; therefore, inhibition of this enzyme causes bacterial cell death [10]. In addition, laser confronts the cytoplasmic membrane through changing the permeability of Cations such as hydrogen and potassium. Outlet of these ions disrupts vital processes in the cell and leads to exudation of main cellular components, water imbalance, destruction of membrane potential, prevention of ATP synthesis and eventually cell death [23]. Such an interaction changes the membrane structure and consequently mobilizes and expands the membrane. It has been established that PDT imposes its effect through changing the proteoglycans of bacterial cell membrane, as researchers have proclaimed that low-power laser irradiation at 660 nm wavelength with MB or toluidine blue influences the proteoglycans of bacterial cell membrane and eliminates the bacteria. MB is a reductase activated by laser irradiation depending on the laser intensity, and inhibits influenza by destructing membranous proteoglycans [15].
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Membrane instability is correlated with membrane ions, and reduces their ion diffusion gradient. In many cases, bacteria neutralize these effects with the use of ion pump; therefore, cell death is prevented. However, more energy is spent during this process, leading to delayed bacterial growth [14]. It seems that the strain in the present study is resistant against the laser type, irradiation duration and intensity and even the PS type. According to the results, it seems that further research is necessary for determining the exact mechanism by which laser therapy exerts its antibacterial impacts against L. acidophilus growth. Conclusion
The results of the present study indicated that MB dye with and without 660-nm laser (continuous-wave or pulsed) decreases L. acidophilus colonies. On the other hand, solitary use of ICG, 808-nm laser, 660-nm laser or their combined application have no effect on L. acidophilus colonies. Conversely, CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 can inhibit the growth of L. acidophilus.
References:
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1. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J. 2001;34(6):429-34. 2. Twetman S, Stecksen-Blicks C. Probiotics and oral health effects in children. Int J Paediatr Dent. 2008;18(1):3-10. 3. Meurman J, Stamatova I. Probiotics: contributions to oral health. Oral diseases. 2007;13(5):443-51. 4. Estrela C, Ribeiro RG, Estrela CR, Pecora JD, Sousa-Neto MD. Antimicrobial effect of 2% sodium hypochlorite and 2% chlorhexidine tested by different methods. Braz Dent J. 2003;14(1):58-62. 5. Wilson M. Bactericidal effect of laser light and its potential use in the treatment of plaque-related diseases. Int Dent J. 1994;44(2):181-9. 6. Garcez AS, Ribeiro MS, Tegos GP, Nunez SC, Jorge AO, Hamblin MR. Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med. 2007;39(1):59-66. 7. Azizi A, Amirzadeh Z, Rezai M, Lawaf S, Rahimi A. Effect of photodynamic therapy with two photosensitizers on Candida albicans. J Photochem Photobiol B Biol.2016;158:26773. 8. Fekrazad R, Seraj B, Chiniforush N, Rokouei M, Mousavi N, Ghadimi S. Effect of antimicrobial photodynamic therapy on the counts of salivary Streptococcus mutans in children with severe early childhood caries. Photodiagnosis Photodyn Ther. 2017;18:319-22. 9. Diogo P, Gonçalves T, Palma P, Santos JM. Photodynamic antimicrobial chemotherapy for root canal system asepsis: a narrative literature review. Int J Dent. 2015;269205. 10. Khosravi AD, Rostamian J, Moradinegad P. Investigation of bactericidal effect of low level laser of Galium-Aluminium-Arsenide on cariogenic species of streptococci and lactobacillus. J Med Sci. 2008;8(6):579-82. 11. Ferraro MJ. Performance standards for antimicrobial susceptibility testing: ninth informational supplement: NCCLS document M100-S9. National Committee for Clinical Laboratory Standards. 1999;19(1). 12. Usacheva MN, Teichert MC, Biel MA. Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram‐positive and gram‐negative microorganisms. Lasers Surg Med. 2001;29(2):165-73. 13. Souza LC, Brito PR, de Oliveira JCM, Alves FR, Moreira EJ, Sampaio-Filho HR, et al. Photodynamic therapy with two different photosensitizers as a supplement to instrumentation/irrigation procedures in promoting intracanal reduction of Enterococcus faecalis. J Endod. 2010;36(2):292-6. 14. Park JH, Moon YH, Bang IS, Kim YC, Kim SA, Ahn SG, et al. Antimicrobial effect of photodynamic therapy using a highly pure chlorin e6. Lasers Med Sci. 2010;25(5):705-10. 15. Rajesh S, Koshi E, Philip K, Mohan A. Antimicrobial photodynamic therapy: An overview. J Indian Soc Periodontol. 2011;15(4):323-327.
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16. Guglielmi Cde A, Simionato MR, Ramalho KM, Imparato JC, Pinheiro SL, Luz MA. Clinical use of photodynamic antimicrobial chemotherapy for the treatment of deep carious lesions. J Biomed Opt. 2011;16(8):088003. 17. Ricatto LG, Conrado LA, Turssi CP, França FM, Basting RT, Amaral FL. Comparative evaluation of photodynamic therapy using LASER or light emitting diode on cariogenic bacteria: An in vitro study. Eur J Dent. 2014;8(4):509-514. 18. Melo MA, Rolim JP, Passos VF, Lima RA, Zanin IC, Codes BM, et al. Photodynamic antimicrobial chemotherapy and ultraconservative caries removal linked for management of deep caries lesions. Photodiagnosis Photodyn Ther. 2015;12(4):581-6. 19. Neves PA, Lima LA, Rodriguez FC, Leitao TJ, Ribeiro CC. Clinical effect of photodynamic therapy on primary carious dentin after partial caries removal. Braz Oral Res. 2016;30(1):e47. 20. Azizi A, Shademan S, Rezai M, Rahimi A, Lawaf S. Effect of photodynamic therapy with two photosensitizers on Streptococcus mutants: In vitro study. Photodiagnosis Photodyn Ther. 2016;16:66-71. 21. Araújo N, Fontana CR, Bagnato V, Gerbi M. Photodynamic antimicrobial therapy of curcumin in biofilms and carious dentine. Lasers Med Sci. 2014;29(2):629-35. 22. Hope CK, Wilson M. Induction of lethal photosensitization in biofilms using a confocal scanning laser as the excitation source. J Antimicrob Chemother. 2006;57(6):12271230. 23. Tuchin VV, Genina EA, Bashkatov AN, Simonenko GV, Odoevskaya OD, Altshuler GB. A pilot study of ICG laser therapy of acne vulgaris: photodynamic and photothermolysis treatment. Lasers Surg Med. 2003;33(5):296-310.
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Figure legends
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Fig. 1. Mean amount of L. acidophilus colonies in the group treated with 660-nm laser and MB Fig. 2. Mean amount of L. acidophilus colonies in the group treated with 808-nm laser and ICG
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Fig. 3. Mean amount of L. acidophilus colonies in the groups treated with ICG or MB
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Fig. 4. Mean amount of L. acidophilus colonies in the groups treated with CHX, NaOCl or penicillin
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CFU (x103)
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Pulsed, 74 seconds
660-nm laser with MB dye
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Figure 1- Mean number of L. acidophilus colonies in the group of 660-nm laser with methylene blue (MB) dye
120
100
CFU (x103)
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0 Control group
Continuous-wave, 37 seconds
Pulsed, 74 seconds
880-nm laser with ICG dye
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Figure 2- Mean number of L. acidophilus colonies in the group of 880-nm laser with Indocyanine green (ICG) dye
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CFU (x103)
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0 Control group
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ICG
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Figure 3- Mean number of L. acidophilus colonies in the methylene blue (MB) and Indocyanine green (ICG) groups
120
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CFU (x103)
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0 0.2% Chlorhexidine
2.5% Sodium hypochlorite
Penicillin
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Figure 4- Mean number of L. acidophilus colonies in the Penicillin, 2.5% Sodium hypochlorite, and 0.2% Chlorhexidine groups
S1572-1000(17)30488-X https://doi.org/10.1016/j.pdpdt.2018.01.014 PDPDT 1105
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
27-10-2017 3-1-2018 22-1-2018
Please cite this article as: Azizi Arash, Mousavian Shiva, Taheri Soudabeh, Lawaf Shirin, Gonoudi Elnaz, Rahimi Arash.Comparison of the antimicrobial efficacy of photodynamic therapy with two mediators against Lactobacillus acidophilus in vitro.Photodiagnosis and Photodynamic Therapy https://doi.org/10.1016/j.pdpdt.2018.01.014 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.
Comparison of the antimicrobial efficacy of photodynamic therapy with two mediators against Lactobacillus acidophilus in vitro
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1-professor/first name:Arash/last name:Azizi/email: [email protected]./ oral medicine department. Islamic Azad University, Dental Branch, Tehran, Iran
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2-dr/first name:Shiva /last name: Mousavian/ email: [email protected]/ oral medicine specialist
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3-mrs/ first name: Soudabeh /last name: Taheri / email: [email protected] / Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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4-dr/ first name: Shirin/ last name: Lawaf/ email: [email protected] / prosthodontics department. Islamic Azad University, Dental Branch, Tehran, Iran 5 -⃰ (Corresponding)
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dr/ firstname :Elnaz / last name: Gonoudi/ email: [email protected]/ . oral medicine department. Islamic Azad University, Dental Branch, Tehran, Iran
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6-dr/ first name:Arash/ last name: Rahimi/ email: [email protected] / Biophysics Department, Islamic Azad University, Science and Research Branch, Tehran, Iran
Highlights
The separate use of MB and combined use of 660-nm laser and MB have a significant inhibitory effect on L. acidophilus growth.
Photodynamic therapy is the good way to eliminate microorganism of oral cavity. Photodynamic therapy is the conservative method to eliminate microorganism of oral cavity. Photodynamic therapy can be replace instead of antibiotic therapy.
Abstract Background and objectives: Lactobacillus is a cariogenic microorganism. Different therapeutic approaches including photodynamic therapy (PDT) have been suggested for treatment of bacterial infection. The purpose of the current study was to compare the effects of PDT with Indocyanine green (ICG) and Methylene blue (MB) photosensitizers (PSs) on Lactobacillus acidophilus (L. acidophilus).
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Materials and methods: In this in-vitro experimental study, 84 samples of L. acidophilus (1 McFarland standard) were compared in 14 experimental groups including: MB, ICG, 660-nm laser, 808-nm laser (pulsed, 74s/continuous-wave, 37s), different combinations of lasers and PSs, Chlorhexidine (CHX) 0.2%, sodium hypochlorite (NaOCl) 2.5%, penicillin 6.3.3 and control group. The samples were cultured in microplates containing blood agar culture medium. After incubation at 37°C for 48 hours, colony forming units (CFUs) of L. acidophilus were counted and compared before and after therapeutic interventions. Data were analyzed using SPSS19 software program according to one-way ANOVA and Kruskal-Wallis test.
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Results: This study showed that the separate use of ICG, 660- and 808-nm lasers (pulsed, 74s/continuous-wave, 37s), and the combined use of 808-nm laser (pulsed, 74s/continuouswave, 37s) and ICG have no significant inhibitory effect on L. acidophilus colonies (P>0.05), whereas the separate use of MB and the combined use of 660-nm laser (continuous-wave, 37s/pulsed, 74s) and MB significantly inhibited the growth of L. acidophilus in comparison with the control group (p<0.05). Likewise, CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 significantly inhibited the bacterial growth (p<0.05).
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Conclusion: The results showed that separate use of MB and combined use of 660-nm laser and MB have a significant inhibitory effect on L. acidophilus growth.
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Introduction
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Keywords: Photodynamic Therapy; Indocyanine Green; Methylene Blue; Chlorhexidine; Sodium Hypochlorite; Penicillin; Lactobacillus Acidophilus
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Lactobacilli, of the lactobacillaceae family, are gram-positive, rod-shaped, none-sporeforming and immobile bacteria. They produce energy through fermentation of sugar, which at least half of its products is comprised of lactic acid. In the oral cavity, lactic acid causes dental damage and induces dental caries [1-3]. Sodium hypochlorite (NaOCl) 2.5% and Chlorhexidine (CHX) 0.2% are antibacterial agents used regularly against different bacterial species; although they have some disadvantages, and in some cases, contradictory results have been reported after their implementation [4]. When chemical antibacterial agents are included in different preventive and therapeutic procedures, microorganisms may neutralize their action through developing resistance towards these agents; therefore, it is necessary to find alternative therapeutic approaches to substitute these chemicals [5].
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Photodynamic therapy (PDT) is a relatively novel therapeutic method based on the interaction between a safe light source and a light-sensitive substance or photosensitizer (PS) such as methylene blue (MB), toluidine blue, aluminum disulfonated phthalocyanine, chlorine derivatives and nontoxic Indocyanine green (ICG). This interaction, in the presence of oxygen, develops a special sequence of biologic events through producing different species of active oxygen, and as a result, microorganism apoptosis and death will occur [6]. Azizi et al in 2016 evaluated the effect of PDT with MB and ICG PSs on Candida albicans (C. albicans), and concluded that PDT with ICG resulted in the lowest amount of Candida albicans colonies [7]. Fekrazad and colleagues in 2017 assessed the antimicrobial effects of PDT with MB on the amount of salivary Streptococcus mutans (S. mutans) in children with severe early caries, and concluded that PDT decreases the amount of salivary S. mutans immediately after the treatment [8].
Materials and Methods
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Lactobacillus acidophilus (L. acidophilus) standard strain
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Considering the favorable effects of PDT established by previous studies, the aim of the present experiment was to determine the antimicrobial effect of PDT with 808- and 660-nm lasers mediated with MB and ICG PSs.
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Standard strain of L. acidophilus PTCC 1643 was procured from Iranian Research Organization for Science and Technology. L. acidophilus bacteria were cultured in brain heart infusion (BHI) liquid medium, and were incubated at 37°C with 10% CO2 for 48 hours. The solution holding L. acidophilus bacteria in the BHI liquid medium was diluted to 1 McFarland standard (approximately 3×108 bacteria/ml). For precise colony counting, the bacterial suspensions in the liquid medium were cultured on the surface of blood agar medium, and the number of colonies was counted after 48 hours (pre-intervention colony count) [4]. Lasers
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Diode lasers (Polaris Co., Poland) with the wavelengths of 808 nm (energy density of 9.48 J/cm2), and 660 nm (energy density of 1.88 J/cm2) were used in the present study. The diameter of the probe was 1cm for both lasers. The irradiation durations in pulsed and continuous-wave modes were 74 and 37 seconds, respectively. Duty cycle (DC) for continuous-wave mode was considered to be 50% with 100Hz frequency [9]. PSs and other antimicrobial agents
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MB 1% (Merck, Germany), which is activated at 660 nm wavelength and causes photooxidation, was used as a PS in the current study. It was prepared at the microbiology laboratory of School of Dentistry of Shahid Beheshti University of Medical Sciences. The second PS used in the current study was ICG (Arcos, New Jersey, USA), which was prepared in dimethyl sulfoxide (DMSO) solvent at the concentration of 0.01%. It triggers photooxidation at 808 nm wavelength. Other antimicrobial agents used in the present study include: CHX 0.2% mouthwash (Behsa Pharmaceuticals Co., Tehran, Iran), NaOCl 2.5% (Bojne Co., Tehran, Iran) and Penicillin 6.3.3.
The therapeutic groups (Table 1) 1- Control group 10µl of bacterial suspension was placed inside a 96-well microplate. The microplate was incubated at 37°C for 24 hours [4]. Afterwards, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted after 48 hours according to colony forming unit (CFU). 2- MB group (2 minutes)
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10µl of bacterial suspension was mixed with 100µl of MB (final concentration of 1%), and the mixture was placed inside a 96-well microplate (at the bottom of flat wells). The microplate was kept in a dark room for 2 minutes to allow adequate exposure of MB to the bacteria. Afterwards, the samples were cultured on blood agar medium, and the number of L. acidophilus bacterial colonies was counted after 48 hours (post-intervention colony count) [10]. 3- 660-nm continuous-wave laser (37 seconds)
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10µl of bacterial suspension was placed inside a 96-well microplate. Afterwards, continuouswave diode laser was irradiated with 40mW output power at 660 nm wavelength and energy density of 1.88 J/cm2 for 37 seconds. After 48 hours, the total number of L. acidophilus bacterial colonies was counted according to CFU [9].
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4- 660-nm pulsed laser (74 seconds)
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10µl of bacterial suspension was placed in a 96-well microplate. Afterwards, pulsed diode laser irradiation was performed with 40mW output power at 660 nm wavelength and energy density of 1.88 J/cm2 for 74 seconds. After 48 hours, the total number of L. acidophilus bacterial colonies was counted according to CFU [9]. 5- 660-nm continuous-wave laser (37 seconds) and MB
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10µl of bacterial suspension was mixed with 100µl of MB at the concentration of 1%. With the use of a sampler, the mixture was placed at the bottom of 6 wells of a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of MB to the bacteria. Afterwards, continuous-wave diode laser was irradiated in each well with 40mW output power at 660 nm wavelength and energy density of 1.88 J/cm2 for 37 seconds. Next, using an inoculating needle or a standard probe, the contents of each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar containing 3 to 5% CO2, and after 48 hours, the total number of L. acidophilus colonies was counted (CFU) [9]. 6- 660-nm pulsed laser (74 seconds) and MB 10µl of bacterial suspension was mixed with 100µl of MB at the concentration of 1%. With the use of a sampler, the mixture was placed at the bottom of 6 wells in a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of MB to the bacteria. Afterwards, 660-nm laser in pulsed mode was irradiated with 40mW output power and energy density of 1.88 J/cm2 for 74 seconds in each well. Next, using an inoculating needle or a standard probe, the contents of
each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar containing 3 to 5% CO2, and after 48 hours, the total number of L. acidophilus bacterial colonies (CFU) was counted [9]. 7- ICG group (2 minutes)
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10 µl of bacterial suspension was mixed with 100µl of ICG (final concentration of 0.01%), and the mixture was placed inside a 96-well microplate (at the bottom of flat wells). The microplate was kept in a dark room for 2 minutes to allow adequate exposure of ICG to the bacteria. Afterwards, the samples were cultured on blood agar medium, and the number of L. acidophilus bacteria was counted after 48 hours (post-intervention colony count) [10]. 8- 808-nm continuous-wave laser (37 seconds)
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10µl of bacterial suspension was placed in a 96-well microplate. Afterwards, continuouswave diode laser was irradiated with 200mW output power at 808 nm wavelength and energy density of 9.48 J/cm2 for 37 seconds. After 48 hours, the total number of L. acidophilus bacterial colonies (CFU) was counted [9]. 9- 808-nm pulsed laser (74 seconds)
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10µl of bacterial suspension was placed in a 96-well microplate. Afterwards, diode laser in pulsed mode was irradiated with 200mW output power at 808 nm wavelength and energy density of 9.48 J/cm2. After 48 hours, the total number of L. acidophilus colonies was counted [9].
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10- 808-nm continuous-wave laser (37 seconds) and ICG
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10µl of bacterial suspension was mixed with 100µl of ICG in DMSO at the final concentration of 0.01%. Using a sampler, the mixture was placed at the bottom of 6 wells in a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of ICG to the bacteria. Afterwards, 808nm continuous-wave diode laser was irradiated with 200mW output power and energy density of 9.48 J/cm2 for 74 seconds. Next, using an inoculating needle or a standard probe, the contents of each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar with 3 to 5% CO2, and after 48 hours, the total number of L. acidophilus colonies was counted [9]. 11- 808-nm pulsed laser (74 seconds) and ICG
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10µl of bacterial suspension was mixed with 100µl of ICG in DMSO at the final concentration of 0.01%. With the use of a sampler, the mixture was placed at the bottom of 6 wells in a microplate, which were located horizontally and sequentially. The microplate was kept in a dark room for 5 minutes to allow adequate exposure of ICG to the bacteria. Afterwards, pulsed diode laser was irradiated with 200mW output power at 808 nm wavelength and energy density of 9.48 J/cm2 for 74 seconds in each well. Next, using an inoculating needle or a standard probe, the contents of each of the 6 wells were separately cultured on blood agar medium in 6 separate plates. The culture plates were placed inside a jar containing 3 to 5% CO2. After 48 hours, the total number of L. acidophilus colonies (CFU) was counted [9]. 12- CHX 0.2%
10µl of bacterial suspension was mixed with 100µl of CHX 0.2%, and the mixture was placed in a 96-well microplate. The microplate was incubated at 37°C for 24 hours [4]. Next, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted 48 hours later. 13- NaOCl 2.5%
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10µl of bacterial suspension was mixed with 100µl of NaOCl 2.5%, and the mixture was placed in a 96-well microplate. The microplate was incubated at 37°C for 24 hours [4]. Afterwards, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted after 48 hours. 14- Penicillin 6.3.3
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10µl of bacterial suspension was mixed with 100µl of penicillin 6.3.3, and the mixture was placed in a 96-well microplate (at the bottom of flat wells). The microplate was incubated at 37°C for 24 hours. Before and after the intervention, the samples were cultured on blood agar medium, and L. acidophilus colonies were counted 48 hours later [11].
Control
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Table 1: Experimental groups
Number of strains 6 6 6
Red laser
Pulsed 74s
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Red laser and MB
Continuous-wave 37s
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Pulsed 74s
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Continuous-wave 37s
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IR laser
Continuous-wave 37s
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IR laser
Pulsed 74s
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IR laser and ICG
Continuous-wave 37s
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11
IR laser and ICG
Pulsed 74s
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12
CHX 0.2%
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NaOCl 2.5%
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14
Penicillin
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Data collection method
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Data were statistically analyzed with SPSS19 software program. Central dispersion indices of L. acidophilus colonies after different therapeutic procedures were determined and reported accompanied by logarithmic values of colony numbers. The differences of CFUs of L. acidophilus among different groups were statistically analyzed using one-way analysis of variance (ANOVA) and Kruskal-Wallis test. Pairwise comparisons between the groups with regards to the number of CFUs of L. acidophilus were performed according to Dunn's test. The type1 error rate was considered to be 0.05 in the current experiment.
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Results
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The mean amounts of L. acidophilus bacteria in the 14 experimental groups are presented in Table 2. According to the results, the separate use of ICG, 660- and 808-nm lasers (continuous-wave, 37 seconds), 660- and 808-nm lasers (pulsed, 74 seconds), the combined use of 808-nm laser (continuous-wave, 37 seconds) and ICG and the combined application of 808-nm laser (pulsed, 74 seconds) and ICG had no significant inhibitory effect on L. acidophilus colonies (P>0.05).
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In contrast, MB 1% dye significantly decreased the number of bacterial colonies (p<0.001). Likewise, the combined use of 660-nm laser (continuous-wave, 37seconds/pulsed, 74 seconds) and MB significantly decreased the number of L. acidophilus in comparison with the control group (p<0.001). CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 also significantly inhibited the growth of L. acidophilus in comparison with the control group (p<0.001).
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Table 2: Mean amount of L. acidophilus colonies in 14 experimental groups after intervention* Number of strains
CFU
Control
6
100×103>
MB
6
49800±5303
660-nm continuous-wave laser (37s)
6
100×103>
660-nm pulsed laser (74s)
6
100×103>
660-nm continuous-wave laser (37s) and MB
6
11167±9280
660-nm pulsed laser (74s) and MB
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7167±3710
ICG
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100×103>
808-nm continuous-wave laser (37s)
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100×103>
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100×103>
808-nm continuous-wave laser (37s) and ICG
6
100×103>
808-nm pulsed laser (74s) and ICG
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100×103>
CHX 0.2%
6
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NaOCl 2.5%
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Penicillin
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*The average number of lactobacilli was equal to 106 before intervention.
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808-nm pulsed laser (74s)
As shown in Figure 1, the combined use of 660-nm laser (continuous-wave, 37 seconds/pulsed, 74 seconds) and MB dye significantly inhibited the growth of L. acidophilus in comparison with the control group (p<0.001).
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Figure 2 shows that different modes of 808-nm laser (continuous-wave, 37 seconds/pulsed, 74 seconds) accompanied by ICG dye had no significant inhibitory effect on L. acidophilus growth rate in comparison with the control group (p>0.05).
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According to Figure 3, MB showed a significant inhibitory effect on L. acidophilus growth rate in comparison with the control group and ICG (p<0.05).
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According to Figure 4, CHX, NaOCl and penicillin significantly decreased the growth rate of L. acidophilus in comparison with the control group (p<0.05).
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Discussion
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As demonstrated by the results, irradiation with 660-nm laser (pulsed, 74 seconds/ continuous-wave, 37 seconds) in combination with MB dye significantly decreased the number of L. acidophilus. In contrast, the separate use of ICG and 808-nm laser (pulsed, 74 seconds/continuous-wave, 37 seconds) and the combined use of 808-nm laser (pulsed, 74 seconds/ continuous-wave 37 seconds) and ICG dye did not show any significant inhibitory effects, whereas CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 significantly decreased the growth of L. acidophilus. Likewise, PDT with MB dye was effective against L. acidophilus. In a study by Usacheva et al in 2001, the clinical antibacterial characteristics of toluidine blue and MB against different microbial species were evaluated in light and darkness. The results showed that all the bacterial species were eliminated after laser irradiation; however, complete photo-degradation of the microorganisms exposed to MB dye was 2 to 7 times higher than that after solitary laser application. Also, bacterial death rate increased with prolonged incubation time. The results indicated that laser therapy has a positive inhibitory impact on bacterial growth [12]. In a study by Souza et al (2010) the impact of laser irradiation at 685 nm wavelength in combination with PSs was assessed on the viability of different C. albicans species. The authors concluded that laser and MB dye decreased the
number of fungal colonies by 88.6, 84.8 and 91.6%. Solitary use of laser decreased the number of all candida species expect for C. albicans. Therefore, photo activation of MB dye with red laser at the wavelength of 685 nm exhibited antifungal effects [13].
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Park et al (2010) assessed the antibacterial efficacy of PDT with pure chlorine against staphylococcus aureus, pseudomonas aeruginosa, Escherichia coli (E. coli) and salmonella enterica. They evaluated the inhibition zone and bacterial viability with the use of red and green fluorescence method. The results of bacterial inhibition zone assessment after PDT with chlorine indicated growth inhibition of staphylococcus aureus and pseudomonas aeruginosa; however, the inhibition was limited with respect to E. coli and salmonella, which shows the dependency of the results on laser energy density and chlorine concentration [14]. Rajesh and colleagues (2011) evaluated the influence of the proprietary parameters of visible low-power laser on biofilm S. mutans and C. albicans, and demonstrated that low-power laser irradiation leads to decreased cell viability and biofilm growth. The reaction of S. mutans species against laser irradiation was similar in all laser doses, and the biofilm growth rate was also dose-dependent. Albeit, when accompanied by C. albicans species, S. mutans bacteria showed higher resistance against low-power laser. The morphology of S. mutans species after low-power laser irradiation was not changed, but the association of microbial species leaded to decreased formation of C. albicans hyphae [15].
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In 2011, Guglielmi et al studied the effects of PDT mediated with MB on deep carious lesions, and showed that PDT significantly decreased S. mutans, lactobacillus and all other viable bacteria. They concluded that this treatment can be a suitable minimally-invasive therapy for deep carious lesions [16].
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In 2014, Ricatto and colleagues compared the effect of PDT with laser and MB dye on cariogenic bacteria (S. mutans and lactobacillus) in bovine dentin in vitro, and concluded that in S. mutans groups, the use of CCLED (dye/LED at 94 J/cm2) and CCLASER (dye/LASER at 94 J/cm2) showed significant reduction of CFUs, which was statistically higher than that in the control, SCLED (no dye/ LED at 94 J/cm2) and SCLASER (no dye/ LASER at 94 J/cm2) groups. Regarding lactobacillus, CCLASER and CCLED groups showed significant reduction of CFUs in comparison with SCLASER which showed moderate results. SCLED and control groups showed limited CFU reduction. Overall, they concluded that PDT with laser or LED mediated with MB dye has significant antimicrobial impacts on cariogenic bacteria in dentin [17]. In a study by Melo et al in 2015 on the antimicrobial effect of PDT mediated with toluidine blue on dentinal carious lesions, it was demonstrated that PDT significantly decreases S. mutans, lactobacillus and all other viable bacteria in comparison with control group [18].
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In a study by Neves et al in 2016 on the clinical impacts of PDT with MB dye on disinfection of deep dentinal layers of deciduous molars after relative caries removal, it was shown that PDT at 120 J/cm2 with 0.01% MB was unable to reduce bacterial contamination in deep dentinal layers. The difference between the conditions of clinical studies and those of the studies performed under completely controlled conditions can be an explanation for the controversy among the results [19].
In 2016, Azizi et al studied PDT with MB and ICG dyes against S. mutans, and concluded that this treatment and also the use of CHX mouthwash can completely eliminate S. mutans colonies [20]. In 2017, Araújo et al assessed the growth capacity of S. mutans and lactobacillus against PDT mediated with Curcumin in dentinal carious lesions. The findings indicated that both light intensities of 19 mW/cm2 and 47.5 mW/cm2 needed 5g/l of Curcumin to significantly reduce bacterial growth, and also high concentrations of Curcumin is toxic for microorganisms [21].
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According to our results, separate use of ICG dye and its combination with laser have no inhibitory effect against L. acidophilus. To date, no other study has evaluated the inhibitory effect of laser mediated with photoactive substances on L. acidophilus growth. Therefore, we found no study to compare our results with. It is recommended to further investigate the exact mechanism of laser irradiation mediated with ICG against L. acidophilus growth.
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According to previous studies, various mechanisms have been reported for the impact of laser irradiation mediated with ICG and MB dyes on inhibiting the growth of bacteria and fungi. The membranes of bacteria and fungi are cellular structures which separate the cell from its surroundings, and due to their specific structure, they are different among various species. Variances have been detected not only between gram-positive and gram-negative bacteria, but also among fungi, bacteria and viruses. Studies have shown that laser irradiation destroys the membrane of bacteria and leads to release of bacterial intracellular contents [22]. Also, it is possible that PDT mediated with PSs inhibits bacterial growth by changing the activity of ionic channels, surface receptors and even bacterial enzymes [10]. It has been stated that PDT acts through inhibition of dihydrofolate reductase. With the use of this enzyme, bacteria synthesize purine and pyrimidine to thicken their cell walls; therefore, inhibition of this enzyme causes bacterial cell death [10]. In addition, laser confronts the cytoplasmic membrane through changing the permeability of Cations such as hydrogen and potassium. Outlet of these ions disrupts vital processes in the cell and leads to exudation of main cellular components, water imbalance, destruction of membrane potential, prevention of ATP synthesis and eventually cell death [23]. Such an interaction changes the membrane structure and consequently mobilizes and expands the membrane. It has been established that PDT imposes its effect through changing the proteoglycans of bacterial cell membrane, as researchers have proclaimed that low-power laser irradiation at 660 nm wavelength with MB or toluidine blue influences the proteoglycans of bacterial cell membrane and eliminates the bacteria. MB is a reductase activated by laser irradiation depending on the laser intensity, and inhibits influenza by destructing membranous proteoglycans [15].
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Membrane instability is correlated with membrane ions, and reduces their ion diffusion gradient. In many cases, bacteria neutralize these effects with the use of ion pump; therefore, cell death is prevented. However, more energy is spent during this process, leading to delayed bacterial growth [14]. It seems that the strain in the present study is resistant against the laser type, irradiation duration and intensity and even the PS type. According to the results, it seems that further research is necessary for determining the exact mechanism by which laser therapy exerts its antibacterial impacts against L. acidophilus growth. Conclusion
The results of the present study indicated that MB dye with and without 660-nm laser (continuous-wave or pulsed) decreases L. acidophilus colonies. On the other hand, solitary use of ICG, 808-nm laser, 660-nm laser or their combined application have no effect on L. acidophilus colonies. Conversely, CHX 0.2%, NaOCl 2.5% and penicillin 6.3.3 can inhibit the growth of L. acidophilus.
References:
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1. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J. 2001;34(6):429-34. 2. Twetman S, Stecksen-Blicks C. Probiotics and oral health effects in children. Int J Paediatr Dent. 2008;18(1):3-10. 3. Meurman J, Stamatova I. Probiotics: contributions to oral health. Oral diseases. 2007;13(5):443-51. 4. Estrela C, Ribeiro RG, Estrela CR, Pecora JD, Sousa-Neto MD. Antimicrobial effect of 2% sodium hypochlorite and 2% chlorhexidine tested by different methods. Braz Dent J. 2003;14(1):58-62. 5. Wilson M. Bactericidal effect of laser light and its potential use in the treatment of plaque-related diseases. Int Dent J. 1994;44(2):181-9. 6. Garcez AS, Ribeiro MS, Tegos GP, Nunez SC, Jorge AO, Hamblin MR. Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med. 2007;39(1):59-66. 7. Azizi A, Amirzadeh Z, Rezai M, Lawaf S, Rahimi A. Effect of photodynamic therapy with two photosensitizers on Candida albicans. J Photochem Photobiol B Biol.2016;158:26773. 8. Fekrazad R, Seraj B, Chiniforush N, Rokouei M, Mousavi N, Ghadimi S. Effect of antimicrobial photodynamic therapy on the counts of salivary Streptococcus mutans in children with severe early childhood caries. Photodiagnosis Photodyn Ther. 2017;18:319-22. 9. Diogo P, Gonçalves T, Palma P, Santos JM. Photodynamic antimicrobial chemotherapy for root canal system asepsis: a narrative literature review. Int J Dent. 2015;269205. 10. Khosravi AD, Rostamian J, Moradinegad P. Investigation of bactericidal effect of low level laser of Galium-Aluminium-Arsenide on cariogenic species of streptococci and lactobacillus. J Med Sci. 2008;8(6):579-82. 11. Ferraro MJ. Performance standards for antimicrobial susceptibility testing: ninth informational supplement: NCCLS document M100-S9. National Committee for Clinical Laboratory Standards. 1999;19(1). 12. Usacheva MN, Teichert MC, Biel MA. Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram‐positive and gram‐negative microorganisms. Lasers Surg Med. 2001;29(2):165-73. 13. Souza LC, Brito PR, de Oliveira JCM, Alves FR, Moreira EJ, Sampaio-Filho HR, et al. Photodynamic therapy with two different photosensitizers as a supplement to instrumentation/irrigation procedures in promoting intracanal reduction of Enterococcus faecalis. J Endod. 2010;36(2):292-6. 14. Park JH, Moon YH, Bang IS, Kim YC, Kim SA, Ahn SG, et al. Antimicrobial effect of photodynamic therapy using a highly pure chlorin e6. Lasers Med Sci. 2010;25(5):705-10. 15. Rajesh S, Koshi E, Philip K, Mohan A. Antimicrobial photodynamic therapy: An overview. J Indian Soc Periodontol. 2011;15(4):323-327.
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16. Guglielmi Cde A, Simionato MR, Ramalho KM, Imparato JC, Pinheiro SL, Luz MA. Clinical use of photodynamic antimicrobial chemotherapy for the treatment of deep carious lesions. J Biomed Opt. 2011;16(8):088003. 17. Ricatto LG, Conrado LA, Turssi CP, França FM, Basting RT, Amaral FL. Comparative evaluation of photodynamic therapy using LASER or light emitting diode on cariogenic bacteria: An in vitro study. Eur J Dent. 2014;8(4):509-514. 18. Melo MA, Rolim JP, Passos VF, Lima RA, Zanin IC, Codes BM, et al. Photodynamic antimicrobial chemotherapy and ultraconservative caries removal linked for management of deep caries lesions. Photodiagnosis Photodyn Ther. 2015;12(4):581-6. 19. Neves PA, Lima LA, Rodriguez FC, Leitao TJ, Ribeiro CC. Clinical effect of photodynamic therapy on primary carious dentin after partial caries removal. Braz Oral Res. 2016;30(1):e47. 20. Azizi A, Shademan S, Rezai M, Rahimi A, Lawaf S. Effect of photodynamic therapy with two photosensitizers on Streptococcus mutants: In vitro study. Photodiagnosis Photodyn Ther. 2016;16:66-71. 21. Araújo N, Fontana CR, Bagnato V, Gerbi M. Photodynamic antimicrobial therapy of curcumin in biofilms and carious dentine. Lasers Med Sci. 2014;29(2):629-35. 22. Hope CK, Wilson M. Induction of lethal photosensitization in biofilms using a confocal scanning laser as the excitation source. J Antimicrob Chemother. 2006;57(6):12271230. 23. Tuchin VV, Genina EA, Bashkatov AN, Simonenko GV, Odoevskaya OD, Altshuler GB. A pilot study of ICG laser therapy of acne vulgaris: photodynamic and photothermolysis treatment. Lasers Surg Med. 2003;33(5):296-310.
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Fig. 1. Mean amount of L. acidophilus colonies in the group treated with 660-nm laser and MB Fig. 2. Mean amount of L. acidophilus colonies in the group treated with 808-nm laser and ICG
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Fig. 3. Mean amount of L. acidophilus colonies in the groups treated with ICG or MB
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Fig. 4. Mean amount of L. acidophilus colonies in the groups treated with CHX, NaOCl or penicillin
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CFU (x103)
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Continuous-wave, 37 seconds
Pulsed, 74 seconds
660-nm laser with MB dye
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Figure 1- Mean number of L. acidophilus colonies in the group of 660-nm laser with methylene blue (MB) dye
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CFU (x103)
80
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IP T
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0 Control group
Continuous-wave, 37 seconds
Pulsed, 74 seconds
880-nm laser with ICG dye
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Figure 2- Mean number of L. acidophilus colonies in the group of 880-nm laser with Indocyanine green (ICG) dye
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60
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CFU (x103)
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0 Control group
MB
ICG
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Figure 3- Mean number of L. acidophilus colonies in the methylene blue (MB) and Indocyanine green (ICG) groups
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CFU (x103)
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0 0.2% Chlorhexidine
2.5% Sodium hypochlorite
Penicillin
Control group
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Figure 4- Mean number of L. acidophilus colonies in the Penicillin, 2.5% Sodium hypochlorite, and 0.2% Chlorhexidine groups