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Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms
Accepted Manuscript Title: Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms Author: Emilia Pithan Prochnow Maritieli Righi Martins Cibele Bruno Campagnolo Roberto Christ Vianna Marcos Antonio Villetti Karla Zanini Kantorski PII: DOI: Reference:
S1572-1000(15)30024-7 http://dx.doi.org/doi:10.1016/j.pdpdt.2015.08.008 PDPDT 691
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
21-5-2015 5-8-2015 20-8-2015
Please cite this article as: Prochnow Emilia Pithan, Martins Maritieli Righi, Campagnolo Cibele Bruno, Vianna Roberto Christ, Villetti Marcos Antonio, Kantorski Karla Zanini.Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2015.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms Emilia Pithan Prochnow DDS MScDa [email protected], Maritieli Righi Martins DDS MScDa [email protected], Cibele Bruno Campagnolo DDS MScDa [email protected], Roberto Christ Vianna PhDb [email protected], Marcos Antonio Villetti PhDc [email protected], Karla Zanini Kantorski DDS MScD PhDd* [email protected] a
Graduate Program in Oral Science, Periodontology Unit, School of Dentistry, Federal
University of Santa Maria, Santa Maria, Rio Grande do Sul State, Brazil. Marechal Floriano Peixoto Street 1184, 97015-372, Santa Maria/RS, Brazil. Tel. +55.55.3220.9210. b
Graduate Program in Nanoscience, Franciscan University Center, Santa Maria, Rio Grande
do Sul, Brazil. Andradas Street 1614, 97010-491, Santa Maria/RS, Brazil. Tel.: +55.55.3220.9210 c
Graduate Program in Chemistry, Chemical-Physical Unit, Federal University of Santa Maria,
Santa Maria, Rio Grande do Sul State, Brazil. Endereço: Roraima Avenue 1000, 97105-900, Santa Maria/RS, Brazil. Tel.: +55.55.3220.9258. d
Graduate Program in Oral Science, Periodontology Unit, Stomatology Department, School of
Dentistry, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul State, Brazil. Address: Marechal Floriano Peixoto Street 1184, 97015-372, Santa Maria/RS, Brazil. *
Corresponding author at: Marechal Floriano Peixoto Street 1184, 97015-372, Santa
Maria/RS, Brazil. Tel.: +55.55.3220.9284.
HIGHLIGHTS MB formulations produced more singlet oxygen than TB formulations. MB/ethanol formulations produced more singlet oxygen than MB/water formulation. MB/ethanol formulations promoted a statistical reduction of P. aeruginosa. TB/ethanol formulations promoted no significant reduction of P. aeruginosa.
Abstract Background data: methylene blue (MB) and toluidine blue (TB) are recognized as safe photosensitizers (Ps) for use in humans. The clinical effectiveness of the antimicrobial photodynamic therapy with MB and TB needs to be optimized, and ethanol can increase their antimicrobial effect. Formulations of MB and TB containing ethanol were evaluated for their ability to produce singlet oxygen and their antibacterial effect on Pseudomonas aeruginosa biofilms. Methods: Photoactivated formulations were prepared by diluting the Ps (250 μM) in buffered water (pH 5.6, sodium acetate/acetic acid), 10% ethanol (buffer: ethanol, 90:10), or 20% ethanol (buffer: ethanol, 80:20). Biofilms also were exposed to the buffer, 10% ethanol, or 20% ethanol without photoactivation. Untreated biofilm was considered the control group. The production of singlet oxygen in the formulations was measured based on the photooxidation of 1,3-diphenylisobenzofuran. The photo-oxidation and CFU (log10) data were evaluated by two-way ANOVA and post-hoc Tukey’s tests. Results: In all the formulations, compared to TB, MB showed higher production of singlet oxygen. In the absence of photoactivation, neither the buffer nor the 10% ethanol solution showed any antimicrobial effect, while the 20% ethanol solution significantly reduced bacterial viability (P = 0.009). With photoactivation, only the formulations containing MB and both 10% and 20% ethanol solutions significantly reduced the viability of P. aeruginosa biofilms when compared with the control. Conclusions: MB formulations containing ethanol enhanced the antimicrobial effect of the photodynamic therapy against P. aeruginosa biofilms in vitro.
Keywords: Laser in Dentistry; Photodynamic Therapy (PDT); Microbiology; Photodynamic inactivation.
Introduction Pseudomonas aeruginosa is a pathogen associated with nosocomial infections, pneumonia in patients with cystic fibrosis, and bacteremia in immunocompromised patients1. In the United States, 60–80% of patients with cystic fibrosis are infected with P. aeruginosa2, and approximately 1,400 deaths due to nosocomial pneumonia per year are caused by the bacterium 3. It is the second most frequent pathogen associated with ventilator-associated pneumonia, and the third or fourth most common cause of septicemia, urinary tract infections, and surgical site infections4. In Brazil, it is the third most isolated bacterium in nosocomial infections according to epidemiological surveillance programs5. P. aeruginosa is capable of expressing different phenotypes due to its high degree of genomic flexibility6 and is recognized for its intrinsic resistance to antibiotics. This resistance can be attributed to the low permeability of its cell wall, the action of efflux pumps that remove the antibiotic molecules that penetrate the intramembranous channels7, the mutations that may change the expression and function of the chromosomes, the acquisition of resistant genes by mobile genetic elements such as plasmids, bacteriophages, and transposons8, and the ability of this microorganism to invade eukaryotic cells and become an opportunistic intracellular pathogen10,11. In biofilms, P. aeruginosa resistance to antibiotics can be 10 to 1,000 times greater than that in planktonic culture12, which is suggestive of the distinct mechanisms for resistance established in biofilms13. These mechanisms include the super expression of efflux pumps, the presence of an extracellular matrix of polysaccharides that surround the microbial colonies forming a protective barrier against antimicrobials14,15,16, and the presence of small subpopulations of P. aeruginosa that are metabolically inactive and therefore unaffected by antimicrobials17. Owing to these resistance mechanisms, chronic and difficult-to-eradicate infections are associated with P. aeruginosa biofilms18. In this context, some authors have been reporting that the development of alternative
supportive therapies, such as antimicrobial photodynamic therapy (aPDT), is of great importance for treatment of P. aeruginosa infections, because its mechanism of action is completely different from the current antibiotic therapy19,20. Also, aPDT has been demonstrated low probability of developing bacterial resistance21-24. Some clinical studies have been reporting the aPDT effect for treatment of chronic skin ulcers25 and a number of skin lesions26 infected with P. aeruginosa. Besides, previous study demonstrated that higher concentrations of photosensitizer (Ps) and intensity of light are required for killing of P. aeruginosa27. Therefore, the P. aeruginosa biofilm consists in an important model for evaluating the antimicrobial effect of the photodynamic therapy. aPDT involves application of a Ps, which, when activated with light of the appropriate wavelength, performs electronic transfers with neighboring molecules, generating reactive oxygen species (ROS), and energy transfers with molecular oxygen, forming singlet oxygen23,28-31. Phenothiazine-derived Ps such as methylene blue (MB) and toluidine blue (TB) are recognized as safe for use in humans32. There is evidence that aPDT with MB can be effective in reducing the viability of P. aeruginosa biofilms33. The formulations commonly employed use water as a solvent for MB. Previous studies have shown that these formulations result in lower singlet oxygen production34, reduced half-life (4 µs)13, and low diffusion potential35 when compared to experimental formulations containing ethanol. Owing to these changes in the photophysical and photochemical characteristics of MB diluted in ethanol, its presence may enhance the antimicrobial effect of aPDT34. To the best of knowledge, there is no evidence of the aPDT effect using formulations with different phenothizine-derived Ps and with different concentrations of ethanol on bacterial biofilms.
This is the first investigation aimed to evaluate the antimicrobial effect of the aPDT in P. aeruginosa biofilms, using formulations of MB and TB containing ethanol (10% or 20%). The research hypothesis were (a) that prior none difference in the antimicrobial effect of photodynamic therapy would be detected between formulations with ethanol 10% and 20%, and (b) that formulations with toluidine blue would promote higher antimicrobial effect than methylene blue formulation.
Materials and Methods Formulations The compositions of the formulations evaluated are shown in Table 1. MB and TB Ps (Sigma-Aldrich, São Paulo, SP, Brazil) were used. All formulations were prepared using a buffer solution of pH of 5.6 (sodium acetate/acetic acid, Sigma-Aldrich). The pH of the solutions was adjusted by using a DM-20 pH meter (Digimed®, São Paulo, SP, Brazil), Solutions were prepared using ultra-pure water (Milli-Q) and ethanol (Cromoline®, Diadema, SP, Brazil). A diode laser (Thera Lase - DMC, São Carlos, SP, Brazil) with a wavelength of 660 nm, a fiber spot size of 0.02827 cm2, and a continuous emission mode was used as the light activation source for all evaluations.
Photochemical Characterization Measurement of singlet oxygen production by using 1,3-diphenylisobenzofuran (DPBF): The production of singlet oxygen by MB and TB in the different formulations was measured based on the photo-oxidation of 1,3-diphenylisobenzofuran (DPBF) (SigmaAldrich), which is sensitive to the products formed from the type-II reaction.
Solutions containing DPBF (75 µm) and MB (2.5 µm) or DPBF (75 µm) and TB (2.5 µm) were placed in a quartz cuvette, and the absorbance spectra were measured with a UV/Vis spectrophotometer (Varian Cary 50 Bio, Barueri, SP, Brazil) at a temperature of 25°C. After obtaining the initial scan, the formulations were subjected to irradiation with a low-power diode laser (Thera Lase - DMC), with the power output of the emitter set at 30 mW and a power density of 1 W/cm² for up to 30 s (with intervals of 1 to 10 s and 2 to 30 s). Production of singlet oxygen by MB and TB was measured based on the decrease in the intensity of absorbance at 415 nm as a function of the irradiation time. The evaluations were conducted in triplicate.
Antimicrobial photodynamic effects In vitro biofilm formation: Biofilms of standard laboratory strains of Pseudomonas aeruginosa PA01 (gramnegative) were formed in vitro on acrylic specimens with an 8-mm diameter. A standardized suspension of Pseudomonas aeruginosa PA01 containing 107 cells/mL was prepared using a spectrophotometer (Instrutherm, UV-1000A, São Paulo, SP, Brazil). An optical density (OD 600nm)
of 1 was used as the parameter. Sterile acrylic specimens (measuring 8 mm in diameter, 1 mm in height) were placed
in a 24-well plate (Costar Corning, New York, NY, USA). Each well contained 2 mL of brain heart infusion broth (BHI, Difco, Detroit, MI, USA) supplemented with 5% sucrose. The wells containing the specimens submerged in BHI broth were inoculated with 100 µL of the standardized suspension (107 cells/mL) and incubated in an orbital shaker (Novatecnica, Modelo NT712, Piracicaba, SP, Brazil) at 37°C for 5 days at 75 rpm25, 26. The wells were inoculated only once and the broth was changed every 24 h. After the incubation period, the
samples were washed with 2 mL of phosphate buffered saline (PBS) to remove cells that were not adhered to the biofilm.
In vitro photosensitization: The antimicrobial effect was evaluated for formulations that were not photoactivated (buffer, ethanol 10%, ethanol 20%) and for photoactivated formulations (MB/water, MB/ethanol 10%, MB/ethanol 20%, TB/water, TB/ethanol 10%, TB/ethanol 20%) according to Table 1. The biofilms were treated with laser alone. The biofilm that formed on specimens that were not treated served as the control group. The specimens containing the biofilm were transferred to a fresh 24-well plate and immersed in 1 ml of each of the different formulations. In formulations that were not photoactivated, the contact of the formulation with the biofilm was maintained for 15 min. In the aPDT groups, the contact was maintained for 5 min. The biofilms were then photoactivated by low-level laser irradiation at a wavelength of 660 nm, a power intensity of 30 mW, an energy of 18 J, and laser fluence of 40 J/cm2 for 10 min. The treated biofilm specimens from each formulation were placed in individual falcon tubes containing 10 ml of PBS and were homogenized using an ultrasonic homogenizer (Sonopuls HD 2200, Bandelin Electronic, Berlin, Germany) at a power of 50 W per 30 s. Next, serial dilutions (10-3 to 10-4) were prepared, and 100 µL-aliquots of the dilutions were seeded in duplicate onto plates containing MacConkey agar (Himedia Laboratories Pvt. Ltd., Mumbai, Índia). The plates were incubated for 48 h at 37 °C. An examiner blinded to the experimental groups determined the number of colony-forming units (CFU/ml). Five specimens for each formulation were used. The experiment was performed in triplicate.
Statistical analysis The data for CFU/ml were converted to the logarithmic form. The data of photochemical characterization and antimicrobial activity were expressed as mean value with standard deviation. Differences between groups were evaluated by the variance tests two-way ANOVA and post-hoc Tukey’s for the photochemical and antimicrobial effect of the aPDT data. . Differences between groups were evaluated by the variance tests one-way ANOVA and post-hoc Tukey’s for the data of antimicrobial effect of the formulations without irradiation. The level of significance was set at 5%. Statistical analysis was performed with the SPSS software, v.13.0 (SPSS Inc., Chicago, IL, USA).
Results Measurement of singlet oxygen production by using 1,3-diphenylisobenzofuran (DPBF) The photodynamic activity coefficients generated by the photo-oxidation of singlet oxygen-sensitive DPBF are shown in Table 2. Compared to TB, MB showed higher production of singlet oxygen, regardless of the solvent used. Inclusion of ethanol in the TB formulations did not result in a significant increase in singlet oxygen production. In formulations containing MB, inclusion of 10% and 20% ethanol significantly increased singlet oxygen production when compared to that observed with formulations containing water alone. The mean values of the DPBF photolytic decomposition kinetic constants (s-1), in different formulations are represented graphically in Figure 1 and 2.
In vitro photodynamic inactivation of biofilms Table 3 shows the values of CFU/ml obtained in the formulations without photoactivation. No antimicrobial effect was observed with the buffer solution or the 10%
ethanol solution. With the 20% ethanol solution, a significant microbial reduction (P = 0.009) was observed compared to that observed in the control (no treatment). The mean CFU log10 values and the standard deviation in the formulations subjected to photoactivation are shown in Table 4. None statistical difference was verified among formulations containing ethanol or water, independently of the photosensitizer type. Formulations containing MB and ethanol showed a statistically significant reduction in the viability of P. aeruginosa biofilms. In the other formulations, the reductions were not statistically significant. The antimicrobial behavior of MB and TB diluted in water was similar. In contrast, MB seemed to be more sensitive to the inclusion of ethanol in the formulation, thus showing lower CFU log10 mean values compared to TB, although these differences were not statistically significant.
Discussion The results of the present study demonstrated that the use of ethanol in the formulation containing MB promoted a statistically significant reduction in the viability of P. aeruginosa biofilms compared with untreated biofilm. These effects were not observed when ethanol was used with TB and when water was used independently of the Ps type. Similar antimicrobial effects were noted between formulations with 10% and 20% ethanol, supporting our first research hypothesis. None statistical difference was verified between MB and TB independently of the formulations, refuting our second hypothesis. The average microbial reduction observed with MB/10% ethanol and MB/20% ethanol was approximately 2.58 log10 and 2.66 log10 compared with the control group, respectively. The reduction was relatively lower with TB (0.79 log10 and 1.70 log10 for TB/10% ethanol and TB/20% ethanol, respectively) and without statistical significance. The
relatively stronger effect of antimicrobial formulations containing ethanol and MB can be attributed to the higher production of singlet oxygen. The presence of ethanol in the formulation resulted in lower MB molecular aggregation, i.e., a higher proportion of monomers in relation to dimers34. Dimers are less effective in capturing energy36,37, acting predominantly in reactions involving electronic exchanges with the substrate (type I reaction), with less production of singlet oxygen (type II reaction). Thus, a formulation that stabilizes the monomers is preferable for therapeutic purposes34. Another aspect to consider is the increase in the half-life of the singlet oxygen generated in the presence of ethanol, which is approximately five times greater than that of singlet oxygen generated in the presence of water13. This phenomenon may provide more time for the Ps to interact with bacterial cells. The Figure 1 and 2 demonstrated that the differences between water- and ethanolformulations in the photolytic decomposition of DPBF were more pronounced after 20 seconds of irradiation. Therefore, irradiation time higher than 20 seconds could be evaluated to improve antimicrobial effect of the formulations with ethanol. In this study, using 20% ethanol against the biofilm showed an antimicrobial effect, which confirms the results obtained by Peters et al. (2003)38. These authors found that ethanol in concentrations equal to or greater than 20% can inhibit the growth of monospecies biofilms, whereas this effect is observed in multispecies biofilms at concentrations greater than 30%. The mechanism by which ethanol exerts its antimicrobial action involves damaging the bacterial membrane and the rapid denaturation of proteins, leading to cell lysis39. When 20% or 10% ethanol were used in the aPDT with TB, the mean microbial reduction was similar when compared to that achieved using 20% or 10% ethanol without photoactivation. Therefore, the antimicrobial effect of the formulations containing TB and ethanol were associated with the mechanisms by which ethanol exerts its antimicrobial action and not by increase of the photochemical properties of the formulations. This was confirmed
by findings when the inclusion of ethanol in the TB formulations did not result in a significant increase in singlet oxygen production. aPDT had no effect when TB was used with ethanol. TB is a Ps more hydrophobic than MB, and should interact more easily with the bacterial membrane (in the hydrophobic region) than MB27. In fact, TB is considered as an effective membrane destructive agent40, while MB damages the bacterial cell DNA and to less extent the outer membrane27. The mechanism of the cell death could depend of how of the Ps interacts with the bacteria. It is possible that the mechanism of interaction between TB and bacterial cell had been changed by the ethanol inclusion in the formulations, impairing the antimicrobial photodynamic effect of the TB. The behavior of MB and TB diluted in water was similar with respect to the antimicrobial effect, although MB produced significantly more singlet oxygen. Thus, other factors may be associated with the antimicrobial action, such as the production of ROS, which may play an important role in the antimicrobial action of photodynamic therapy in areas characterized by low availability of oxygen or oxygen depletion. In 2001, Usacheva et al.27 observed a higher antimicrobial effect of the aPDT on the planktonic cells of P. aeruginosa with TB compared to that observed with MB when both were diluted in water. The authors found that the TB interacted more strongly with bacterial lipopolysaccharides, which explained its relatively stronger antimicrobial effect. Nonetheless, this behavior may not be the same against P. aeruginosa biofilms. Previous studies have investigated antimicrobial agents in different models of P. aeruginosa biofilms. Yu et al. 201241 demonstrated that aztreonam reduced the cell viability of P. aeruginosa biofilms formed on epithelial cells by approximately 1 log10. On the other hand, thromycin was more effective, showing reductions above 4 log10. Elkahtib and Noreddin (2014)42 observed that 8 times the minimum inhibitory concentration of ciprofloxacin and 4 times that of clarithromycin were required for a microbial reduction of 2.2
log10 and 2.8 log10, respectively, in biofilms established in 24 h. In this study, reductions of 2.58 log10 and 2.66 log10 in biofilms within 5 days were observed with MB diluted in 10% and 20% ethanol, respectively. Previous investigations have demonstrated that biofilms that formed over longer periods exhibit higher resistance to antimicrobials43,44. Thus, considering that systemic antibiotics may result in opportunistic infections and hypersensitivity reactions, and that the development of bacterial resistance is a growing problem22, aPDT has been identified as a potential alternative to treat infections associated with P. aeruginosa biofilms. Besides, infections caused by other microorganisms could be benefited from formulations evaluated in the present study. Therefore, the aPDT using formulations with ethanol and MB or TB could be evaluated on bacterial biofilms of other species.
Conclusions The antimicrobial photodynamic effect can be optimized with the use of photosensitizers proven to be clinically safe, such as MB, with the inclusion of ethanol in the formulation.
Acknowledgments This study was supported by the Foundation for Post-Graduate Education (CAPES), Brasilia, Brazil.
Author Disclosure Statement No competing financial interests exist.
References 1. Fugitani S, Sun H-Y, Yu VL, Weingarten JA. Pneumonia Due to Pseudomonas aeruginosa. Part I: Epidemiology, Clinical Diagnosis, and Source. Rec Adv in Ch Med 2011;139:909-919. 2. Aaron SD, Vandemheen KL, Ramotar K et al. Infection with Transmissible Strains of Pseudomonas aeruginosa and Clinical Outcomes in Adults with Cystic Fibrosis. JAMA 2010;304:2145-2153. 3. Anaissie E, Penzak SR, Dignani C. The Hospital Water Supply as a Source of Nosocomial Infections. A Plea for Action. Arch Intern Med 2002;162:1483-1492. 4. Falkinham JO, Hilborn ED, Ardulno MJ, Prudlen A, Edwards MA. Epidemiology and Eology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. Envir. Health Persp 2015 [Epub ahead of print]. 5. Sader HS, Jones RN, Gales AC, Silva JB and Pignatari AC. SENTRY antimicrobial surveillance program report: Latin American and Brazilian results for 1997 through 2001. Braz J Infect Dis 2004;8:25-79. 6. Wehmhöner D, Häussler S, Tümmler B, Jänsch L, Bredenbruch F, Wehland J and Steinmetz I. Inter- and intraclonal diversity of the Pseudomonas aeruginosa proteome manifests within the secretome. J Bacteriol 2003;185:5807-5814. 7. Hancock RE and Speert DP. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 2000;3:247-255. 8. Lister PD, Wolter DJ and Hanson N. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microb Rev 2009;22:582-610.
9. Garcia-Medina R, Dunne WM, Singh PK, Brody SL. Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells. Infect. Immun. 2005; 73: 8298-8305. 10. Schmiedl A, Kerber-Momot T, Munder A, Pabst R and Tschernig T. Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa. Cell Tissue Res 2010;342:67-73. 11. Buyck JM, Tuikens PM, Van Bambeke F. Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PA01 in a model of THP-1 Human Monocytes. Antimic Agents and Chem 2013;57:2310-2318. 12. Nickel JC, Ruseska I, Wright JB and Costerton JW. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother 1985;27:619-624. 13. Meisel P, Kocher T. Photodynamic therapy for periodontal diseases: state of the art. J Photochem Photobiol 2005;79:159-170. 14. Ryder C, Byrd M and Wozniak DJ. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr. Opin. Microbiol 2007;10:644-648. 15. Colvin KM, Gordon VD, Murakami K, Borlee BR, Wozniak DJ, Wong GC and Parsek MR. The Pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog 2011;7: e1001264. 16. Nair MGV, Sharma SCD, Sahni BAK, et al. Antimicrobial use and antimicrobial resistance in nosocomial pathogens at a tertiary care hospital in Pune. Medical Jour Armed Forces India 2015;71:112-119. 17. Mulcahy LR, Burns JL, Lory S and Lewis K. Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J Bacteriol 2010;192:6191-6199.
18. Parsek MR and Singh PK. Bacterial biofilms: an emerging link to disease pathogenesis. Annu. Rev. Microbiol 2003;57:677-701. 19. Sharma G, Rao S, Bansal A, Dang S, Gupta S, Gabrani R. Pseudomonas aeruginosa biofilm: Potential therapeutic targets. Biologicals 2014;42:1-7. 20. Dorotkiewicz-Jach A, Augustyniak D, Olszak T, Drulis-Kawa Z. Modern therapeutic approaches
against
Pseudomonas
aeruginosa
infections.
Curr
Med
Chem
2015;22:1642-1664. 21. Wainwright M, Phoenix DA, Marland J, Wareing DR and Bolton FJ. A study of photobactericidal activity in the phenothiazinium series. Immunol Med Microbiol 1997;19:75-80. 22. Hamblin MR and Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem Photobiol Sci 2004;3:436-450. 23. Maisch T. Anti-microbial photodynamic therapy: useful in the future? Lasers Med Sci 2007;22:83-91. 24. Goulart RC, Bolean M, Paulino TP, Thedei G, Souza SL, Tedesco AC and Ciancaglini P. Photodynamic therapy in planktonic and biofilm cultures of Aggregatibacter actinomycetemcomitans. Photomed Laser Surg 2010;28:53-60. 25. Lei X, Liu B, Huang Z, Wu J. A clinical study of photodynamic therapy for chronic skin ulcers in lower limbs infected with Pseudomonas aeruginosa. Arch Dermatol Res 2015;307:49-55. 26. Calzavara-Pinton PG, Rossi MT, Sala R. A retrospective analysis of real-life practice of off-label photodynamic therapy using methyl aminolevulinate (MAL-PDT) in 20 italian dermatology departments. Part 2: oncologic and infectious indications. Photochem Photobiol Sci 2013;12:158-165.
27. Usacheva MN, Teichert MC and Biel MA. Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram-positive and gram-negative microorganisms. Lasers Surg Med 2001;29:165-173. 28. Castano AP, Demidova TN and Hamblin MR. Mechanisms in photodynamic therapy: part one - photosensitizers, photochemistry and cellular localization. Photodiag Photodynam Ther 2004;1:279-293. 29. Ge L, Shu R, Li Y, et al. Adjunctive effect of photodynamic therapy to scaling and root planning in the treatment of chronic periodontitis. Photomed Laser Surg 2011;29:33-37. 30. Soukos SN and Goodson JM. Photodynamic therapy in the control of oral biofilms. Periodontol. 2000 2011;55:143-166. 31. Rolim JP, de-Melo MA, Guedes SF, et al. The antimicrobial activity of photodynamic therapy against Streptococcus mutans using different photosensitizers. J Photochem Photobiol B 2012;106:40-46. 32. National Toxicology Program. Executive summary of safety and toxicity information for methylene blue trihydrate. U.S. Department of Health and Human Services 1990;7220-79-3. 33. Biel MA, Sievert C, Usacheva M, et al. Reduction of Endotracheal Tube Biofilms using Antimicrobial Photodynamic Therapy. Lasers Surg Med 2011;43:586-590. 34. George S and Kishen A. Photophysical, photochemical, and photobiological characterization of methylene blue formulations for light-activated root canal disinfection. J Biomed Opt 2007;12:29-34. 35. Ochsner MJ. Photophysical and photobiological processes in the photodynamic therapy of tumors. Photochem Photobiol 1997;39:1-18.
36. Gabrielli DS, Belisle E, Severino D, Kowaltowski AJ and Baptista MS. Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions. Photochem Photobiol 2004;79:227-232. 37. Patil KR and Talap P. Self-aggregation of methylene blue in aqueous medium and aqueous solutions of Bu4 NBr and urea. Phys Chem Chem Phys 2002;2:4313-4317. 38. Peters MB, Ward RM, Rane HS, Lee SA and Nover MC. Efficacy of ethanol against Candida albicans and Staphylococcus aureus polymicrobial biofilms. Antimicrob Agents Chemoter 2013;57:74-82. 39. Macdonnell G and Russel AD. Antiseptics and disinfectants: activity, action and resistance. Clin Microbiol Rev 1999;12:147-179. 40. Bhatti M, MacRobert A, Meghji S, Henderson B, Wilson M. A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization. Photochem Photobiol 1998;68:370-376. 41. Yu Q, Griffin EF, Moreau-Marquis S, Schwartzman JD, Stanton BA and O’Toole GA. In vitro evaluation of tobramycin and aztreonam versus Pseudomonas aeruginosa biofilms on cystics fibrosis-derived human airway epithelial cells. J Antimicrob Chemother. 2012; 67: 2673-2681. 42. Elkhatib W and Noreddin A. Efficacy of ciprofloxacin-clarithromycin combination against drug-resistant Pseudomonas aeruginosa mature biofilm sing in vitro experimental model. Microbi Drug Resist 2014;20:575-582. 43. Anwar H, Strap JL, Costerton JW. Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob Agents Chemother 1992;36:1347-1351 44. Singla S, Harjai K and Chhibber S. Susceptibility of different phases of biofilm of Klebsiella pneumoniae to three different antibiotics. J Antibiot 2013;66:61-66.
FIGURE CAPTIONS Figure 1 The mean values of the DPBF photolytic decomposition kinetic constants (s-1) in different formulations with methylene blue (MB). Figure 2 The mean values of the DPBF photolytic decomposition kinetic constants (s-1) in different formulations with toluidine blue (TB).
Fig. 1
Fig. 2
Tables Table 1. Formulations of Methylene Blue (MB) and Toluidine Blue (TB) containing water, 10% ethanol, and 20% ethanol submitted to the irradiation. Formulations without photosensitizer did not submitted to the irradiation. Photodynamic Formulations
Formulation compositions
inactivation of biofilm
MB/water
MB dissolved in Milli-Q ultrapure water
MB/ethanol 10%
MB dissolved in 10% ethyl alcohol and Milli-Q ultrapure water (90:10)
MB/ethanol 20%
MB dissolved in 20% ethyl alcohol and Milli-Q ultrapure (80:20)
TB/water
TB dissolved in Milli-Q ultrapure water
TB/ethanol 10%
TB dissolved in 10% ethyl alcohol and Milli-Q
YES
ultrapure water (90:10) TB/ethanol 20%
TB dissolved in 20% ethyl alcohol and Milli-Q ultrapure water (80:20)
Buffer
buffer solution to a pH of 5.6 - sodium acetate/acetic acid NO
Ethanol 10%
10% ethyl alcohol and Milli-Q ultrapure water
Ethanol 20%
20% ethyl alcohol and Milli-Q ultrapure water
All formulations were prepared using a buffer solution of pH of 5.6 (sodium acetate/acetic acid).
Table 2. Mean (standard deviation) of the photodynamic activity coefficient generated by the photo-oxidation of singlet oxygen-sensitive DPBF. Photosensitizers Groups MB
TB
Water
0.044 (0.0005)A
0.104 (0.01)C
Ethanol 10%
0.067 (0.001)B
0.113 (0.009)C
Ethanol 20%
0.063 (0.001)B
0.114 (0.01)C
Parameters: [TB and MB] = 2.5 µM; [DPBF] = 75 µM. Two-way ANOVA e post hoc Tukey tests. Different capital letters represent statistically significant differences among the groups (P<0.05).
Table 3. Mean CFU/ml (log) (standard deviations) of P. aeruginosa in the formulations without photoactivation. Groups
P. aeruginosa (mean ± SD)
Control
6.80 (0.44)A
Buffer
6.22 (0.30)A
Ethanol 10%
6.00 (0.72)A
Ethanol 20%
5.33 (0.45)B
Different capital letters (A and B) represent statistically significant differences among the groups (One-Way ANOVA e post hoc Tukey tests, P<0.05)
Tabela 4. Mean CFU/ml (log) (standard deviations) of P. aeruginosa exposed to different formulations in the photodynamic inactivation. Photosensitizers Groups MB
TB
Control
6.80 (0.44)A
Laser alone
6.99 (0.57)A
Water
5.47 (0.61)AB
5.50 (0.37)AB
Ethanol 10%
4.22 (1.02)B
6.01 (0.67)AB
Ethanol 20%
4.14 (1.26)B
5.10 (2.03)AB
Parameters: [TB and MB] = 250 µM. Irradiation parameters: Low level laser irradiation, wavelight of 660 nm, power intensity of 30 mW, energy of 20 J and laser fluence of 40 J/cm2 for 10 min. Different capital letters (A and B) represent statistically significant differences among the groups (Two-Way ANOVA e post hoc Tukey tests, P<0.05)
S1572-1000(15)30024-7 http://dx.doi.org/doi:10.1016/j.pdpdt.2015.08.008 PDPDT 691
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
21-5-2015 5-8-2015 20-8-2015
Please cite this article as: Prochnow Emilia Pithan, Martins Maritieli Righi, Campagnolo Cibele Bruno, Vianna Roberto Christ, Villetti Marcos Antonio, Kantorski Karla Zanini.Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2015.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms Emilia Pithan Prochnow DDS MScDa [email protected], Maritieli Righi Martins DDS MScDa [email protected], Cibele Bruno Campagnolo DDS MScDa [email protected], Roberto Christ Vianna PhDb [email protected], Marcos Antonio Villetti PhDc [email protected], Karla Zanini Kantorski DDS MScD PhDd* [email protected] a
Graduate Program in Oral Science, Periodontology Unit, School of Dentistry, Federal
University of Santa Maria, Santa Maria, Rio Grande do Sul State, Brazil. Marechal Floriano Peixoto Street 1184, 97015-372, Santa Maria/RS, Brazil. Tel. +55.55.3220.9210. b
Graduate Program in Nanoscience, Franciscan University Center, Santa Maria, Rio Grande
do Sul, Brazil. Andradas Street 1614, 97010-491, Santa Maria/RS, Brazil. Tel.: +55.55.3220.9210 c
Graduate Program in Chemistry, Chemical-Physical Unit, Federal University of Santa Maria,
Santa Maria, Rio Grande do Sul State, Brazil. Endereço: Roraima Avenue 1000, 97105-900, Santa Maria/RS, Brazil. Tel.: +55.55.3220.9258. d
Graduate Program in Oral Science, Periodontology Unit, Stomatology Department, School of
Dentistry, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul State, Brazil. Address: Marechal Floriano Peixoto Street 1184, 97015-372, Santa Maria/RS, Brazil. *
Corresponding author at: Marechal Floriano Peixoto Street 1184, 97015-372, Santa
Maria/RS, Brazil. Tel.: +55.55.3220.9284.
HIGHLIGHTS MB formulations produced more singlet oxygen than TB formulations. MB/ethanol formulations produced more singlet oxygen than MB/water formulation. MB/ethanol formulations promoted a statistical reduction of P. aeruginosa. TB/ethanol formulations promoted no significant reduction of P. aeruginosa.
Abstract Background data: methylene blue (MB) and toluidine blue (TB) are recognized as safe photosensitizers (Ps) for use in humans. The clinical effectiveness of the antimicrobial photodynamic therapy with MB and TB needs to be optimized, and ethanol can increase their antimicrobial effect. Formulations of MB and TB containing ethanol were evaluated for their ability to produce singlet oxygen and their antibacterial effect on Pseudomonas aeruginosa biofilms. Methods: Photoactivated formulations were prepared by diluting the Ps (250 μM) in buffered water (pH 5.6, sodium acetate/acetic acid), 10% ethanol (buffer: ethanol, 90:10), or 20% ethanol (buffer: ethanol, 80:20). Biofilms also were exposed to the buffer, 10% ethanol, or 20% ethanol without photoactivation. Untreated biofilm was considered the control group. The production of singlet oxygen in the formulations was measured based on the photooxidation of 1,3-diphenylisobenzofuran. The photo-oxidation and CFU (log10) data were evaluated by two-way ANOVA and post-hoc Tukey’s tests. Results: In all the formulations, compared to TB, MB showed higher production of singlet oxygen. In the absence of photoactivation, neither the buffer nor the 10% ethanol solution showed any antimicrobial effect, while the 20% ethanol solution significantly reduced bacterial viability (P = 0.009). With photoactivation, only the formulations containing MB and both 10% and 20% ethanol solutions significantly reduced the viability of P. aeruginosa biofilms when compared with the control. Conclusions: MB formulations containing ethanol enhanced the antimicrobial effect of the photodynamic therapy against P. aeruginosa biofilms in vitro.
Keywords: Laser in Dentistry; Photodynamic Therapy (PDT); Microbiology; Photodynamic inactivation.
Introduction Pseudomonas aeruginosa is a pathogen associated with nosocomial infections, pneumonia in patients with cystic fibrosis, and bacteremia in immunocompromised patients1. In the United States, 60–80% of patients with cystic fibrosis are infected with P. aeruginosa2, and approximately 1,400 deaths due to nosocomial pneumonia per year are caused by the bacterium 3. It is the second most frequent pathogen associated with ventilator-associated pneumonia, and the third or fourth most common cause of septicemia, urinary tract infections, and surgical site infections4. In Brazil, it is the third most isolated bacterium in nosocomial infections according to epidemiological surveillance programs5. P. aeruginosa is capable of expressing different phenotypes due to its high degree of genomic flexibility6 and is recognized for its intrinsic resistance to antibiotics. This resistance can be attributed to the low permeability of its cell wall, the action of efflux pumps that remove the antibiotic molecules that penetrate the intramembranous channels7, the mutations that may change the expression and function of the chromosomes, the acquisition of resistant genes by mobile genetic elements such as plasmids, bacteriophages, and transposons8, and the ability of this microorganism to invade eukaryotic cells and become an opportunistic intracellular pathogen10,11. In biofilms, P. aeruginosa resistance to antibiotics can be 10 to 1,000 times greater than that in planktonic culture12, which is suggestive of the distinct mechanisms for resistance established in biofilms13. These mechanisms include the super expression of efflux pumps, the presence of an extracellular matrix of polysaccharides that surround the microbial colonies forming a protective barrier against antimicrobials14,15,16, and the presence of small subpopulations of P. aeruginosa that are metabolically inactive and therefore unaffected by antimicrobials17. Owing to these resistance mechanisms, chronic and difficult-to-eradicate infections are associated with P. aeruginosa biofilms18. In this context, some authors have been reporting that the development of alternative
supportive therapies, such as antimicrobial photodynamic therapy (aPDT), is of great importance for treatment of P. aeruginosa infections, because its mechanism of action is completely different from the current antibiotic therapy19,20. Also, aPDT has been demonstrated low probability of developing bacterial resistance21-24. Some clinical studies have been reporting the aPDT effect for treatment of chronic skin ulcers25 and a number of skin lesions26 infected with P. aeruginosa. Besides, previous study demonstrated that higher concentrations of photosensitizer (Ps) and intensity of light are required for killing of P. aeruginosa27. Therefore, the P. aeruginosa biofilm consists in an important model for evaluating the antimicrobial effect of the photodynamic therapy. aPDT involves application of a Ps, which, when activated with light of the appropriate wavelength, performs electronic transfers with neighboring molecules, generating reactive oxygen species (ROS), and energy transfers with molecular oxygen, forming singlet oxygen23,28-31. Phenothiazine-derived Ps such as methylene blue (MB) and toluidine blue (TB) are recognized as safe for use in humans32. There is evidence that aPDT with MB can be effective in reducing the viability of P. aeruginosa biofilms33. The formulations commonly employed use water as a solvent for MB. Previous studies have shown that these formulations result in lower singlet oxygen production34, reduced half-life (4 µs)13, and low diffusion potential35 when compared to experimental formulations containing ethanol. Owing to these changes in the photophysical and photochemical characteristics of MB diluted in ethanol, its presence may enhance the antimicrobial effect of aPDT34. To the best of knowledge, there is no evidence of the aPDT effect using formulations with different phenothizine-derived Ps and with different concentrations of ethanol on bacterial biofilms.
This is the first investigation aimed to evaluate the antimicrobial effect of the aPDT in P. aeruginosa biofilms, using formulations of MB and TB containing ethanol (10% or 20%). The research hypothesis were (a) that prior none difference in the antimicrobial effect of photodynamic therapy would be detected between formulations with ethanol 10% and 20%, and (b) that formulations with toluidine blue would promote higher antimicrobial effect than methylene blue formulation.
Materials and Methods Formulations The compositions of the formulations evaluated are shown in Table 1. MB and TB Ps (Sigma-Aldrich, São Paulo, SP, Brazil) were used. All formulations were prepared using a buffer solution of pH of 5.6 (sodium acetate/acetic acid, Sigma-Aldrich). The pH of the solutions was adjusted by using a DM-20 pH meter (Digimed®, São Paulo, SP, Brazil), Solutions were prepared using ultra-pure water (Milli-Q) and ethanol (Cromoline®, Diadema, SP, Brazil). A diode laser (Thera Lase - DMC, São Carlos, SP, Brazil) with a wavelength of 660 nm, a fiber spot size of 0.02827 cm2, and a continuous emission mode was used as the light activation source for all evaluations.
Photochemical Characterization Measurement of singlet oxygen production by using 1,3-diphenylisobenzofuran (DPBF): The production of singlet oxygen by MB and TB in the different formulations was measured based on the photo-oxidation of 1,3-diphenylisobenzofuran (DPBF) (SigmaAldrich), which is sensitive to the products formed from the type-II reaction.
Solutions containing DPBF (75 µm) and MB (2.5 µm) or DPBF (75 µm) and TB (2.5 µm) were placed in a quartz cuvette, and the absorbance spectra were measured with a UV/Vis spectrophotometer (Varian Cary 50 Bio, Barueri, SP, Brazil) at a temperature of 25°C. After obtaining the initial scan, the formulations were subjected to irradiation with a low-power diode laser (Thera Lase - DMC), with the power output of the emitter set at 30 mW and a power density of 1 W/cm² for up to 30 s (with intervals of 1 to 10 s and 2 to 30 s). Production of singlet oxygen by MB and TB was measured based on the decrease in the intensity of absorbance at 415 nm as a function of the irradiation time. The evaluations were conducted in triplicate.
Antimicrobial photodynamic effects In vitro biofilm formation: Biofilms of standard laboratory strains of Pseudomonas aeruginosa PA01 (gramnegative) were formed in vitro on acrylic specimens with an 8-mm diameter. A standardized suspension of Pseudomonas aeruginosa PA01 containing 107 cells/mL was prepared using a spectrophotometer (Instrutherm, UV-1000A, São Paulo, SP, Brazil). An optical density (OD 600nm)
of 1 was used as the parameter. Sterile acrylic specimens (measuring 8 mm in diameter, 1 mm in height) were placed
in a 24-well plate (Costar Corning, New York, NY, USA). Each well contained 2 mL of brain heart infusion broth (BHI, Difco, Detroit, MI, USA) supplemented with 5% sucrose. The wells containing the specimens submerged in BHI broth were inoculated with 100 µL of the standardized suspension (107 cells/mL) and incubated in an orbital shaker (Novatecnica, Modelo NT712, Piracicaba, SP, Brazil) at 37°C for 5 days at 75 rpm25, 26. The wells were inoculated only once and the broth was changed every 24 h. After the incubation period, the
samples were washed with 2 mL of phosphate buffered saline (PBS) to remove cells that were not adhered to the biofilm.
In vitro photosensitization: The antimicrobial effect was evaluated for formulations that were not photoactivated (buffer, ethanol 10%, ethanol 20%) and for photoactivated formulations (MB/water, MB/ethanol 10%, MB/ethanol 20%, TB/water, TB/ethanol 10%, TB/ethanol 20%) according to Table 1. The biofilms were treated with laser alone. The biofilm that formed on specimens that were not treated served as the control group. The specimens containing the biofilm were transferred to a fresh 24-well plate and immersed in 1 ml of each of the different formulations. In formulations that were not photoactivated, the contact of the formulation with the biofilm was maintained for 15 min. In the aPDT groups, the contact was maintained for 5 min. The biofilms were then photoactivated by low-level laser irradiation at a wavelength of 660 nm, a power intensity of 30 mW, an energy of 18 J, and laser fluence of 40 J/cm2 for 10 min. The treated biofilm specimens from each formulation were placed in individual falcon tubes containing 10 ml of PBS and were homogenized using an ultrasonic homogenizer (Sonopuls HD 2200, Bandelin Electronic, Berlin, Germany) at a power of 50 W per 30 s. Next, serial dilutions (10-3 to 10-4) were prepared, and 100 µL-aliquots of the dilutions were seeded in duplicate onto plates containing MacConkey agar (Himedia Laboratories Pvt. Ltd., Mumbai, Índia). The plates were incubated for 48 h at 37 °C. An examiner blinded to the experimental groups determined the number of colony-forming units (CFU/ml). Five specimens for each formulation were used. The experiment was performed in triplicate.
Statistical analysis The data for CFU/ml were converted to the logarithmic form. The data of photochemical characterization and antimicrobial activity were expressed as mean value with standard deviation. Differences between groups were evaluated by the variance tests two-way ANOVA and post-hoc Tukey’s for the photochemical and antimicrobial effect of the aPDT data. . Differences between groups were evaluated by the variance tests one-way ANOVA and post-hoc Tukey’s for the data of antimicrobial effect of the formulations without irradiation. The level of significance was set at 5%. Statistical analysis was performed with the SPSS software, v.13.0 (SPSS Inc., Chicago, IL, USA).
Results Measurement of singlet oxygen production by using 1,3-diphenylisobenzofuran (DPBF) The photodynamic activity coefficients generated by the photo-oxidation of singlet oxygen-sensitive DPBF are shown in Table 2. Compared to TB, MB showed higher production of singlet oxygen, regardless of the solvent used. Inclusion of ethanol in the TB formulations did not result in a significant increase in singlet oxygen production. In formulations containing MB, inclusion of 10% and 20% ethanol significantly increased singlet oxygen production when compared to that observed with formulations containing water alone. The mean values of the DPBF photolytic decomposition kinetic constants (s-1), in different formulations are represented graphically in Figure 1 and 2.
In vitro photodynamic inactivation of biofilms Table 3 shows the values of CFU/ml obtained in the formulations without photoactivation. No antimicrobial effect was observed with the buffer solution or the 10%
ethanol solution. With the 20% ethanol solution, a significant microbial reduction (P = 0.009) was observed compared to that observed in the control (no treatment). The mean CFU log10 values and the standard deviation in the formulations subjected to photoactivation are shown in Table 4. None statistical difference was verified among formulations containing ethanol or water, independently of the photosensitizer type. Formulations containing MB and ethanol showed a statistically significant reduction in the viability of P. aeruginosa biofilms. In the other formulations, the reductions were not statistically significant. The antimicrobial behavior of MB and TB diluted in water was similar. In contrast, MB seemed to be more sensitive to the inclusion of ethanol in the formulation, thus showing lower CFU log10 mean values compared to TB, although these differences were not statistically significant.
Discussion The results of the present study demonstrated that the use of ethanol in the formulation containing MB promoted a statistically significant reduction in the viability of P. aeruginosa biofilms compared with untreated biofilm. These effects were not observed when ethanol was used with TB and when water was used independently of the Ps type. Similar antimicrobial effects were noted between formulations with 10% and 20% ethanol, supporting our first research hypothesis. None statistical difference was verified between MB and TB independently of the formulations, refuting our second hypothesis. The average microbial reduction observed with MB/10% ethanol and MB/20% ethanol was approximately 2.58 log10 and 2.66 log10 compared with the control group, respectively. The reduction was relatively lower with TB (0.79 log10 and 1.70 log10 for TB/10% ethanol and TB/20% ethanol, respectively) and without statistical significance. The
relatively stronger effect of antimicrobial formulations containing ethanol and MB can be attributed to the higher production of singlet oxygen. The presence of ethanol in the formulation resulted in lower MB molecular aggregation, i.e., a higher proportion of monomers in relation to dimers34. Dimers are less effective in capturing energy36,37, acting predominantly in reactions involving electronic exchanges with the substrate (type I reaction), with less production of singlet oxygen (type II reaction). Thus, a formulation that stabilizes the monomers is preferable for therapeutic purposes34. Another aspect to consider is the increase in the half-life of the singlet oxygen generated in the presence of ethanol, which is approximately five times greater than that of singlet oxygen generated in the presence of water13. This phenomenon may provide more time for the Ps to interact with bacterial cells. The Figure 1 and 2 demonstrated that the differences between water- and ethanolformulations in the photolytic decomposition of DPBF were more pronounced after 20 seconds of irradiation. Therefore, irradiation time higher than 20 seconds could be evaluated to improve antimicrobial effect of the formulations with ethanol. In this study, using 20% ethanol against the biofilm showed an antimicrobial effect, which confirms the results obtained by Peters et al. (2003)38. These authors found that ethanol in concentrations equal to or greater than 20% can inhibit the growth of monospecies biofilms, whereas this effect is observed in multispecies biofilms at concentrations greater than 30%. The mechanism by which ethanol exerts its antimicrobial action involves damaging the bacterial membrane and the rapid denaturation of proteins, leading to cell lysis39. When 20% or 10% ethanol were used in the aPDT with TB, the mean microbial reduction was similar when compared to that achieved using 20% or 10% ethanol without photoactivation. Therefore, the antimicrobial effect of the formulations containing TB and ethanol were associated with the mechanisms by which ethanol exerts its antimicrobial action and not by increase of the photochemical properties of the formulations. This was confirmed
by findings when the inclusion of ethanol in the TB formulations did not result in a significant increase in singlet oxygen production. aPDT had no effect when TB was used with ethanol. TB is a Ps more hydrophobic than MB, and should interact more easily with the bacterial membrane (in the hydrophobic region) than MB27. In fact, TB is considered as an effective membrane destructive agent40, while MB damages the bacterial cell DNA and to less extent the outer membrane27. The mechanism of the cell death could depend of how of the Ps interacts with the bacteria. It is possible that the mechanism of interaction between TB and bacterial cell had been changed by the ethanol inclusion in the formulations, impairing the antimicrobial photodynamic effect of the TB. The behavior of MB and TB diluted in water was similar with respect to the antimicrobial effect, although MB produced significantly more singlet oxygen. Thus, other factors may be associated with the antimicrobial action, such as the production of ROS, which may play an important role in the antimicrobial action of photodynamic therapy in areas characterized by low availability of oxygen or oxygen depletion. In 2001, Usacheva et al.27 observed a higher antimicrobial effect of the aPDT on the planktonic cells of P. aeruginosa with TB compared to that observed with MB when both were diluted in water. The authors found that the TB interacted more strongly with bacterial lipopolysaccharides, which explained its relatively stronger antimicrobial effect. Nonetheless, this behavior may not be the same against P. aeruginosa biofilms. Previous studies have investigated antimicrobial agents in different models of P. aeruginosa biofilms. Yu et al. 201241 demonstrated that aztreonam reduced the cell viability of P. aeruginosa biofilms formed on epithelial cells by approximately 1 log10. On the other hand, thromycin was more effective, showing reductions above 4 log10. Elkahtib and Noreddin (2014)42 observed that 8 times the minimum inhibitory concentration of ciprofloxacin and 4 times that of clarithromycin were required for a microbial reduction of 2.2
log10 and 2.8 log10, respectively, in biofilms established in 24 h. In this study, reductions of 2.58 log10 and 2.66 log10 in biofilms within 5 days were observed with MB diluted in 10% and 20% ethanol, respectively. Previous investigations have demonstrated that biofilms that formed over longer periods exhibit higher resistance to antimicrobials43,44. Thus, considering that systemic antibiotics may result in opportunistic infections and hypersensitivity reactions, and that the development of bacterial resistance is a growing problem22, aPDT has been identified as a potential alternative to treat infections associated with P. aeruginosa biofilms. Besides, infections caused by other microorganisms could be benefited from formulations evaluated in the present study. Therefore, the aPDT using formulations with ethanol and MB or TB could be evaluated on bacterial biofilms of other species.
Conclusions The antimicrobial photodynamic effect can be optimized with the use of photosensitizers proven to be clinically safe, such as MB, with the inclusion of ethanol in the formulation.
Acknowledgments This study was supported by the Foundation for Post-Graduate Education (CAPES), Brasilia, Brazil.
Author Disclosure Statement No competing financial interests exist.
References 1. Fugitani S, Sun H-Y, Yu VL, Weingarten JA. Pneumonia Due to Pseudomonas aeruginosa. Part I: Epidemiology, Clinical Diagnosis, and Source. Rec Adv in Ch Med 2011;139:909-919. 2. Aaron SD, Vandemheen KL, Ramotar K et al. Infection with Transmissible Strains of Pseudomonas aeruginosa and Clinical Outcomes in Adults with Cystic Fibrosis. JAMA 2010;304:2145-2153. 3. Anaissie E, Penzak SR, Dignani C. The Hospital Water Supply as a Source of Nosocomial Infections. A Plea for Action. Arch Intern Med 2002;162:1483-1492. 4. Falkinham JO, Hilborn ED, Ardulno MJ, Prudlen A, Edwards MA. Epidemiology and Eology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. Envir. Health Persp 2015 [Epub ahead of print]. 5. Sader HS, Jones RN, Gales AC, Silva JB and Pignatari AC. SENTRY antimicrobial surveillance program report: Latin American and Brazilian results for 1997 through 2001. Braz J Infect Dis 2004;8:25-79. 6. Wehmhöner D, Häussler S, Tümmler B, Jänsch L, Bredenbruch F, Wehland J and Steinmetz I. Inter- and intraclonal diversity of the Pseudomonas aeruginosa proteome manifests within the secretome. J Bacteriol 2003;185:5807-5814. 7. Hancock RE and Speert DP. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 2000;3:247-255. 8. Lister PD, Wolter DJ and Hanson N. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microb Rev 2009;22:582-610.
9. Garcia-Medina R, Dunne WM, Singh PK, Brody SL. Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells. Infect. Immun. 2005; 73: 8298-8305. 10. Schmiedl A, Kerber-Momot T, Munder A, Pabst R and Tschernig T. Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa. Cell Tissue Res 2010;342:67-73. 11. Buyck JM, Tuikens PM, Van Bambeke F. Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PA01 in a model of THP-1 Human Monocytes. Antimic Agents and Chem 2013;57:2310-2318. 12. Nickel JC, Ruseska I, Wright JB and Costerton JW. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother 1985;27:619-624. 13. Meisel P, Kocher T. Photodynamic therapy for periodontal diseases: state of the art. J Photochem Photobiol 2005;79:159-170. 14. Ryder C, Byrd M and Wozniak DJ. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr. Opin. Microbiol 2007;10:644-648. 15. Colvin KM, Gordon VD, Murakami K, Borlee BR, Wozniak DJ, Wong GC and Parsek MR. The Pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog 2011;7: e1001264. 16. Nair MGV, Sharma SCD, Sahni BAK, et al. Antimicrobial use and antimicrobial resistance in nosocomial pathogens at a tertiary care hospital in Pune. Medical Jour Armed Forces India 2015;71:112-119. 17. Mulcahy LR, Burns JL, Lory S and Lewis K. Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J Bacteriol 2010;192:6191-6199.
18. Parsek MR and Singh PK. Bacterial biofilms: an emerging link to disease pathogenesis. Annu. Rev. Microbiol 2003;57:677-701. 19. Sharma G, Rao S, Bansal A, Dang S, Gupta S, Gabrani R. Pseudomonas aeruginosa biofilm: Potential therapeutic targets. Biologicals 2014;42:1-7. 20. Dorotkiewicz-Jach A, Augustyniak D, Olszak T, Drulis-Kawa Z. Modern therapeutic approaches
against
Pseudomonas
aeruginosa
infections.
Curr
Med
Chem
2015;22:1642-1664. 21. Wainwright M, Phoenix DA, Marland J, Wareing DR and Bolton FJ. A study of photobactericidal activity in the phenothiazinium series. Immunol Med Microbiol 1997;19:75-80. 22. Hamblin MR and Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem Photobiol Sci 2004;3:436-450. 23. Maisch T. Anti-microbial photodynamic therapy: useful in the future? Lasers Med Sci 2007;22:83-91. 24. Goulart RC, Bolean M, Paulino TP, Thedei G, Souza SL, Tedesco AC and Ciancaglini P. Photodynamic therapy in planktonic and biofilm cultures of Aggregatibacter actinomycetemcomitans. Photomed Laser Surg 2010;28:53-60. 25. Lei X, Liu B, Huang Z, Wu J. A clinical study of photodynamic therapy for chronic skin ulcers in lower limbs infected with Pseudomonas aeruginosa. Arch Dermatol Res 2015;307:49-55. 26. Calzavara-Pinton PG, Rossi MT, Sala R. A retrospective analysis of real-life practice of off-label photodynamic therapy using methyl aminolevulinate (MAL-PDT) in 20 italian dermatology departments. Part 2: oncologic and infectious indications. Photochem Photobiol Sci 2013;12:158-165.
27. Usacheva MN, Teichert MC and Biel MA. Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram-positive and gram-negative microorganisms. Lasers Surg Med 2001;29:165-173. 28. Castano AP, Demidova TN and Hamblin MR. Mechanisms in photodynamic therapy: part one - photosensitizers, photochemistry and cellular localization. Photodiag Photodynam Ther 2004;1:279-293. 29. Ge L, Shu R, Li Y, et al. Adjunctive effect of photodynamic therapy to scaling and root planning in the treatment of chronic periodontitis. Photomed Laser Surg 2011;29:33-37. 30. Soukos SN and Goodson JM. Photodynamic therapy in the control of oral biofilms. Periodontol. 2000 2011;55:143-166. 31. Rolim JP, de-Melo MA, Guedes SF, et al. The antimicrobial activity of photodynamic therapy against Streptococcus mutans using different photosensitizers. J Photochem Photobiol B 2012;106:40-46. 32. National Toxicology Program. Executive summary of safety and toxicity information for methylene blue trihydrate. U.S. Department of Health and Human Services 1990;7220-79-3. 33. Biel MA, Sievert C, Usacheva M, et al. Reduction of Endotracheal Tube Biofilms using Antimicrobial Photodynamic Therapy. Lasers Surg Med 2011;43:586-590. 34. George S and Kishen A. Photophysical, photochemical, and photobiological characterization of methylene blue formulations for light-activated root canal disinfection. J Biomed Opt 2007;12:29-34. 35. Ochsner MJ. Photophysical and photobiological processes in the photodynamic therapy of tumors. Photochem Photobiol 1997;39:1-18.
36. Gabrielli DS, Belisle E, Severino D, Kowaltowski AJ and Baptista MS. Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions. Photochem Photobiol 2004;79:227-232. 37. Patil KR and Talap P. Self-aggregation of methylene blue in aqueous medium and aqueous solutions of Bu4 NBr and urea. Phys Chem Chem Phys 2002;2:4313-4317. 38. Peters MB, Ward RM, Rane HS, Lee SA and Nover MC. Efficacy of ethanol against Candida albicans and Staphylococcus aureus polymicrobial biofilms. Antimicrob Agents Chemoter 2013;57:74-82. 39. Macdonnell G and Russel AD. Antiseptics and disinfectants: activity, action and resistance. Clin Microbiol Rev 1999;12:147-179. 40. Bhatti M, MacRobert A, Meghji S, Henderson B, Wilson M. A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization. Photochem Photobiol 1998;68:370-376. 41. Yu Q, Griffin EF, Moreau-Marquis S, Schwartzman JD, Stanton BA and O’Toole GA. In vitro evaluation of tobramycin and aztreonam versus Pseudomonas aeruginosa biofilms on cystics fibrosis-derived human airway epithelial cells. J Antimicrob Chemother. 2012; 67: 2673-2681. 42. Elkhatib W and Noreddin A. Efficacy of ciprofloxacin-clarithromycin combination against drug-resistant Pseudomonas aeruginosa mature biofilm sing in vitro experimental model. Microbi Drug Resist 2014;20:575-582. 43. Anwar H, Strap JL, Costerton JW. Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob Agents Chemother 1992;36:1347-1351 44. Singla S, Harjai K and Chhibber S. Susceptibility of different phases of biofilm of Klebsiella pneumoniae to three different antibiotics. J Antibiot 2013;66:61-66.
FIGURE CAPTIONS Figure 1 The mean values of the DPBF photolytic decomposition kinetic constants (s-1) in different formulations with methylene blue (MB). Figure 2 The mean values of the DPBF photolytic decomposition kinetic constants (s-1) in different formulations with toluidine blue (TB).
Fig. 1
Fig. 2
Tables Table 1. Formulations of Methylene Blue (MB) and Toluidine Blue (TB) containing water, 10% ethanol, and 20% ethanol submitted to the irradiation. Formulations without photosensitizer did not submitted to the irradiation. Photodynamic Formulations
Formulation compositions
inactivation of biofilm
MB/water
MB dissolved in Milli-Q ultrapure water
MB/ethanol 10%
MB dissolved in 10% ethyl alcohol and Milli-Q ultrapure water (90:10)
MB/ethanol 20%
MB dissolved in 20% ethyl alcohol and Milli-Q ultrapure (80:20)
TB/water
TB dissolved in Milli-Q ultrapure water
TB/ethanol 10%
TB dissolved in 10% ethyl alcohol and Milli-Q
YES
ultrapure water (90:10) TB/ethanol 20%
TB dissolved in 20% ethyl alcohol and Milli-Q ultrapure water (80:20)
Buffer
buffer solution to a pH of 5.6 - sodium acetate/acetic acid NO
Ethanol 10%
10% ethyl alcohol and Milli-Q ultrapure water
Ethanol 20%
20% ethyl alcohol and Milli-Q ultrapure water
All formulations were prepared using a buffer solution of pH of 5.6 (sodium acetate/acetic acid).
Table 2. Mean (standard deviation) of the photodynamic activity coefficient generated by the photo-oxidation of singlet oxygen-sensitive DPBF. Photosensitizers Groups MB
TB
Water
0.044 (0.0005)A
0.104 (0.01)C
Ethanol 10%
0.067 (0.001)B
0.113 (0.009)C
Ethanol 20%
0.063 (0.001)B
0.114 (0.01)C
Parameters: [TB and MB] = 2.5 µM; [DPBF] = 75 µM. Two-way ANOVA e post hoc Tukey tests. Different capital letters represent statistically significant differences among the groups (P<0.05).
Table 3. Mean CFU/ml (log) (standard deviations) of P. aeruginosa in the formulations without photoactivation. Groups
P. aeruginosa (mean ± SD)
Control
6.80 (0.44)A
Buffer
6.22 (0.30)A
Ethanol 10%
6.00 (0.72)A
Ethanol 20%
5.33 (0.45)B
Different capital letters (A and B) represent statistically significant differences among the groups (One-Way ANOVA e post hoc Tukey tests, P<0.05)
Tabela 4. Mean CFU/ml (log) (standard deviations) of P. aeruginosa exposed to different formulations in the photodynamic inactivation. Photosensitizers Groups MB
TB
Control
6.80 (0.44)A
Laser alone
6.99 (0.57)A
Water
5.47 (0.61)AB
5.50 (0.37)AB
Ethanol 10%
4.22 (1.02)B
6.01 (0.67)AB
Ethanol 20%
4.14 (1.26)B
5.10 (2.03)AB
Parameters: [TB and MB] = 250 µM. Irradiation parameters: Low level laser irradiation, wavelight of 660 nm, power intensity of 30 mW, energy of 20 J and laser fluence of 40 J/cm2 for 10 min. Different capital letters (A and B) represent statistically significant differences among the groups (Two-Way ANOVA e post hoc Tukey tests, P<0.05)