Photodynamic and peptide-based strategy to inhibit Gram-positive bacterial biofilm formation.

The self-produced extracellular polymeric matrix of biofilms renders them difficult to eliminate once they are established. This makes the inhibition of biofilm formation key to successful treatment of biofilm infection. Antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides offer a new approach as antibiofilm strategies. In this study sub-lethal doses of aPDT (with chlorin-e6 (Ce6-PDT) or methylene blue (MB-PDT)) and the peptides AU (aurein 1.2 monomer) or (AU)2K (aurein 1.2 C-terminal dimer) were combined to evaluate their ability to prevent biofilm development by Enterococcus faecalis. Biofilm formation was assessed by resazurin reduction, confocal microscopy, and infrared spectroscopy. All treatments successfully prevented biofilm development. The (AU)2K dimer had a stronger effect, both alone and combined with aPDT, while the monomer AU had significant activity when combined with Ce6-PDT. Additionally, it is shown that the peptides bind to the lipoteichoic acid of the E. faecalis cell wall, pointing to a possible key mechanism of biofilm inhibition.

Confocal fluorescence imaging to evaluate the effect of antimicrobial photodynamic therapy depth on P. gingivalis and T. denticola biofilms.

BACKGROUND: Porphyromonas gingivalis and Treponema denticola are both principally implicated in the incidence of both periodontal disease and peri-implantitis. Recent studies have demonstrated that these bacteria exhibit symbiotic growth in vitro and a synergistic virulence in co-infection of animal models. Found at varying depths throughout the biofilm, these bacteria present a significant challenge to traditional antimicrobial treatment modalities. Antimicrobial photodynamic therapy (aPDT) has yielded high success against bacterial biofilms, namely those found in the oral cavity. Data on the use of aPDT against these particular periodontal pathogens is, however, scarce. Here, we studied the qualitative killing efficacy and depth of drug and laser penetration into defined P. gingivalis and T. denticola biofilms. METHODS: P. gingivalis and T. denticola were incubated under anaerobic (10%CO2, 10%H2, 80%N2) conditions for two days in diluted TSB with PBS (TYGVS for T. denticola maintenance) to elicit biofilm growth on coverslip-modified polystyrene dishes. Treated biofilms were exposed to a purpurin-based sensitizer (25 µg/mL in DMSO) for 30 min, and then aPDT was carried out using a diode laser at 664 nm. Light doses of 15 and 45 J/cm2 were used. All biofilms were then exposed to Filmtracer™ LIVE/DEAD® Biofilm Viability Kit (Cat No. L10316). Qualitative analysis was performed using a Zeiss LSM 510 Meta NLO Confocal Microscope with attached Zeiss Axioimager Z1 and Axiovert 200 M for visual data collection, and images were processed using the ZEN Digital Imaging for Light Microscopy software suite. Analysis was performed in 2 × 3 stacks to assess the entire depth of both the biofilm and presumed drug/laser penetration. RESULTS: Initial planktonic studies confirmed that the bacteria in question were present in the grown cultures and susceptible to aPDT exposure. Biofilm control groups were found to have significant levels of surviving bacterial colonies. Both treatment groups featured complete bacterial kill throughout the entirety of the biofilm (average: 23.17 µm; range: 18.13-27.20 µm). CONCLUSIONS: The efficacy of the purpurin-based PS and aPDT is demonstrated to be effective at both high and low light doses. Bacterial kill was fully efficacious at each visualized biofilm layer (1.01 µm/z-level). This study serves as a proof of concept for future studies that must consider appropriate treatment parameters, including the amount of applied PS, and laser dose. These findings indicate that aPDT is a method that can be used to eliminate microorganisms associated with biofilms implicated in the etiology of peri-implantitis and periodontitis at large.

Antimicrobial photodynamic therapy of S. mutans biofilms attached to relevant dental materials.

BACKGROUND: Antimicrobial Photodynamic therapy (aPDT) has demonstrated efficacy in situations where conventional antibiotic therapies can be challenged such as biofilms, gram-negative bacteria, and antimicrobial resistant organisms. Surface characteristics can affect biofilm adherence and integrity and so may modify the effectiveness of aPDT. This study investigates the killing efficacy of aPDT on S. mutans biofilms grown on relevant dental substrata, examining the killing efficacy and specifically the effects of aPDT on the biofilm matrix architecture. MATERIALS AND METHODS: S. mutans (NCTC 10449) was grown in 48 hours biofilms on different substrata, specifically glass, titanium, and denture acrylic. During aPDT assays, the biofilms were treated with a purpurin based sensitizer ([25 ug/ml] in DMSO) for 30 minutes, then exposed to a 664 nm diode laser at light doses of 15, 30, and 45 J/cm2 . Colony forming unit assays were performed to determine survival following treatment. Controls for comparison in survival assays consisted of (No light/No PS; No light/PS; and No light/DMSO). MAIR-IR spectroscopy analysis was performed to investigate aPDT effects on biofilm composition before and after jet impingement. RESULTS: Survival was greatly reduced in the biofilm cultures following the aPDT assays. All light doses achieved a greater then 3-log inactivation on 48 hours biofilms grown on polished denture acrylic. The higher light doses (45 and 30 J) achieved greater than 3-log inactivation in 48 hours biofilms grown on glass. The higher light doses (30 and 45 J/cm2 ) produced a 2-log inactivation in 48 hours biofilms grown on titanium. Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy data demonstrates enhanced loss of exopolysaccharide (EPS) and Amide in the aPDT treated biofilms following jet impingement. CONCLUSION: Antimicrobial PDT experiments using a purpurin based sensitizer and laser light doses of 15, 30, and 45 J/cm2 , against S. mutans biofilm grown on different surfaces, show the effectiveness of this therapy. In CFU survival assays, a dose response to the laser is evident. While considerable disinfection was achieved on all surfaces compared to the controls, not all surfaces could be disinfected equally. MAIR-IR spectroscopy showed that aPDT groups lost more EPS and Amide versus controls, suggesting aPDT induced biofilm embrittlement, which was revealed by jet impingement. With demonstrated efficacy against various microbes and on different substrata, antimicrobial aPDT shows potential for clinical application in biofilm-mediated diseases such as peri-implantitis and periodontitis. Lasers Surg. Med. 48:995-1005, 2016. © 2016 Wiley Periodicals, Inc.

Photodynamic therapy (PDT) for malignant brain tumors--where do we stand?

INTRODUCTION: What is the current status of photodynamic therapy (PDT) with regard to treating malignant brain tumors? Despite several decades of effort, PDT has yet to achieve standard of care. PURPOSE: The questions we wish to answer are: where are we clinically with PDT, why is it not standard of care, and what is being done in clinical trials to get us there. METHOD: Rather than a meta-analysis or comprehensive review, our review focuses on who the major research groups are, what their approaches to the problem are, and how their results compare to standard of care. Secondary questions include what the effective depth of light penetration is, and how deep can we expect to kill tumor cells. CURRENT RESULTS: A measurable degree of necrosis is seen to a depth of about 5mm. Cavitary PDT with hematoporphyrin derivative (HpD) results are encouraging, but need an adequate Phase III trial. Talaporfin with cavitary light application appears promising, although only a small case series has been reported. Foscan for fluorescence guided resection (FGR) plus intraoperative cavitary PDT results were improved over controls, but are poor compared to other groups. 5-Aminolevulinic acid-FGR plus postop cavitary HpD PDT show improvement over controls, but the comparison to standard of care is still poor. CONCLUSION: Continued research in PDT will determine whether the advances shown will mitigate morbidity and mortality, but certainly the potential for this modality to revolutionize the treatment of brain tumors remains. The various uses for PDT in clinical practice should be pursued.

Moraxella catarrhalis is susceptible to antimicrobial photodynamic therapy with Photofrin.

BACKGROUND AND OBJECTIVE: Moraxella catarrhalis is a significant cause of pediatric otitis media (OM), which is the most prevalent bacterial infection in children and primary reason for antibiotic administration in this population. Moreover, biofilm formation has been implicated as a primary mechanism of chronic or recurrent OM disease. As bacterial biofilms are inherently resistant to most antibiotics and these complex structures also present a significant challenge to the immune system, there is a clear need to identify novel antimicrobial approaches to treat OM infections. In this study, we evaluated the potential efficacy of antibacterial photodynamic therapy (aPDT) with porfimer sodium (Photofrin (PF)) against planktonic as well as biofilm-associated M. catarrhalis. MATERIALS AND METHODS: The bactericidal activity of aPDT with PF was assessed against multiple recent clinical isolates of M. catarrhalis grown planktonically as well as in biofilms. The bactericidal activity of PF-aPDT was quantified by enumeration of colony forming units post-treatment. The effect of aPDT on M. catarrhalis biofilms was further investigated with scanning electron microscopy (SEM) imaging. RESULTS: aPDT with PF significantly reduced M. catarrhalis viability. Although PF-aPDT caused higher killing in planktonic grown organisms (5-6 log kill), biofilm grown bacteria also demonstrated a statistically significant reduction in viable organisms (3-4 log decrease in recoverable bacteria) following treatment as compared to saline only controls (P < 0.01). SEM studies indicated the PF-aPDT treated bacteria exhibited prominent morphological changes with visibly distorted cell membranes. CONCLUSIONS: aPDT with PF elicits significant bactericidal activity against both planktonic and biofilm-associated M. catarrhalis, suggesting this technology warrants further analysis as a potential novel antimicrobial treatment for acute or recurrent OM.

Photodynamic therapy as an alternative treatment for disinfection of bacteria in oral biofilms.

BACKGROUND AND OBJECTIVES: Biofilm-related diseases such as caries and periodontal disease are prevalent chronic oral infections which pose significant oral and general health risks. Biofilms are sessile communities attached to surfaces. Photodynamic therapy (PDT) has been demonstrated to have a significant anti-microbial effect and presents as an alternative to treating biofilm-related disease. The aim of this study was to determine the ability of porfimer sodium induced PDT to treat localized infections of Streptococcus mutans in biofilm communities. MATERIALS AND METHODS: Reproducible biofilms were formed by S. mutans strain ATCC 27351 growing in log phase at 37°C in Brain Heart Infusion medium, circulating through flow cells at 3 ml/minute for 36-48 hours. The photosensitizer used was porfimer sodium (Photofrin®) at 125 µg/ml with biofilm immersion times of 5 minutes and increasing energy density of post-immersion laser illumination at 630 nm (100 mW/cm(2) ). Resulting effects on bacterial viability in the biofilms were tracked by monitoring alamarBlue® conversion. Supplementary data characterizing the biofilms before and after exposure to PDT were acquired by Multiple Attenuated Internal Reflection Infrared Spectroscopy (MAIR-IR). RESULTS: The results of this study show that PDT using porfimer sodium and 630 nm laser light was effective in significantly reducing the viability of S. mutans biofilms. Maximum effectiveness was seen when biofilms were exposed to both photosensitizer and light versus controls. Porfimer sodium incubation times as short as 5 minutes in solutions as dilute as 25 µg/ml and illuminated with as little as 30 J/cm(2) resulted in significant decreases in viability of bacteria in biofilms. Optimum parameters appear to be 125 µg/ml porfimer sodium concentration and incubated for 5 minutes and 60 J/cm(2) of light energy density. CONCLUSIONS: This study has demonstrated that significant killing of the cariogenic organism S. mutans by the combination of a photosensitizer and the appropriate wavelength of laser light was possible even when the bacteria are embedded in an extracellular matrix.

Autofluorescence-guided surveillance for oral cancer.

Early detection of oral premalignant lesions (OPL) and oral cancers (OC) is critical for improved survival. We evaluated if the addition of autofluorescence visualization (AFV) to conventional white-light examination (WLE) improved the ability to detect OPLs/OCs. Sixty high-risk patients, with suspicious oral lesions or recently diagnosed untreated OPLs/OCs, underwent sequential surveillance with WLE and AFV. Biopsies were obtained from all suspicious areas identified on both examinations (n = 189) and one normal-looking control area per person (n = 60). Sensitivity, specificity, and predictive values were calculated for WLE, AFV, and WLE + AFV. Estimates were calculated separately for lesions classified by histopathologic grades as low-grade lesions, high-grade lesions (HGL), and OCs. Sequential surveillance with WLE + AFV provided a greater sensitivity than WLE in detecting low-grade lesions (75% versus 44%), HGLs (100% versus 71%), and OCs (100% versus 80%). The specificity in detecting OPLs/OCs decreased from 70% with WLE to 38% with WLE + AFV. Thirteen of the 76 additional biopsies (17%) obtained based on AFV findings were HGLs/OCs. Five patients (8%) were diagnosed with a HGL/OC only because of the addition of AFV to WLE. In seven patients, additional HGL/OC foci or wider OC margins were detected on AFV. Additionally, AFV aided in the detection of metachronous HGL/OC in 6 of 26 patients (23%) with a history of previously treated head and neck cancer. Overall, the addition of AFV to WLE improved the ability to detect HGLs/OCs. In spite of the lower specificity, AFV + WLE can be a highly sensitive first-line surveillance tool for detecting OPLs/OCs in high-risk patients.

A dose ranging study of photodynamic therapy with porfimer sodium (Photofrin) for treatment of basal cell carcinoma.

BACKGROUND AND OBJECTIVES: While basal cell carcinoma (BCC) is effectively treated by several methods, many patients with numerous or frequently occurring lesions seek alternatives that can treat multiple cancers, with improved cosmetic outcome. PDT for esophageal and lung carcinomas is approved at a porfimer sodium (Photofrin) dose of 2 mg/kg, but lower doses increase selectivity and decrease both cutaneous phototoxicity and cost. We evaluated low doses of porfimer sodium PDT for treatment of multiple BCC. MATERIALS AND METHODS: Seventy-seven patients with 2,041 BCC were injected with 0.75, 0.875, or 1.0 mg/kg porfimer sodium and treated 2 days later with 630-nm light. Clinical responses were determined at 6 months, then periodically to 5 years. RESULTS: Increasing porfimer sodium dose increased complete responses (CR), with initial CR rates of 72.7% (66-78%, 95% CI), 79.9% (73-86%, 95% CI), and 92.2% (91-93%, 95% CI), albeit with some lower selectivity at the highest dose. At 1 mg/kg, 5-year recurrence rates were 28% (21-35%, 95% CI) and 15% (11-18%, 95% CI) for sporadic and nevoid basal cell carcinoma syndrome (NBCCS) lesions, respectively. CONCLUSIONS: This is the largest dose-ranging study of porfimer sodium, and the largest number of lesions treated in a single study. We found that with 1 mg/kg porfimer sodium, PDT can be a selective and durable treatment for sporadic and NBCCS-associated BCC.

Lasers and light sources for PDT: past, present and future.

The more recent use of Photodynamic therapy in Oncology dates to the early 1970's, when Dr. Thomas J. Dougherty, began his investigations into the mechanisms and clinical uses hematoporphyrin derivative (HpD). Since then the therapy has found its way through the regulatory process in numerous countries throughout the world. In many of these locales as it was in the United States, this was the first drug device approval, for oncology, that had been undertaken and ultimately approved, by the regulatory agencies in the respective countries. Throughout this time changes occurred in the formulation of HpD as well as the development of other photosensitizers. The more difficult aspect, however, of this modality has been the availability of reliable, affordable and appropriate devices for the production and delivery of light to the targeted areas. In the last 10 years, however, there has been a slow yet improving landscape in the development of devices for PDT that ultimately will provide the impetus for greater acceptance of PDT in the medical community.