Edited by: Rustam Aminov, Technical University of Denmark, Denmark
Reviewed by: Ali Al-Ahmad, Universitätsklinikum Freiburg, Germany; Daniela Vecchio, Massachusetts General Hospital, USA; Heinrich Walt, University Hospital Zürich, Switzerland; Joanna Nakonieczna, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Poland
*Correspondence: Fabian Cieplik, Department of Conservative Dentistry and Periodontology, University Medical Center Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Due to increasing resistance of pathogens toward standard antimicrobial procedures, alternative approaches that are capable of inactivating pathogens are necessary in support of regular modalities. In this instance, the photodynamic inactivation of bacteria (PIB) may be a promising alternative. For clinical application of PIB it is essential to ensure appropriate comparison of given photosensitizer (PS)-light source systems, which is complicated by distinct absorption and emission characteristics of given PS and their corresponding light sources, respectively. Consequently, in the present study two strategies for adjustment of irradiation parameters were evaluated: (i) matching energy doses applied by respective light sources (common practice) and (ii) by development and application of a formula for adjusting the numbers of photons absorbed by PS upon irradiation by their corresponding light sources. Since according to the photodynamic principle one PS molecule is excited by the absorption of one photon, this formula allows comparison of photodynamic efficacy of distinct PS per excited molecule. In light of this, the antimicrobial photodynamic efficacy of recently developed PS SAPYR was compared to that of clinical standard PS Methylene Blue (MB) regarding inactivation of monospecies biofilms formed by
In a current assessment the Centers for Disease Control and Prevention of the United States of America stated that antibiotic-resistant bacteria are causative for at least two million illnesses and 23,000 deaths per year in the U.S. alone (Centers for Disease Control and Prevention,
In particular in the field of dentistry, it should be a major goal to moderate the application of systemic antibiotics. For example, it is common practice to deliver Amoxicillin and Metronidazol combined for 1 week in addition to conventional scaling and root planing for treatment of severe forms of chronic periodontitis (Winkelhoff et al.,
In light of this, a promising alternative may be the photodynamic inactivation of bacteria (PIB). PIB is an antimicrobial approach consisting of the application of a
Currently, in dental practice predominantly PS based on a phenothiazinium structure are clinically used like Methylene Blue (MB) (Gursoy et al.,
For appropriate comparison of antimicrobial photodynamic efficacy rates of given PS-light source systems, it is essential to adjust irradiation parameters, which is complexed by distinct absorption and emission characteristics of given PS and their corresponding light sources. Hereby, it is common practice to match the energy doses applied by the respective light sources. In this instance, emission and absorption characteristics are mostly left without consideration. However, – according to the photodynamic principle – one PS molecule is excited by the absorption of one photon, wherefore the numbers of photons absorbed by PS upon irradiation by their corresponding light sources should be calculated allowing comparison of photodynamic efficacy rates of distinct PS per excited molecule.
Aim of the present study was to evaluate two strategies for adjustment of irradiation parameters: (I) adjusting the energy doses applied by the respective light sources irrespective of spectral properties and (II) by development and application of a formula for calculating the numbers of absorbed photons for each PS upon irradiation by its corresponding light source. Applying these strategies, SAPYR and MB were exemplarily compared regarding inactivation of monospecies biofilms formed by oral key pathogens
The phenalen-1-one PS SAPYR [2-((4-pyridinyl)methyl)-1H-phenalen-1-one chloride; Figure
For irradiation of SAPYR, a Waldmann PIB 3000 blue light source (λ em = 380–600 nm) was used, whereas MB was irradiated with a Waldmann PDT 1200L red light source (λ em = 580–750 nm) (both: Waldmann Medizintechnik, Villingen-Schwenningen, Germany). Emission spectra of the light sources were recorded by means of a monochromator with a CCD detection system (SPEX 232, HORIBA Jobin Yvon, Longjumeau Cedex, France). Figure
For PIB experiments, the light intensities obtained from both light sources at the level of the samples were measured with a thermal low-power sensor (Nova 30 A-P-SH, Ophir-Spiricon, North Logan, UT).
For calculation of the numbers of absorbed photons, the extinction coefficients of the PS at distinct wavelengths [ε (λ)] and the spectral radiant power emitted by the respective light sources Pem (λ) were measured. For determination of the spectral resolved absorption, the concentration of the PS (c, here: 100 μM) and the thickness of the solution (d, here: 1.3 mm) are necessary parameters. Moreover, the universal physical constant of the velocity of light (c0) and the Planck constant (h) are required.
ε (λ) | Extinction coefficient [(mol×cm)−1] | Measured spectrally resolved by a photospectrometer |
Pem(λ) | Spectral radiant power of the respective light source [mW/nm] | Measured spectrally resolved by a CCD detector system |
c | Concentration of the PS [μM] | Here: 100 μM |
d | Thickness of the solution [mm] | Here: 1.3 mm |
c0 | Velocity of light [m/s] | 299,792,458 m/s |
h | Planck constant [J×s] | 6.62606957×10−34 Js |
λ | Wavelength [nm] |
First, irradiation parameters for SAPYR were determined to irradiation with PIB 3000 for 600 s at an irradiance at sample- level of 50 mW/cm2, corresponding to an energy dose of 30 J/cm2.
In order to compare antimicrobial photodynamic efficacy of SAPYR and MB at corresponding energy dose, irradiation of MB was with PDT 1200L at 50 mW/cm2 for 600 s.
For comparing antimicrobial photodynamic efficacy of both PS at matching numbers of absorbed photons, the constants, the experimental data and the measured values were inserted in the formula outlined above, resulting in a number of 1.13 × 1016 photons/second absorbed by SAPYR for irradiation with PIB 3000 (irradiance at sample-level: 50 mW/cm2). Likewise, a number of 3.75 × 1016 photons/second absorbed by MB for irradiation with PDT 1200L (irradiance at sample-level: 20 mW/cm2) was calculated. Consequently, in order to adjust the numbers of absorbed photons for both PS, a 3.32 times longer irradiation period was necessary for SAPYR with PIB 3000 compared to MB with PDT 1200L. Therefore, irradiation periods were determined to be 600 s for SAPYR with PIB 3000 (energy dose: 30 J/cm2) and 181 s for MB with PDT 1200L (energy dose: 3.6 J/cm2).
The presence of EPS in
Peroxidase-labeled Con A (Sigma-Aldrich, St. Louis, MO) was diluted in PBS containing 0.05% (vol/vol) Tween 20 (Merck, Darmstadt, Germany) (diluting buffer; DB) obtaining final concentrations of 10.0 and 12.5 μg/ml of Con A.
After a cultivation period of 72 h, biofilms were washed twice with sterile PBS to remove non-adherent bacteria and were incubated either with 50 μl of the respective PS at a concentration of 100 μM (experimental group PS+L+:
Immediately afterwards PS or PBS was carefully removed and each biofilm was brought to suspension with 200 μl of PBS by multiple up-and-down-pipetting and transferred to a 1.5 ml Eppendorf tube. These were placed in an ultrasonic water-bath chamber (35 kHz; Qualilab USR30H, Merck Eurolab, Bruchsal, Germany) for 5 min and then vigorously vortexed (REAX top, Heidolph Instruments, Schwabach, Germany) for 5 s for separation of aggregated bacteria. Serial tenfold dilutions (10−2 to 10−7) were prepared in BHI-broth and aliquots (3 × 20 μl) were plated on Mueller-Hinton-agar (
For ELLA experiments (data from 12 independent samples), OD at 405 nm was plotted versus the logarithm of Con A concentrations. Values were fitted in a dose-response curve including 95% confidence intervals using Table Curve 2D (Systat Software Inc., San Jose, CA).
All results of PIB experiments are graphically shown as medians, including 25 and 75% quantiles and were calculated using SPSS for Windows, version 20 (SPSS Inc., Chicago, IL) from the values of at least six independent experiments, each performed in duplicate. Horizontal solid and dashed lines in the figures depict reductions of 3 and 5 log10 steps CFU respectively, compared to untreated control groups PS-L-. Medians on these lines exhibit inactivation efficacy rates of 99.9% (3 log10) or 99.999% (5 log10). According to the infection control guidelines, this is declared as biologically relevant antimicrobial activity or disinfectant effect, respectively (Boyce and Pittet,
D-glucose and D-mannose residues in the EPS of
SAPYR and MB were evaluated against
For comparing MB at adjusted energy dose compared to SAPYR (30 J/cm2; number of absorbed photons: 56.5 × 1018), MB was irradiated with PDT 1200L for 600 s at an irradiance of 50 mW/cm2 reaching the samples, too. Here, CFU of
In contrast, for comparing MB at adjusted numbers of absorbed photons compared to SAPYR (6.78 × 1018; energy dose: 3.6 J/cm2), MB was irradiated with PDT 1200L for 181 s at an irradiance at sample-level of 20 mW/cm2, which resulted in no reduction of CFU-median against both,
In all cases there was no reduction of CFU after treatment with PS only (PS+L−). Treatment with light only (PS−L+) had no effect on CFU of
Aim of the present study was to compare the antimicrobial photodynamic efficacy of given PS by using two strategies for adjustment of irradiation parameters. Since different PS exhibit distinct absorption characteristics, distinct light sources have to be used and their irradiation parameters have to be adjusted in order to ensure appropriate comparison of antimicrobial photodynamic efficacy. Usually, for adjustment of irradiation parameters only the energy doses applied by the respective light sources are matched. In this instance neither the molar extinction coefficients of a given PS nor the energy per wavelength emitted by the respective light source are factored in. Furthermore, the number of emitted photons of a given light source not only depends on the power but also on the wavelength of the emitted photons. In this study, a formula is presented considering all these aspects in order to calculate the numbers of photons, which are absorbed per second by a given PS when irradiated by its corresponding light source:
For a certain wavelength λ0,
Depending on the mechanism of photodynamic action and based on the Jablonski diagram (Baier et al.,
So, adjusting the numbers of absorbed photons of distinct PS-light source systems ensures equal numbers of activated PS-molecules, thus allowing comparison of the antimicrobial photodynamic efficacy rates per excited PS-molecule. Therefore, distinct photodynamic antimicrobial efficacy despite adjusted numbers of absorbed photons may not be caused by photophysical properties but rather by PS-inherent features like mechanism of action (type I or type II; singlet oxygen quantum yield), attachment, uptake, intracellular localization etc.
For experimental validation of this formula, we exemplarily compared antimicrobial photodynamic efficacy rates of phenalen-1-one PS SAPYR to that of clinical standard PS MB (adjusted either by energy doses or by numbers of absorbed photons) regarding inactivation of monospecies biofilms formed by oral key pathogens
Irradiation parameters for SAPYR were designated and for irradiation of MB either the energy dose applied by the respective light sources or the numbers of absorbed photons were adjusted compared to SAPYR. For the latter, we inserted the constants, the experimental data and the measured values in the formula presented above in order to calculate the numbers of photons, which were absorbed per second by each PS when irradiated by its corresponding light source.
Under these conditions, SAPYR was able to inactivate monospecies biofilms of
Despite adjusted numbers of absorbed photons, there were distinct antimicrobial photodynamic efficacy rates of SAPYR and MB in this study, which may be explained by PS-inherent aspects like mentioned above: Since there is no remarkable difference in molecular dimension between SAPYR and MB (272.3 and 284.4 g/mol without counterions, respectively), the extent of steric hindrance for penetration through EPS may be similar and unlikely accounts for the observed difference in the PIB efficacy. As both, SAPYR and MB, are single positively charged, electrostatic interactions with negatively charged EPS molecules should be similar, too. Though, it is known that SAPYR may act like a tenside due to its chemical structure comprising the combination of a hydrophilic pyridinium unit and a large hydrophobic tail (Cieplik et al.,
Since 1O2 is known to be the main ROS in the mechanism of PIB (Maisch et al.,
In a former study, SAPYR was evaluated against monospecies and polyspecies biofilms formed by
When comparing the PIB efficacy rates of SAPYR here to those of the former study,
Apart from that, it was found that irradiation of
Taken together, the results of this study provide evidence that PIB with SAPYR may be a promising approach for inactivation of biofilms, whereas PIB with MB is ineffective in this regard. However, before any clinical application of SAPYR, its toxicity against host cells has to be examined in subsequent studies. Furthermore, the capability of blue light to penetrate dental hard and gingival tissues has to be investigated for proper light-activation of SAPYR like it has already been shown for red light and MB (Ronay et al.,
In general, a PS should feature high binding affinity for microorganisms (positive charge for adherence to negatively charged bacterial cell walls) (Alves et al.,
The data of this study clearly demonstrates that adjusting the numbers of absorbed photons is crucial for comparing antimicrobial photodynamic efficacy rates of distinct PS. This is essential for further development and/or optimization of both, PS and light sources.
In this study, a formula is presented for calculation of the number of photons absorbed by a given PS upon irradiation by its corresponding light source. In this way, appropriate comparison of PIB efficacy of given PS-light source systems is ensured because according to the photodynamic principle one PS molecule can only be excited by the absorption of one photon.
For experimental validation of this formula, antimicrobial photodynamic efficacy of the phenalen-1-one PS SAPYR was exemplarily compared to that of clinical standard PS Methylene Blue regarding inactivation of monospecies biofilms formed by
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Isabelle Tabenski is gratefully acknowledged for helpful discussions. FC thanks for financial support from the University Medical Center Regensburg (ReForm A program) and for a travel grant from the German Society of Periodontology (DG PARO). JR was funded by a grant of the German Research Foundation (DFG-RE-3323/2-1). Publication of this work was supported by the German Research Foundation (DFG) within the funding program Open Access Publishing.