Fabian Cieplik

Antimicrobial photodynamic therapy for inactivation of biofilms formed by oral key pathogens

With increasing numbers of antibiotic-resistant pathogens all over the world there is a pressing need for strategies that are capable of inactivating biofilm-state pathogens with less potential of developing resistances in pathogens. Antimicrobial strategies of that kind are especially needed in dentistry in order to avoid the usage of antibiotics for treatment of periodontal, endodontic or mucosal topical infections caused by bacterial or yeast biofilms. One possible option could be the antimicrobial photodynamic therapy (aPDT), whereby the lethal effect of aPDT is based on the principle that visible light activates a photosensitizer (PS), leading to the formation of reactive oxygen species, e.g., singlet oxygen, which induce phototoxicity immediately during illumination. Many compounds have been described as potential PS for aPDT against bacterial and yeast biofilms so far, but conflicting results have been reported. Therefore, the aim of the present review is to outline the actual state of the art regarding the potential of aPDT for inactivation of biofilms formed in vitro with a main focus on those formed by oral key pathogens and structured regarding the distinct types of PS.

Photodynamic biofilm inactivation by SAPYR—An exclusive singlet oxygen photosensitizer

Prevention and control of biofilm-growing microorganisms are serious problems in public health due to increasing resistances of some pathogens against antimicrobial drugs and the potential of these microorganisms to cause severe infections in patients. Therefore, alternative approaches that are capable of killing pathogens are needed to supplement standard treatment modalities. One alternative is the photodynamic inactivation of bacteria (PIB). The lethal effect of PIB is based on the principle that visible light activates a photosensitizer, leading to the formation of reactive oxygen species, e.g., singlet oxygen, which induces phototoxicity immediately during illumination. SAPYR is a new generation of photosensitizers. Based on a 7-perinaphthenone structure, it shows a singlet oxygen quantum yield ΦΔ of 99% and is water soluble and photostable. Moreover, it contains a positive charge for good adherence to cell walls of pathogens. In this study, the PIB properties of SAPYR were investigated against monospecies and polyspecies biofilms formed in vitro by oral key pathogens. SAPYR showed a dual mechanism of action against biofilms: (I) it disrupts the structure of the biofilm even without illumination; (II) when irradiated, it inactivates bacteria in a polymicrobial biofilm after one single treatment with an efficacy of ≥ 99.99%. These results encourage further investigation on the potential of PIB using SAPYR for the treatment of localized infectious diseases.

Improving photodynamic inactivation of bacteria in dentistry: highly effective and fast killing of oral key pathogens with novel tooth-colored type-II photosensitizers.

Increasing antibiotic resistances in microorganisms create serious problems in public health. This demands alternative approaches for killing pathogens to supplement standard treatment methods. Photodynamic inactivation of bacteria (PIB) uses light activated photosensitizers (PS) to generate reactive oxygen species immediately upon illumination, inducing lethal phototoxicity. Positively charged phenalen-1-one derivatives are a new generation of PS for light-mediated killing of pathogens with outstanding singlet oxygen quantum yield ΦΔ of >97%. Upon irradiation with a standard photopolymerizer light (bluephase C8, 1260 ± 50 mW/cm(2)) the PS showed high activity against the oral key pathogens Enterococcus faecalis, Actinomyces naeslundii, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans. At a concentration of 10 μM, a maximum efficacy of more than 6 log10 steps (≥ 99.9999%) of bacteria killing is reached in less than 1 min (light dose 50 J/cm(2)) after one single treatment. The pyridinium substituent as positively charged moiety is especially advantageous for antimicrobial action.

The impact of absorbed photons on antimicrobial photodynamic efficacy.

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 Enterococcus faecalis and Actinomyces naeslundii whereby evaluating both adjustment strategies. PIB with SAPYR exhibited CFU-reductions of 5.1 log10 and 6.5 log10 against E. faecalis and A. naeslundii, respectively, which is declared as a disinfectant efficacy. In contrast, the effect of PIB with MB was smaller when the applied energy dose was adjusted compared to SAPYR (CFU-reductions of 3.4 log10 and 4.2 log10 against E. faecalis and A. naeslundii), or there was even no effect at all when the number of absorbed photons was adjusted compared to SAPYR. Since adjusting the numbers of absorbed photons is the more precise and adequate method from a photophysical point of view, this strategy should be considered in further studies when antimicrobial efficacy rates of distinct PS-light source systems are compared.

Photodynamic Inactivation of Root Canal Bacteria by Light Activation through Human Dental Hard and Simulated Surrounding Tissue.

Introduction: Photodynamic inactivation of bacteria (PIB) may be a supportive antimicrobial approach for use in endodontics, but sufficient activation of photosensitizers (PS) in root canals is a critical point. Therefore, aim of this study was to evaluate the ability of PS absorbing blue (TMPyP) or red light (Methylene Blue; MB) for light activation through human dental hard and simulated surrounding tissue to inactivate root canal bacteria. Methods: A tooth model was fabricated with a human premolar and two molars in an acrylic resin bloc simulating the optical properties of a porcine jaw. The distal root canal of the first molar was enlarged to insert a glass tube (external diameter 2 mm) containing PS and stationary-phase Enterococcus faecalis. Both PS (10 μM) were irradiated for 120 s with BlueV (20 mW/cm2; λem = 400–460 nm) or PDT 1200L (37.8 mW/cm2; λem = 570–680 nm; both: Waldmann Medizintechnik), respectively. Irradiation parameters ensured identical numbers of photons absorbed by each PS. Three setups were chosen: irradiating the glass pipette only (G), the glass pipette inside the single tooth without (GT) and with (GTM) simulated surrounding tissues. Colony forming units (CFU) were evaluated. Transmission measurements of the buccal halves of hemisected mandibular first molars were performed by means of a photospectrometer. Results: PIB with both PS led to reduction by ≥ 5 log10 of E. faecalis CFU for each setup. From transmission measurements, a threshold wavelength λth for allowing an amount of light transmission for sufficient activation of PS was determined to be 430 nm. Conclusion: This study can be seen as proof of principle that light activation of given intra-canal PS from outside a tooth may be possible at wavelengths ≥ 430 nm, facilitating clinical application of PIB in endodontics.

Phenalen-1-one-Mediated Antimicrobial Photodynamic Therapy: Antimicrobial Efficacy in a Periodontal Biofilm Model and Flow Cytometric Evaluation of Cytoplasmic Membrane Damage

In light of increasing resistance toward conventional antibiotics and antiseptics, antimicrobial photodynamic therapy (aPDT) may be a valuable alternative, especially for use in dentistry. In this regard, photosensitizers (PS) based on a phenalen-1-one structure seem to be especially favorable due to their high singlet oxygen quantum yield. However, the actual target structures of phenalen-1-one-mediated aPDT are still unclear. The aim of the present study was to investigate the antimicrobial efficacy of aPDT mediated by phenalen-1-one derivatives SAPYR and SAGUA for inactivation of a polymicrobial biofilm consisting of three putative periodontal pathogens in vitro and to get first insights in the mechanism of action of phenalen-1-one-mediated aPDT by assessing damage of cytoplasmic membranes. aPDT with SAPYR exhibited identical antimicrobial efficacy as compared to chlorhexidine (CHX) [4.4–6.1 log10 reduction of colony forming units (CFUs) depending on bacterial species] while aPDT with SAGUA was less effective (2.0–2.8 log10). Flow cytometric analysis combined with propidium iodide (PI) staining revealed no damage of cytoplasmic membranes after aPDT with both phenalen-1-one derivatives, which was confirmed by spectroscopic measurements for release of nucleic acids after treatment. Spectrophotometric PS-uptake measurements showed no uptake of SAPYR by bacterial cells. Despite the inability to pinpoint the actual target of phenalen-1-one-mediated aPDT, this study shows the high antimicrobial potential of phenalen-1-on mediated aPDT (especially when using SAPYR) and represents a first step for getting insights in the mechanism and damage patterns of aPDT with this class of PS.

Phenalen-1-One-Mediated Antimicrobial Photodynamic Therapy and Chlorhexidine Applied to a Novel Caries Biofilm Model

Antimicrobial photodynamic therapy (aPDT) may be useful as a supportive antimicrobial measure for caries-active subjects. In this study, the antimicrobial efficacy of aPDT with a phenalen-1-one photosensitizer was evaluated in a novel in vitro biofilm model comprising Actinomyces naeslundii, Actinomyces odontolyticus, and Streptococcus mutans and was compared to chlorhexidine. The proposed biofilm model allows high-throughput screening for antimicrobial efficacy while exhibiting a differentiated response to different antimicrobial approaches. While chlorhexidine 0.2% showed a reduction of ≈4 log10 for all species, aPDT led to a more pronounced reduction of S. mutans (2.8 log10) than of Actinomyces spp. (1.2 or 1.3 log10). A similar effect was also observed in monospecies biofilms. Therefore, aPDT may be more effective against S. mutans than against Actinomyces spp. when in biofilms, and this antimicrobial approach merits further investigations.

Antimicrobial efficacy of irradiation with visible light on oral bacteria in vitro: a systematic review

AIM Resistances to antibiotics employed for treatment of infectious diseases have increased to alarming numbers making it more and more difficult to treat diseases caused by microorganisms resistant to common antibiotics. Consequently, novel methods for successful inactivation of pathogens are required. In this instance, one alternative could be application of light for treatment of topical infections. Antimicrobial properties of UV light are well documented, but due to its DNA-damaging properties use for medical purposes is limited. In contrast, irradiation with visible light may be more promising. METHODS Literature was systematically screened for research concerning inactivation of main oral bacterial species by means of visible light. RESULTS Inactivation of bacterial species, especially pigmented ones, in planktonic state showed promising results. There is a lack of research examining the situation when organized as biofilms. CONCLUSION More research concerning situation in a biofilm state is required.

Antimicrobial photodynamic therapy – what we know and what we don’t

Abstract Considering increasing number of pathogens resistant towards commonly used antibiotics as well as antiseptics, there is a pressing need for antimicrobial approaches that are capable of inactivating pathogens efficiently without the risk of inducing resistances. In this regard, an alternative approach is the antimicrobial photodynamic therapy (aPDT). The antimicrobial effect of aPDT is based on the principle that visible light activates a per se non-toxic molecule, the so-called photosensitizer (PS), resulting in generation of reactive oxygen species that kill bacteria unselectively via an oxidative burst. During the last 10–20 years, there has been extensive in vitro research on novel PS as well as light sources, which is now to be translated into clinics. In this review, we aim to provide an overview about the history of aPDT, its fundamental photochemical and photophysical mechanisms as well as photosensitizers and light sources that are currently applied for aPDT in vitro. Furthermore, the potential of resistances towards aPDT is extensively discussed and implications for proper comparison of in vitro studies regarding aPDT as well as for potential application fields in clinical practice are given. Overall, this review shall provide an outlook on future research directions needed for successful translation of promising in vitro results in aPDT towards clinical practice.

Antimicrobial efficacy of alternative compounds for use in oral care toward biofilms from caries-associated bacteria in vitro

For caries‐active patients, antimicrobial measures may be useful in addition to mechanical biofilm removal. The aim of this study was to investigate the antimicrobial efficacy of alternative compounds for use in oral care from two main categories (i.e., preservatives and natural compounds) toward biofilms from caries‐associated bacteria as compared to oral care gold‐standards chlorhexidine digluconate (CHX), cetylpyridinium chloride (CPC), and zinc. Compounds were screened in initial Streptococcus mutans biofilms. Then, the most effective compounds were further investigated in mature S. mutans and polymicrobial biofilms comprising Actinomyces naeslundii, Actinomyces odontolyticus, and S. mutans. Here, distinct treatment periods and concentrations were evaluated. Biofilms were visualized by scanning electron microscopy and bacterial membrane damage was evaluated by means of flow cytometry and staining with SYBR Green and propidium iodide. Citrus extract was the only compound exhibiting similar antimicrobial efficacy in initial S. mutans biofilms (>5 log10) as compared to CHX and CPC, but its effect was clearly inferior in mature S. mutans and polymicrobial biofilms. Flow cytometric data suggested that the mechanism of antimicrobial action of citrus extract may be based on damage of bacterial membranes similar to CHX and CPC. From all alternative compounds investigated in this study, citrus extract exhibited the highest antimicrobial efficacy toward in vitro biofilms from caries‐associated bacteria, but still was less effective than oral care gold‐standard antiseptics CHX and CPC. Nevertheless, citrus extract may be a valuable antimicrobial compound for use in oral care for caries‐active patients.