Effect of photodynamic therapy on clinical isolates of Staphylococcus spp

Staphylococcus spp. are opportunistic microorganisms known for their capacity to develop resistance against antimicrobial agents. The objective of this study was to evaluate the effect of photodynamic therapy (PDT) on 20 Staphylococcus strains isolated from the human oral cavity, including S. aureus, S. schleiferi, S. epidermidis, S. capitis, S. haemolyticus, and S. lentus. A suspension of each Staphylococcus strain (106 cells/ mL) was submitted to PDT using methylene blue and a low power laser. The isolated effects of methylene blue, laser treatment and ciprofloxacin were also evaluated. After the experimental treatments, 0.1 mL aliquots of the suspensions were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were analyzed by analysis of variance and Tukey’s test (p ≤ 0.05). The mean reduction in bacterial counts of the strains submitted to PDT ranged from 4.89 to 6.83 CFU (log10)/mL, with the observation of a decreasing susceptibility to treatment of S. schleiferi, S. haemolyticus, S. epidermidis, S. capitis, S. aureus, and S. lentus. The results showed that PDT was effective in reducing the number of viable cells of all clinical Staphylococcus isolates studied. Descriptors: Staphylococcus; Photochemotherapy; Methylene Blue; Ciprofloxacin. Introduction Several species of the genus Staphylococcus can be isolated from the human oral cavity and are part of the transient microbiota.1 These microorganisms are found in the oral cavity of approximately 95% of healthy individuals and have become a matter of concern because of their capacity to develop resistance against antimicrobial agents.2,3 In addition to their involvement in oral diseases, such as angular cheilitis, parotiditis, staphylococcal mucositis and periodontitis, these bacteria are the causative agents of severe skin and soft tissue infections that are associated with an increased risk of complications, such as bacteremia, pneumonia and endocarditis.1,4-7 Methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from the oral cavity of healthy individuals, a finding that indicates an increased risk of dissemination to other sites.7,8 Other Staphylococcus species have also been recognized as important etiological agents of opportunistic infections in individuals with differing predisposing factors, and some isolates have shown a reduced susceptibility to β-lactam antibiotics.3,9 Vancomycin is the antibiotic of choice in the case of reduced Miyabe M, Junqueira JC, Costa ACBP, Jorge AOC, Ribeiro MS, Feist IS Braz Oral Res. 2011 May-Jun;25(3):230-4 231 β-lactam susceptibility. However, although resistance is rare, therapeutic failure is increasingly being reported.4 Photodynamic therapy (PDT) has emerged as a therapeutic option for the treatment of infectious diseases. This therapy consists of the activation of a photosensitive dye with light of an appropriate wavelength, consequently resulting in the production of reactive oxygen species, such as free radicals and singlet oxygen. These reactive oxygen species can damage DNA and the cell membrane, resulting in the leakage of cell components, inactivation of transport systems and cell death.10,11 The objective of the present study was to evaluate the effect of photodynamic antimicrobial chemotherapy on different Staphylococcus strains isolated from the human oral cavity. Materials and Methods Staphylococcus strains The study was approved by the Ethics Committee of the School of Dentistry of São José dos Campos, UNESP (07/2007-PH/CEP). Twenty Staphylococcus strains isolated from the human oral cavity (S. aureus, n  =  5; S. schleiferi, n = 5; S. epidermidis, n = 6; S. capitis, n = 2; S. haemolyticus, n = 1, and S. lentus, n = 1) were studied. These strains were obtained from the Laboratory of Microbiology and Immunology, School of Dentistry of São José dos Campos, UNESP. Twenty-four-hour cultures, incubated at 37°C on mannitol agar (Difco, Detroit, MI, USA), were inoculated into brain-heart infusion broth (Difco) and incubated for a further 18 h at 37°C. The cultures were then centrifuged at 1300 x g for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 5  mL sterile saline (0.85% NaCl). This procedure was repeated once. The suspension was standardized in a spectrophotometer (B582, Micronal, São Paulo, Brazil) to an optical density of 0.374 at a wavelength of 490 nm, corresponding to 106 cells /mL. Photosensitizer, laser and antibiotic treatment Methylene blue (MB), at a concentration of 3 mM, was used for photosensitization of the Staphylococcus strains. The dye solution was prepared by dissolving the powder (Sigma-Aldrich, Steinheim, Germany) in 0.85% NaCl and filtering it through a 0.22-μm membrane filter (Millipore, São Paulo, Brazil). After filtration, the dye solution was stored in the dark. A gallium-aluminum-arsenide laser (Easy Laser, Clean Line, Taubaté, Brazil), emitting light at 660 nm (visible red), was used as the light source. The wavelength of the laser corresponds to the maximum absorption of MB. The laser settings were as follows: power of 35 mW, energy of 10 J, time of 285 s, fluence rate of 92 mW/cm2, and fluence of 26.3 J/cm2. Ciprofloxacin (Vivência Compounding Pharmacy, Pindamonhangaba, SP, Brazil) was used as an antibiotic at a concentration of 1.6 mg/mL. In vitro photosensitization Thirty assays were carried out for each suspension for a total of 600 assays. The Staphylococcus spp. strains were submitted to the following experimental conditions (n = 6): a. photosensitization with MB and laser irradiation (P+L+); b. photosensitization with MB in the absence of light (P+L-); c. treatment with saline and laser irradiation (PL+); d. treatment with the antibiotic (AB); and e. treatment with saline in the absence of light as a control group (P-L-). Sterile 96-well flat-bottom microtiter plates with a lid (Costar Corning, New York, USA) were used. An aliquot (0.1 mL) of the bacterial suspension was added to each well (area of 0.38 cm2). Next, 0.1 mL of the photosensitizer was added for groups P+L+ and P+L-, 0.1  mL of saline for groups P-L+ and P-L-, and 0.1 mL of the antibiotic for group AB. The plates were then shaken for 5 min in an orbital shaker (Solab, Piracicaba, Brazil). After this period, the contents of the wells of groups P+L+ and P-L+ was irradiated as described above. The samples were irradiated under aseptic conditions in a laminar flow chamber in the dark. Effect of photodynamic therapy on clinical isolates of Staphylococcus spp Braz Oral Res. 2011 May-Jun;25(3):230-4 232 After the experimental treatments, serial dilutions were prepared and 0.1-mL aliquots of the dilutions were seeded in duplicate onto brain-heart infusion agar plates (Difco). After incubation for 24 h at 37°C, the number of colony-forming units (CFU/ mL) was determined, and the results were log-transformed (log10). Statistical analysis The results (log10 CFU/mL) were analyzed by analysis of variance (ANOVA) and the Tukey test, with the level of significance set at 5%, using the Minitab 14.0 program. Results Figures 1 and 2 show the mean and standard deviation of CFU/mL obtained for the different Staphylococcus species studied under each experimental condition. A significant reduction in bacterial counts was observed for the groups submitted to PDT (P+L+) when compared to the other groups (P-L-, P-L+ and P+L-). Ciprofloxacin treatment resulted in a reduction of 100% for all the groups except for S. schleiferi, which presented a reduction of 5.97 log10 CFU/mL. A small reduction in bacterial counts was observed for the P+Lgroups in comparison to the P-Lgroups for all species. However, the difference was only significant for two species: S. epidermidis and S. haemolyticus. The number of bacterial cells was higher in the P-L+ groups compared to the P-Lgroups, but the difference was not significant. Table 1 shows the log10 CFU/mL reduction observed for the P+L+ groups compared to the P-Lgroups. Among the strains tested, S. lentus was the species most resistant to PDT, followed by S. aureStrains CFU (log10)/mL P-LP-L+ P+LP+L+ Reduction in log of P+L+ S. aureus (n = 5) 7.46A 7,51A 6.51A 1.19B 6.27 S.epidermidis (n = 6) 7.49A 7.53A 6.33B 0.86C 6.63 S. schleiferi (n = 5) 7.44A 7.59A 6.32A 0.61C 6.83 S. capitis (n = 2) 7.10A 7.60A 5.68A 0.71B 6.39 S. haemolyticus (n = 1) 6.71A 7.06A 6.15B 0.00C 6.71 S. lentus (n = 1) 7.44A 7.53A 7.26A 2.55B 4.89


Introduction
Several species of the genus Staphylococcus can be isolated from the human oral cavity and are part of the transient microbiota. 1 These microorganisms are found in the oral cavity of approximately 95% of healthy individuals and have become a matter of concern because of their capacity to develop resistance against antimicrobial agents. 2,3][6][7] Methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from the oral cavity of healthy individuals, a finding that indicates an increased risk of dissemination to other sites. 7,8Other Staphylococcus species have also been recognized as important etiological agents of opportunistic infections in individuals with differing predisposing factors, and some isolates have shown a reduced susceptibility to β-lactam antibiotics. 3,9Vancomycin is the antibiotic of choice in the case of reduced β-lactam susceptibility.However, although resistance is rare, therapeutic failure is increasingly being reported. 4hotodynamic therapy (PDT) has emerged as a therapeutic option for the treatment of infectious diseases.This therapy consists of the activation of a photosensitive dye with light of an appropriate wavelength, consequently resulting in the production of reactive oxygen species, such as free radicals and singlet oxygen.These reactive oxygen species can damage DNA and the cell membrane, resulting in the leakage of cell components, inactivation of transport systems and cell death. 10,11he objective of the present study was to evaluate the effect of photodynamic antimicrobial chemotherapy on different Staphylococcus strains isolated from the human oral cavity.

Staphylococcus strains
The study was approved by the Ethics Committee of the School of Dentistry of São José dos Campos, UNESP (07/2007-PH/CEP).
Twenty-four-hour cultures, incubated at 37°C on mannitol agar (Difco, Detroit, MI, USA), were inoculated into brain-heart infusion broth (Difco) and incubated for a further 18 h at 37°C.The cultures were then centrifuged at 1300 x g for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 5 mL sterile saline (0.85% NaCl).This procedure was repeated once.The suspension was standardized in a spectrophotometer (B582, Micronal, São Paulo, Brazil) to an optical density of 0.374 at a wavelength of 490 nm, corresponding to 10 6 cells /mL.

Photosensitizer, laser and antibiotic treatment
Methylene blue (MB), at a concentration of 3 mM, was used for photosensitization of the Staphylococcus strains.The dye solution was prepared by dissolving the powder (Sigma-Aldrich, Steinheim, Germany) in 0.85% NaCl and filtering it through a 0.22-µm membrane filter (Millipore, São Paulo, Brazil).After filtration, the dye solution was stored in the dark.
A gallium-aluminum-arsenide laser (Easy Laser, Clean Line, Taubaté, Brazil), emitting light at 660 nm (visible red), was used as the light source.The wavelength of the laser corresponds to the maximum absorption of MB.The laser settings were as follows: power of 35 mW, energy of 10 J, time of 285 s, fluence rate of 92 mW/cm², and fluence of 26.3 J/cm².
Ciprofloxacin (Vivência Compounding Pharmacy, Pindamonhangaba, SP, Brazil) was used as an antibiotic at a concentration of 1.6 mg/mL.

In vitro photosensitization
Thirty assays were carried out for each suspension for a total of 600 assays.The Staphylococcus spp.strains were submitted to the following experimental conditions (n = 6): a. photosensitization with MB and laser irradiation (P+L+); b. photosensitization with MB in the absence of light (P+L-); c. treatment with saline and laser irradiation (P-L+); d. treatment with the antibiotic (AB); and e. treatment with saline in the absence of light as a control group (P-L-).
Sterile 96-well flat-bottom microtiter plates with a lid (Costar Corning, New York, USA) were used.An aliquot (0.1 mL) of the bacterial suspension was added to each well (area of 0.38 cm²).Next, 0.1 mL of the photosensitizer was added for groups P+L+ and P+L-, 0.1 mL of saline for groups P-L+ and P-L-, and 0.1 mL of the antibiotic for group AB.The plates were then shaken for 5 min in an orbital shaker (Solab, Piracicaba, Brazil).After this period, the contents of the wells of groups P+L+ and P-L+ was irradiated as described above.The samples were irradiated under aseptic conditions in a laminar flow chamber in the dark.
After the experimental treatments, serial dilutions were prepared and 0.1-mL aliquots of the dilutions were seeded in duplicate onto brain-heart infusion agar plates (Difco).After incubation for 24 h at 37°C, the number of colony-forming units (CFU/ mL) was determined, and the results were log-transformed (log 10 ).

Statistical analysis
The results (log 10 CFU/mL) were analyzed by analysis of variance (ANOVA) and the Tukey test, with the level of significance set at 5%, using the Minitab 14.0 program.

Results
Figures 1 and 2 show the mean and standard deviation of CFU/mL obtained for the different Staphylococcus species studied under each experi-mental condition.A significant reduction in bacterial counts was observed for the groups submitted to PDT (P+L+) when compared to the other groups (P-L-, P-L+ and P+L-).Ciprofloxacin treatment resulted in a reduction of 100% for all the groups except for S. schleiferi, which presented a reduction of 5.97 log 10 CFU/mL.
A small reduction in bacterial counts was observed for the P+L-groups in comparison to the P-L-groups for all species.However, the difference was only significant for two species: S. epidermidis and S. haemolyticus.The number of bacterial cells was higher in the P-L+ groups compared to the P-L-groups, but the difference was not significant.
Table 1 shows the log 10 CFU/mL reduction observed for the P+L+ groups compared to the P-Lgroups.Among the strains tested, S. lentus was the species most resistant to PDT, followed by S. aure-

Discussion
3][14] Grinholc et al. 12 , treating MRSA and methicillin-sensitive S. aureus (MSSA) strains with 25 µM protoporphyrin irradiated with 624 nm light, observed a reduction of 0 to 3 log for MRSA and of 0.2 to 3 log for MSSA, demonstrating a similar sensitivity to PDT for β-lactamresistant and -sensitive strains.Wainwright et al. 13 studied the effect of phenothiazine dyes irradiated with visible light (350-800 nm) on an S. aureus reference strain and on MRSA.The reduction in bacterial counts achieved with PDT was 4 log 10 when 5 µM MB was used for the reference strain and 10 to 50 µM MB for MRSA.In another study, Tubby et al. 14 were able to kill approximately 6 log MRSA cells with PDT, using 20 µM MB and a laser light dose of 9.65 J/cm².In addition, PDT reduced the activity of V8 protease and α-hemolysin by 100% and the activity of sphingomyelinase by 92%.The authors suggested that the reactive oxygen species formed during PDT interact with, and inactivate, bacterial virulence factors, providing an additional mechanism for the antibacterial action of this therapy.
The formation of a biofilm, observed in Staphylococcus strains, increases their resistance to antibiotics and protects the cells against the action of the immune system. 15Sharma et al. 16 studied the effects of PDT on biofilm formation in MRSA and S. epidermidis and observed that toluidine blue, combined with a diode laser, showed low antibacterial activity.In addition, S. aureus was more resistant to PDT than S. epidermidis.
Zolfaghari et al. 17 evaluated the in vivo effect of PDT in mice with excisional and superficial skin wounds inoculated with a suspension of MRSA.The wounds were treated with 0.3 µM MB and irradiated with 360 J/cm² of diode laser light for 30 min.Treatment resulted in a reduction of 1.4 log 10 CFU/ mL in excision wounds and of 1.15 log 10 CFU/mL in superficial wounds.The authors emphasized the importance of these results because they represent the first report of the in vivo killing of MRSA and raise the possibility of attempting to reproduce this treatment in humans.
In the present study, administration of the dye alone exerted a low antimicrobial effect, with a significant difference being observed only for S. epidermidis and S. haemolyticus.][20] Gois et al. 21also reported a reduction in the number of S. aureus cells of 0.97, 0.97 and 1.05 log 10 when irradiated with red LED (20 J/cm²) and diode laser light (20 and 40 J/cm²), respectively, in the absence of a photosensitizer.
Ciprofloxacin used in the present study is a broad spectrum antibiotic with potent activity against bacteria of the genus Staphylococcus. 22This antibiotic killed all of the strains studied, except for one S. schleiferi isolate.This latter finding may be due to previous exposure of this microorganism to the antibiotic or to the development of primary resistance.However, among the Staphylococcus species submitted to PDT, S. schleiferi was the most sensitive, with a mean reduction of 6.83 log 10 CFU/mL, suggesting that PDT might be used as an alternative for the treatment of antibiotic-resistant strains.

Conclusion
PDT was effective in reducing the number of viable cells of all clinical Staphylococcus isolates studied.

Table 1 -
Mean log 10 CFU/mL of Staphylococcus spp.under the