In vitro photodynamic eradication of Pseudomonas aeruginosa in planktonic and biofilm culture.

Photodynamic disinfection (PDD) is a nonantibiotic approach to treating drug-resistant bacterial infections. Pseudomonas aeruginosa, an opportunistic pathogen, is problematic because of its propensity to develop antibiotic resistance and its ability to secrete a protective biofilm matrix. This study examined the ability of PDD to eradicate planktonic and biofilm cultures of P. aeruginosa in vitro. Planktonic P. aeruginosa cultures were briefly exposed to a methylene blue-based photosensitizer formulation and subjected to energy doses ranging from 1.7 to 20.6 J cm(-2) using a 670 nm nonthermal diode laser. Biofilms were grown for 24 and 48 h and exposed to photosensitizer for 30 s before illumination with 13.2 or 26.4 J of energy. A single exposure of planktonic P. aeruginosa to photosensitizer at >15.5 J cm(-2) resulted in 100% eradication (>7 log(10) reduction from control), an effect that could be decreased significantly in the presence of the singlet oxygen quenchers l-tryptophan and sodium azide. Decreasing the energy dose below this threshold by varying both power density and illumination duration resulted in a dose-dependent decrease in bacterial kill. In addition, 24 h biofilm viability was reduced by 99% with single exposure and 99.9% with double exposure, while 48 h biofilm viability was reduced by >99.999% with both single and double exposures. This study shows that PDD is effective in eradicating planktonic and biofilm cultures of P. aeruginosa, supporting the concept that translation into clinical practice for indications such as otitis externa and wound disinfection is a viable option.

Photodynamic therapy of antibiotic-resistant biofilms in a maxillary sinus model.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common chronic conditions in the United States. There is a significant subpopulation of CRS patients who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. Antimicrobial photodynamic therapy (aPDT) is a noninvasive nonantibiotic broad spectrum antimicrobial treatment. Our previous in vitro studies demonstrated that aPDT reduced CRS polymicrobial planktonic bacteria and fungi by >99.9% after a single treatment. However, prior to human treatment, the effectiveness of aPDT to eradicate polymicrobial biofilms in a maxillary sinus cavity must be demonstrated. The objective of this study was to demonstrate the effectiveness of a noninvasive aPDT treatment of antibiotic resistant biofilms known to cause CRS in a novel anatomically correct maxillary sinus in vitro model using an enhanced photosensitizer solution. METHODS: Antibiotic resistant polymicrobial biofilms of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) were grown in an anatomically correct novel maxillary sinus model and treated with a methylene blue/ethylenediamine tetraacetic acid (EDTA) photosensitizer and 670-nm nonthermal activating light. Cultures of the biofilms were obtained before and after light treatment to determine efficacy of biofilm reduction. RESULTS: The in vitro maxillary sinus CRS biofilm study demonstrated that aPDT reduced the CRS polymicrobial biofilm by >99.99% after a single treatment. CONCLUSION: aPDT can effectively treat CRS polymicrobial antibiotic resistant Pseudomonas aeruginosa and MRSA biofilms in a maxillary sinus cavity model.

The effect of antimicrobial photodynamic therapy on human ciliated respiratory mucosa.

BACKGROUND: Chronic recurrent sinusitis (CRS) is one of the most common chronic conditions in the United States. There is a significant subpopulation of CRS patients who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. Antimicrobial photodynamic therapy (aPDT) is a noninvasive nonantibiotic broad spectrum antimicrobial treatment. Our previous in vitro studies demonstrated that aPDT reduced CRS polymicrobial biofilm and planktonic bacteria and fungi by > 99.9% after a single treatment. Prior to human treatment however, aPDT treatment must be demonstrated to not result in histologic damage to the sinus ciliated respiratory epithelium. The objective of this study was to demonstrate the safety of aPDT treatment on a living human ciliated respiratory mucosal model (EpiAirway). METHODS: A study of aPDT treatment of EpiAirway was performed. Treatment groups included a nontreatment control, laser light alone, photosensitizer alone, and therapeutic photosensitizer and light combination (aPDT). At completion of treatment, the EpiAirway tissue was fixed in 10% formalin, paraffin-embedded, sectioned, H&E stained and mounted. All samples were blinded and microscopically examined by a human pathologist to assess any effect of aPDT on the tissue, cilia, or mucosal glands. The results were correlated with the treatment parameters. RESULTS: The EpiAirway histologic study demonstrated no histologic alteration of the respiratory cilia or mucosal epithelium in any of the treatment groups. CONCLUSIONS: aPDT is a safe treatment for CRS resulting in no histologic alteration of human ciliated respiratory mucosa as is found in the human sinuses.

Energy dose parameters affect antimicrobial photodynamic therapy-mediated eradication of periopathogenic biofilm and planktonic cultures.

OBJECTIVE: This study evaluated the in vitro efficacy of a commercially available aPDT system in eradication of the periopathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans in both planktonic and biofilm cultures. BACKGROUND DATA: Antimicrobial photodynamic therapy (aPDT) is an effective antibacterial approach in vitro; however, few data are available regarding effective light-energy parameters. MATERIALS AND METHODS: Planktonic and biofilm cultures of periopathogens were exposed to a methylene blue-based formulation and irradiated with a 670-nm nonthermal diode laser. Energy doses were varied from 2.3 to 9.4 J/cm(2) through adjustments in illumination time and a constant power density. Controls consisted of no treatment, light only, and photosensitizer only. Temperature changes were recorded in experimental samples before and after illumination. RESULTS: aPDT with an energy dose of 9.4 J/cm(2) was effective in eradicating P. gingivalis, F. nucleatum, and A. actinomycetemcomitans in biofilm and planktonic form. Reductions from control in planktonic cultures at this energy dose were 6.8 +/- 0.7, 5.2 +/- 0.6, and 1.9 +/- 0.6 log(10), respectively, whereas biofilm reductions were 4.5 +/- 1.2, 3.4 +/- 1.1, and 4.9 +/- 1.4 log(10). Decreasing the treatment time produced an energy dose-dependent killing effect in both models. Changes in sample temperature did not exceed 3 degrees C under these exposure parameters. CONCLUSION: This study demonstrated that three important periopathogens are susceptible to aPDT-mediated killing, regardless of whether they are present in planktonic or biofilm form. Furthermore, a clear energy dose-dependence exists with this treatment that should to be taken into account when determining optimal treatment times in clinical application.