Colonization of Helicobacter pylori in the oral cavity - an endless controversy?

Helicobacter pylori is associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer. Since the oral cavity is the entry port and the first component of the gastrointestinal system, the oral cavity has been discussed as a potential reservoir of H. pylori. Accordingly, a potential oral-oral transmission route of H. pylori raises the question concerning whether close contact such as kissing or sharing a meal can cause the transmission of H. pylori. Therefore, this topic has been investigated in many studies, applying different techniques for detection of H. pylori from oral samples, i.e. molecular techniques, immunological or biochemical methods and traditional culture techniques. While molecular, immunological or biochemical methods usually yield high detection rates, there is no definitive evidence that H. pylori has ever been isolated from the oral cavity. The specificity of those methods may be limited due to potential cross-reactivity, especially with H. pylori-like microorganisms such as Campylobacter spp. Furthermore, the influence of gastroesophageal reflux has not been investigated so far. This review aims to summarize and critically discuss previous studies investigating the potential colonization of H. pylori in the oral cavity and suggest novel research directions for targeting this critical research question.

Limited antimicrobial efficacy of oral care antiseptics in microcosm biofilms and phenotypic adaptation of bacteria upon repeated exposure.

OBJECTIVES: The aims of this study were to investigate the antimicrobial efficacy of antiseptics in saliva-derived microcosm biofilms, and to examine phenotypic adaption of bacteria upon repeated exposure to sub-inhibitory antiseptic concentrations. METHODS: Saliva-derived biofilms were formed mimicking caries- or gingivitis-associated conditions, respectively. Microbial compositions were analyzed by semiconductor-based 16S rRNA sequencing. Biofilms were treated with CHX, CPC, BAC, ALX, and DQC for 1 or 10 min, and colony forming units (CFU) were evaluated. Phenotypic adaptation of six selected bacterial reference strains toward CHX, CPC, and BAC was assessed by measuring minimum inhibitory concentrations (MICs) over 10 passages of sub-inhibitory exposure. Protein expression profiles were investigated by SDS-PAGE. RESULTS: Both biofilms showed outgrowth of streptococci and Veillonella spp., while gingivitis biofilms also showed increased relative abundances of Actinomyces, Granulicatella, and Gemella spp. Antiseptic treatment for 1 min led to no relevant CFU-reductions despite for CPC. When treated for 10 min, CPC was most effective followed by BAC, ALX, CHX, and DQC. Stable adaptations with up to fourfold MIC increases were found in E. coli toward all tested antiseptics, in E. faecalis toward CHX and BAC, and in S. aureus toward CPC. Adapted E. coli strains showed different protein expression as compared with the wildtype strain. CONCLUSION: Antiseptics showed limited antimicrobial efficacy toward mature biofilms when applied for clinically relevant treatment periods. Bacteria showed phenotypic adaptation upon repeated sub-inhibitory exposure. CLINICAL RELEVANCE: Clinicians should be aware that wide-spread use of antiseptics may pose the risk of inducing resistances in oral bacteria.

Cetylpyridinium Chloride: Mechanism of Action, Antimicrobial Efficacy in Biofilms, and Potential Risks of Resistance.

Antimicrobial resistance is a serious issue for public health care all over the world. While resistance toward antibiotics has attracted strong interest among researchers and the general public over the last 2 decades, the directly related problem of resistance toward antiseptics and biocides has been somewhat left untended. In the field of dentistry, antiseptics are routinely used in professional care, but they are also included in lots of oral care products such as mouthwashes or dentifrices, which are easily available for consumers over-the-counter. Despite this fact, there is little awareness among the dental community about potential risks of the widespread, unreflected, and potentially even needless use of antiseptics in oral care. Cetylpyridinium chloride (CPC), a quaternary ammonium compound, which was first described in 1939, is one of the most commonly used antiseptics in oral care products and included in a wide range of over-the-counter products such as mouthwashes and dentifrices. The aim of the present review is to summarize the current literature on CPC, particularly focusing on its mechanism of action, its antimicrobial efficacy toward biofilms, and on potential risks of resistance toward this antiseptic as well as underlying mechanisms. Furthermore, this work aims to raise awareness among the dental community about the risk of resistance toward antiseptics in general.

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.

A comparative study on the antibacterial photodynamic efficiency of a curcumin derivative and a formulation on a porcine skin model.

INTRODUCTION: The propagation of pathogens resistant to antibiotics around the globe has induced an urgent call for action: alternatives to conventional antibiotic therapy have to be developed to prevent a post-antibiotic catastrophe. This study focuses on the enhancement of Photodynamic Inactivation (PDI) of Gram(+) versus Gram(-) bacteria comparing a cationic derivative of curcumin (SACUR-3) to curcumin bound to polyvinylpyrrolidone (PVP-CUR) using an ex vivo porcine skin model to simulate an application on the human skin and foodstuff. EXPERIMENTAL: Porcine skin samples were inoculated with either Staphylococcus aureus or Escherichia coli and treated with either SACUR-3 or PVP-CUR at concentrations of 50 or 100 µM, respectively. Subsequent to blue light illumination (435 nm, 33.8 J cm(-2)) quantitative analyses were performed by counting the colony forming units. Furthermore, the localization of both photoactive compounds in the porcine skin was determined by fluorescence microscopy. PDI of S. aureus resulted in a reduction of 2.2 log10 steps if employing 50 µM of SACUR-3 and of 1.7 log10 steps with 50 µM of PVP-CUR. Phototoxicity towards E. coli was 3.3 log10 steps using 100 µM of SACUR-3 and 0.3 log10 steps for 100 µM of PVP-CUR. Both compounds do not exceed the stratum corneum of the skin. CONCLUSION: A direct comparison of both approaches yields that the cationic curcumin derivative SACUR-3 is effective against Gram(+) and Gram(-) pathogens, whereas the formulation of PVP-CUR has a photokilling effect on the Gram(+) model strain only, but leaves the approval of curcumin as a food additive E100 unaffected. Our results suggest the applicability of SACUR-3-based PDI in dermatology, hand hygiene and food production.

Fast and effective inactivation of Bacillus atrophaeus endospores using light-activated derivatives of vitamin B2.

Highly resistant endospores may cause severe problems in medicine as well as in the food and packaging industries. We found that bacterial endospores can be inactivated quickly with reactive oxygen species (ROS) that were generated by a new generation of flavin photosensitizers. Flavins like the natural compound vitamin B2 are already known to produce ROS but they show a poor antimicrobial photodynamic killing efficacy due to the lack of positive charges. Therefore we synthesized new flavin photosensitizers that have one (FLASH-01a) or eight (FLASH-07a) positive charges and can hence attach to the negatively charged surface of endospores. In this study we used standardized Bacillus atrophaeus endospores (ATCC 9372) as a biological surrogate model for a proof-of-concept study of photodynamic inactivation experiments using FLASH-01a and FLASH-07a. After incubation of spores with different flavin concentrations, the flavin derivatives were excited with blue light at a light dose of 70 J cm(-2). The inactivation of spores was investigated either in suspension or after attachment to polyethylene terephthalate (PET) surfaces. Incubation of spores suspended in Millipore water with 4 mM FLASH-01a for 10 seconds and irradiation with blue light for 10 seconds caused a biologically relevant decrease of spore survival of 3.5 log10 orders. Using FLASH-07a under the same conditions we achieved a decrease of 4.4 log10 orders. Immobilized spores on PET surfaces were efficiently killed with 7.0 log10 orders using 8 mM FLASH-07a. The total treatment time (incubation + irradiation) was as short as 20 seconds. The results of this study show evidence that endospores can be fastly and effectively inactivated with new generations of flavin photosensitizers that may be useful for industrial or medical applications in the future.

Singlet oxygen generation in porphyrin-doped polymeric surface coating enables antimicrobial effects on Staphylococcus aureus.

Surfaces can be coated with photosensitizer molecules, which generate singlet oxygen ((1)O2) when the surface is exposed to light. (1)O2 may diffuse from the coating and has the potential to kill microorganisms present on the surface. In the present study a derivative of the meso-tetraphenylporphyrin (TPP) was immobilized onto polyurethane (PU) after being sprayed and polymerized as a thin layer onto poly-methylmethacrylate (PMMA). PU is gas permeable and thus a sufficient amount of oxygen reaches the photosensitizer in this coating. The surface generation of (1)O2 and its diffusion were investigated by detecting its luminescence at 1270 nm and a tri-iodide assay. Antimicrobial photodynamic surface effects were tested on Staphylococcus aureus. The spectrally resolved detection of (1)O2 luminescence yielded a clear peak at 1275 nm. The time-resolved luminescence showed multi-exponential decay, revealing rise and decay times in the range of 5-2 × 10(2)µs. The photodynamic inactivation of S. aureus was monitored at different photosensitizer concentrations and radiant exposures of light. A photodynamic killing of >99.9% (>3log10-steps) was achieved within an irradiation time of 30 min. The photodynamic killing on the bioactive surface confirmed the antimicrobial effect of (1)O2 that was generated in the PU-coating and reached the bacteria by diffusion.

Blue light kills Aggregatibacter actinomycetemcomitans due to its endogenous photosensitizers.

OBJECTIVES: The aim of this study was to demonstrate that the periodontal pathogen Aggregatibacter actinomycetemcomitans (AA) can be killed by irradiation with blue light derived from a LED light-curing unit due to its endogenous photosensitizers. MATERIALS AND METHODS: Planktonic cultures of AA and Escherichia coli were irradiated with blue light from a bluephase® C8 light-curing unit with an emission peak at 460 nm, which is usually applied for polymerization of dental resins. A CFU-assay was performed for the analysis of viable bacteria after treatment. Moreover, bacterial cells were lysed and the lysed AA and E. coli were investigated for generation of singlet oxygen. Spectroscopic measurements of lysed AA and E. coli were performed and analyzed for characteristic absorption and emission peaks. RESULTS: A light dose of 150 J/cm(2) induced a reduction of ≥5 log10 steps of viable AA, whereas no effect of blue light was found against E. coli. Spectrally resolved measurements of singlet oxygen luminescence showed clearly that a singlet oxygen signal is generated from lysed AA upon excitation at 460 nm. Spectroscopic measurements of lysed AA exhibited characteristic absorption and emission peaks similar to those of known porphyrins and flavins. CONCLUSIONS: AA can be inactivated by irradiation with blue light only, without application of an exogenous photosensitizer. CLINICAL RELEVANCE: These results encourage further studies on the potential use of these blue light-mediated auto-photosensitization processes in the treatment of periodontitis for the successful inactivation of Aggregatibacter actinomycetemcomitans.

Phenotypic Adaptation to Antiseptics and Effects on Biofilm Formation Capacity and Antibiotic Resistance in Clinical Isolates of Early Colonizers in Dental Plaque.

Despite the wide-spread use of antiseptics in dental practice and oral care products, there is little public awareness of potential risks associated with antiseptic resistance and potentially concomitant cross-resistance. Therefore, the aim of this study was to investigate potential phenotypic adaptation in 177 clinical isolates of early colonizers of dental plaque (Streptococcus, Actinomyces, Rothia and Veillonella spp.) upon repeated exposure to subinhibitory concentrations of chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) over 10 passages using a modified microdilution method. Stability of phenotypic adaptation was re-evaluated after culture in antiseptic-free nutrient broth for 24 or 72 h. Strains showing 8-fold minimal inhibitory concentration (MIC)-increase were further examined regarding their biofilm formation capacity, phenotypic antibiotic resistance and presence of antibiotic resistance genes (ARGs). Eight-fold MIC-increases to CHX were detected in four Streptococcus isolates. These strains mostly exhibited significantly increased biofilm formation capacity compared to their respective wild-type strains. Phenotypic antibiotic resistance was detected to tetracycline and erythromycin, consistent with the detected ARGs. In conclusion, this study shows that clinical isolates of early colonizers of dental plaque can phenotypically adapt toward antiseptics such as CHX upon repeated exposure. The underlying mechanisms at genomic and transcriptomic levels need to be investigated in future studies.

Ecological Effects of Daily Antiseptic Treatment on Microbial Composition of Saliva-Grown Microcosm Biofilms and Selection of Resistant Phenotypes.

Antiseptics are widely used in dental practice and included in numerous over-the-counter oral care products. However, the effects of routine antiseptic use on microbial composition of oral biofilms and on the emergence of resistant phenotypes remain unclear. Microcosm biofilms were inoculated from saliva samples of four donors and cultured in the Amsterdam Active Attachment biofilm model for 3 days. Then, they were treated two times daily with chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) for a period of 7 days. Ecological changes upon these multiple antiseptic treatments were evaluated by semiconductor-based sequencing of bacterial 16S rRNA genes and identification of amplicon sequence variants (ASVs). Furthermore, culture-based approaches were used for colony-forming units (CFU) assay, identification of antiseptic-resistant phenotypes using an agar dilution method, and evaluation of their antibiotic susceptibilities. Both CHX and CPC showed only slight effects on CFU and could not inhibit biofilm growth despite the two times daily treatment for 7 days. Both antiseptics showed significant ecological effects on the microbial compositions of the surviving microbiota, whereby CHX led to enrichment of rather caries-associated saccharolytic taxa and CPC led to enrichment of rather gingivitis-associated proteolytic taxa. Antiseptic-resistant phenotypes were isolated on antiseptic-containing agar plates, which also exhibited phenotypic resistance to various antibiotics. Our results highlight the need for further research into potential detrimental effects of antiseptics on the microbial composition of oral biofilms and on the spread of antimicrobial resistance in the context of their frequent use in oral healthcare.

Transcriptomic Stress Response in Streptococcus mutans following Treatment with a Sublethal Concentration of Chlorhexidine Digluconate.

Despite the widespread use of antiseptics such as chlorhexidine digluconate (CHX) in dental practice and oral care, the risks of potential resistance toward these antimicrobial compounds in oral bacteria have only been highlighted very recently. Since the molecular mechanisms behind antiseptic resistance or adaptation are not entirely clear and the bacterial stress response has not been investigated systematically so far, the aim of the present study was to investigate the transcriptomic stress response in Streptococcus mutans after treatment with CHX using RNA sequencing (RNA-seq). Planktonic cultures of stationary-phase S. mutans were treated with a sublethal dose of CHX (125 µg/mL) for 5 min. After treatment, RNA was extracted, and RNA-seq was performed on an Illumina NextSeq 500. Differentially expressed genes were analyzed and validated by qRT-PCR. Analysis of differential gene expression following pathway analysis revealed a considerable number of genes and pathways significantly up- or downregulated in S. mutans after sublethal treatment with CHX. In summary, the expression of 404 genes was upregulated, and that of 271 genes was downregulated after sublethal CHX treatment. Analysis of differentially expressed genes and significantly regulated pathways showed regulation of genes involved in purine nucleotide synthesis, biofilm formation, transport systems and stress responses. In conclusion, the results show a transcriptomic stress response in S. mutans upon exposure to CHX and offer insight into potential mechanisms that may result in development of resistances.

2D luminescence imaging of physiological wound oxygenation.

In cutaneous wound healing, the role of oxygen in vivo is poorly understood. We studied wound surface pO(2) during physiological wound healing in humans. Split-thickness skin graft donor sites (n=12) served as standardized wound models. Wound surface pO(2) was measured at 1, 6 and 14days after split-skin harvesting using two-dimensional luminescence lifetime imaging (2D-LLI) of palladium(II)-meso-tetraphenyl-tetrabenzoporphyrin (Pd-TPTBP) in polystyrene-co-acrylonitrile (PSAN) particles on transparent foils. In another experiment, we removed the stratum corneum (SC) on the volar forearm (n=10) by tape strippings to study the impact of the SC on the epidermal oxygen barrier. Split-skin donor site pO(2) significantly decreased during the time course of physiological healing. Regional differences in pO(2) within donor site wounds were visualized for the first time in literature. No difference was found in pO(2) before and after SC removal, showing that the SC is not a major constituent of the epidermal oxygen barrier.

Insights Into Mechanisms of Antimicrobial Photodynamic Action Toward Biofilms Using Phenalen-1-One Derivatives as Photosensitizers.

INTRODUCTION: In view of increasing resistance against antibiotics and antiseptics, antimicrobial photodynamic therapy (aPDT) may be a promising approach for use in dentistry. The aim of this study was to investigate the mechanism of action of aPDT with the phenalene-1-one derivatives SAPYR and SA-PN-05 as photosensitizers by evaluating bacterial ability to replicate, membrane integrity, metabolic activity, and formation of reactive oxygen species (ROS) in biofilms of Actinomyces naeslundii, Streptococcus mutans, and Escherichia coli. MATERIALS AND METHODS: Single-species biofilms (A. naeslundii, S. mutans, and E. coli) were cultured under aerobic conditions for 48 h followed by treatment with the photosensitizers SAPYR and SA-PN-05 at various concentrations (0, 50, 100, 500 µM) and different incubation periods of 5, 10, 20, and 30 min and subsequent irradiation for 10 min (Waldmann PIB 3000; λem = 360-600 nm; 50 mW/cm2; 30 J/cm2). Control samples were treated with dH2O and kept in dark for the same periods. Bacterial ability to replicate was evaluated by colony forming unit (CFU) assay. The cytoplasmic membrane integrity was investigated by flow cytometry using SYBR Green and propidium iodide and visualized by scanning and transmission electron microscopy. For SAPYR, metabolic activity and formation of intracellular ROS after irradiation were evaluated via luminescence and fluorometric assays, respectively. RESULTS: SAPYR showed antimicrobial effects (>3 log10 CFU reduction) on S. mutans after 5 min and on A. naeslundii after 20 min incubation and light activation. For E. coli, CFU reduction was >2 log10 after 30 min of incubation. SA-PN-05 showed an antimicrobial effect after 5 min for all bacteria. Membrane damage upon aPDT with SAPYR was observed for E. coli, but not for S. mutans and A. naeslundii. Following treatment with SA-PN-05, irradiated samples and dark controls of all three species showed loss of membrane integrity. Luminescence and fluorometric assays showed a reduction in metabolic activity and an increase in formation of intracellular ROS in all three species upon aPDT treatment with SAPYR. CONCLUSION: The observed loss in ability to replicate upon aPDT with SAPYR in single-species biofilms may be due to an increase in formation of intracellular ROS upon photodynamic treatment.

Resistance Toward Chlorhexidine in Oral Bacteria - Is There Cause for Concern?

The threat of antibiotic resistance has attracted strong interest during the last two decades, thus stimulating stewardship programs and research on alternative antimicrobial therapies. Conversely, much less attention has been given to the directly related problem of resistance toward antiseptics and biocides. While bacterial resistances toward triclosan or quaternary ammonium compounds have been considered in this context, the bis-biguanide chlorhexidine (CHX) has been put into focus only very recently when its use was associated with emergence of stable resistance to the last-resort antibiotic colistin. The antimicrobial effect of CHX is based on damaging the bacterial cytoplasmic membrane and subsequent leakage of cytoplasmic material. Consequently, mechanisms conferring resistance toward CHX include multidrug efflux pumps and cell membrane changes. For instance, in staphylococci it has been shown that plasmid-borne qac ("quaternary ammonium compound") genes encode Qac efflux proteins that recognize cationic antiseptics as substrates. In Pseudomonas stutzeri, changes in the outer membrane protein and lipopolysaccharide profiles have been implicated in CHX resistance. However, little is known about the risk of resistance toward CHX in oral bacteria and potential mechanisms conferring this resistance or even cross-resistances toward antibiotics. Interestingly, there is also little awareness about the risk of CHX resistance in the dental community even though CHX has been widely used in dental practice as the gold-standard antiseptic for more than 40 years and is also included in a wide range of oral care consumer products. This review provides an overview of general resistance mechanisms toward CHX and the evidence for CHX resistance in oral bacteria. Furthermore, this work aims to raise awareness among the dental community about the risk of resistance toward CHX and accompanying cross-resistance to antibiotics. We propose new research directions related to the effects of CHX on bacteria in oral biofilms.

Antibacterial efficacy of cold atmospheric plasma against Enterococcus faecalis planktonic cultures and biofilms in vitro.

Nosocomial infections have become a serious threat in our times and are getting more difficult to handle due to increasing development of resistances in bacteria. In this light, cold atmospheric plasma (CAP), which is known to effectively inactivate microorganisms, may be a promising alternative for application in the fields of dentistry and dermatology. CAPs are partly ionised gases, which operate at low temperature and are composed of electrons, ions, excited atoms and molecules, reactive oxygen and nitrogen species. In this study, the effect of CAP generated from ambient air was investigated against Enterococcus faecalis, grown on agar plates or as biofilms cultured for up to 72 h. CAP reduced the colony forming units (CFU) on agar plates by > 7 log10 steps. Treatment of 24 h old biofilms of E. faecalis resulted in CFU-reductions by ≥ 3 log10 steps after CAP treatment for 5 min and by ≥ 5 log10 steps after CAP treatment for 10 min. In biofilm experiments, chlorhexidine (CHX) and UVC radiation served as positive controls and were only slightly more effective than CAP. There was no damage of cytoplasmic membranes upon CAP treatment as shown by spectrometric measurements for release of nucleic acids. Thus, membrane damage seems not to be the primary mechanism of action for CAP towards E. faecalis. Overall, CAP showed pronounced antimicrobial efficacy against E. faecalis on agar plates as well as in biofilms similar to positive controls CHX or UVC.

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.

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.

Antimicrobial photodynamic therapy as an adjunct for treatment of deep carious lesions-A systematic review.

For deep carious lesions, a more conservative treatment modality ("selective caries removal") has been proposed, where only the heavily contaminated dentine is removed. In this regard, effective adjuncts for cavity disinfection such as the antimicrobial photodynamic therapy (aPDT) can be valuable clinically prior to definitive restoration. Therefore, the aim of this study was to systematically assess clinical studies on the effectiveness of aPDT as a supplementary tool in the treatment of deep caries lesions. Searches were performed in four databases (PubMed, EMBASE, ISI Web of Science, ClinicalTrials.gov) from 1st January, 2011 until 21st June, 2016 for search terms relevant to the observed parameters, pathological condition, intervention and anatomic entity. The pooled information was evaluated according to PRISMA guidelines. At first, 1651 articles were recovered, of which 1249 full-text articles were evaluated, 270 articles thereof were reviewed for eligibility and finally 6 articles met all inclusion criteria. The aPDT protocols involved Methylene Blue, Toluidine Blue and aluminium-chloride-phthalocyanine as photosensitizers and diode lasers, light-emitting diodes and halogen light-sources. The data from five reports, utilizing both culture-dependent and -independent methods, disclosed significant reduction of cariogenic bacterial load after mechanical caries removal with adjunct aPDT. As these studies exhibit some methodological limitations, e.g. lack of positive controls, this systematic review can support the application of aPDT to a limited extent only in terms of reducing the microbial load in deep carious lesions before restorative treatment.

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/cm(2); λem = 400-460 nm) or PDT 1200L (37.8 mW/cm(2); 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.

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.