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JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):190-192; doi:10.1093/jac/dkl205
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Enhancement of erythrosine-mediated photodynamic therapy of Streptococcus mutans biofilms by light fractionation

Daniel Metcalf{dagger}, Colin Robinson, Deirdre Devine and Simon Wood*

Division of Oral Biology, Leeds Dental Institute, University of Leeds, Clarendon Way Leeds LS2 9LU, UK

*Corresponding author. Tel: +44-113-233-6159; Fax: +44-113-233-6548; E-mail: s.r.wood{at}leeds.ac.uk

Received 14 February 2006; returned 15 April 2006; revised 24 April 2006; accepted 27 April 2006


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Objectives: We aimed to increase the bacterial cell killing efficacy of erythrosine-mediated photodynamic therapy (PDT) of Streptococcus mutans biofilms by fractionating the delivered light dose into a series of shorter pulses.

Methods: S. mutans biofilms of 200 µm thickness were grown in a constant-depth film fermenter (CDFF). Biofilms were incubated with 22 µM erythrosine before being irradiated with white light for increasing periods of time. We also used light dose fractionation to deliver the same overall dose of light in a series of shorter pulses separated by dark periods. Bacterial cell killing as a result of each killing protocol was quantified by colony counting.

Results: A 2 log10 of bacterial cell killing was achieved with 5 min of continuous white light irradiation. For time periods longer than 5 min the amount of killing increased more slowly, which was probably due to photobleaching of the erythrosine. Fractionation of the light dose into 5x 1 min doses separated by dark recovery periods of 5 min increased the amount of bacterial killing by 1 log10 compared with 5 min continuous irradiation. Further fractionation of the light dose into 10x 30 s doses separated by 2 min recovery periods resulted in 3.7 log10 of cell kill, an improvement of 1.7 log10 compared with the continuous irradiation protocol.

Conclusions: Erythrosine-mediated PDT of S. mutans biofilms can be further enhanced by fractionation of the applied light dose.

Keywords: plaque , photosensitizers , PDT


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Antibacterial agents are widely used in the treatment of oral diseases, but problems of development of bacterial resistance1 mean alternative strategies are required to control bacterial plaque biofilms and treat caries, gingivitis and periodontal disease.

Photodynamic therapy (PDT) is a promising antibacterial treatment.2 Upon irradiation with light corresponding to an absorption maximum of the photosensitizer, cytotoxic reactive oxygen species are produced that can cause rapid oxidation of cellular constituents and cell death.3

Since they are localized infections plaque-related diseases would be well suited to PDT. In addition, local administration of both photosensitizer and light is relatively straightforward in the oral cavity.4 Antibacterial photosensitizers currently under investigation for use in the mouth include toluidine blue O (TBO)5 and chlorin e6.6 These agents show great promise but will, of necessity, be subject to lengthy clinical and regulatory assessment. However, more immediate benefit could be derived from photosensitizers already approved for oral use. One such photosensitizer is erythrosine, currently used clinically as a plaque-disclosing agent. We recently reported the successful use of erythrosine-mediated PDT in the treatment of Streptococcus mutans biofilms.7

The aim of the present study was to optimize bacterial killing and reduce light irradiation times by fractionating the applied light dose, a technique that has been shown to enhance PDT in cancer treatment.8,9


   Materials and methods
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Bacterial culture

S. mutans (NCTC 10449) was maintained by weekly subculture on Columbia agar (Oxoid, Basingstoke, UK) supplemented with 5% (v/v) horse blood.

S. mutans biofilms were grown in a constant-depth film fermenter (CDFF) as described previously.7 Briefly, the CDFF was inoculated with overnight cultures of S. mutans in brain heart infusion broth (BHI) (Oxoid). After inoculation, the CDFF was fed with BHI at a rate of 0.7 L per day. Waste was withdrawn from the CDFF by another peristaltic pump. Each of the five plugs in the CDFF pans were recessed by 200 µm and biofilm thickness was maintained at 200 µm by means of a scraper bar. For the treatment of biofilms, pans were aseptically removed through the sampling port in the top plate of the CDFF.

Photodynamic therapy of biofilms

Biofilm-containing pans were removed and incubated with 22 µM erythrosine in ringers saline (RS; Lab M, Bury, UK) for 15 min, in the dark, at room temperature. The biofilm pans were then placed into fresh RS and irradiated using the previously described light system.7 Average light intensity was 22.7 mW/cm2 in the region of maximum absorption by erythrosine (500–550 nm). This represented a total light dose of 6.75 J/cm2 after 5 min of irradiation. Continuous irradiation was carried out for 0, 1, 2, 5, 10, 15 or 30 min. For light fractionation, biofilms were pulsed under the light system for 5x 1 min periods or for 10x 30 s periods. Between light pulses the samples were left in darkness for a 5 min (for 1 min pulses) or 2 min (for 30 s pulses) recovery period at room temperature. Two pans were used per time point, and following irradiation three biofilm-containing plugs (each of 5 mm diameter) were removed from each pan. The biofilms were disrupted from the plugs by vortexing with sterile glass beads (3 mm diameter) for 1 min and were serially diluted in RS. Surviving cells were counted by serial dilution and plating in triplicate on Columbia agar supplemented with 5% (v/v) horse blood. Plates were incubated aerobically in 5% CO2 at 37°C for 48 h. In each case, n = 3–6 and the entire experiment was repeated twice. Statistical analysis was performed using two-sample t-tests.


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S. mutans biofilms typically contained 1010–1011 cfu per plug before treatment. Figure 1 shows that the killing of S. mutans biofilm cells with erythrosine-mediated PDT and continuous irradiation occurred in a light dose-dependent manner up to 5 min of irradiation.


Figure 1
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  		Figure 1. Effect of continuous irradiation time on biofilm cell survival. Seven day biofilms were incubated with 22 µM erythrosine for 15 min and then irradiated with white light for 0–30 min. Error bars indicate the SD of 6–12 determinations.

 
As ~98% of the cell killing occurred in the first 5 min of irradiation, we decided to fractionate a 5 min irradiation period. This also prevented significant photobleaching, which we have shown occurs at irradiation times of longer than 15 min.7

The results for the light fractionation regimes compared with continuous irradiation are shown in Figure 2. Continuous irradiation for 5 min resulted in 2 ± 0.2 log10 cell kill. The dose–response curve was biphasic, plateauing after 3 min of irradiation. Five times 1 min light pulses of the erythrosine-treated biofilms, with 5 min recovery periods between pulses, resulted in a 3.0 ± 0.3 log10 kill of biofilm bacteria. Ten times 30 s light pulses, with 2 min recovery periods, again improved cell killing to 3.7 ± 0.3 log10 (although this was not significantly different to the cell killing seen with 5x 1 min pulses). Both of the light fractionation regimes caused statistically significantly more cell killing (P < 0.005) when compared with the 5 min continuous irradiation regime.


Figure 2
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  		Figure 2. A comparison of continuous irradiation and light fractionation in the erythrosine-mediated PDT of S. mutans biofilm cells. Biofilms were incubated with 22 µM erythrosine for 15 min and then: irradiated with 0, 1, 2, 3 or 5 min of continuous white light (filled squares); subjected to 5 x 1 min white light pulses separated by dark recovery periods of 5 min (open circles); or subjected to 10x 30 s white light pulses separated by dark recovery periods of 2 min (filled triangles). Error bars indicate the SD of 6–12 determinations.

 
The two light fractionation regimes were also superior to longer continuous irradiation times of up to 30 min. The 10 x 30 s light pulse regime (3.7 ± 0.3 log10 kill) gave almost a 10-fold increase in cell killing compared with 30 min continuous irradiation (2.8 ± 0.1 log10). This increase was statistically significant (P < 0.005).


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The present study has shown that the fractionation of white light during the erythrosine-mediated PDT of S. mutans biofilms grown in vitro results in increased cell killing compared with continuous irradiation. This may be due to the replenishment, during dark periods, of target molecules (such as oxygen) for the excited photosensitizer. The general replenishment or redistribution of the photosensitizer itself during dark periods, in both aerobic and anaerobic cases, is also a possibility. The photodynamic process also leads to diminished erythrosine levels due to photobleaching,7 so any photosensitizer concentration gradient might be equilibrated during dark periods.

Overall, this work further highlights the clinical potential of erythrosine-mediated PDT. In conjunction with light dose fractionation, irradiation times can be reduced to clinically acceptable levels. Further work is now being undertaken to determine the efficacy of erythrosine-PDT on natural oral biofilms formed in vivo.


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None to declare.


   Footnotes
 
{dagger}Present address. Continuous Improvement, Johnson & Johnson Wound Management, Airebank Mill, Skipton, North Yorkshire BD23 3RX, UK Back


   Acknowledgements
 
This work was supported by a BBSRC Research Studentship.


   References
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1 Courvalin P. (1996) Evasion of antibiotic action by bacteria. J Antimicrob Chemother 37:855–69.[Abstract/Free Full Text]

2 Wainwright M. (1998) Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 42:13–28.[Abstract/Free Full Text]

3 Dougherty TJ, Gomer CJ, Henderson BW, et al. (1998) Photodynamic therapy. J Natl Cancer Inst 90:889–905.[Abstract/Free Full Text]

4 Löe H. (2000) Oral hygiene in the prevention of caries and periodontal disease. Int Dent J 50:129–39.[Web of Science][Medline]

5 Bhatti M, MacRobert A, Meghji S, et al. (1998) A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization. Photochem Photobiol 68:370–6.[CrossRef][Web of Science][Medline]

6 Pfitzner A, Sigusch BW, Albrecht V, et al. (2004) Killing of periodontopathogenic bacteria by photodynamic therapy. J Periodontol 75:1343–9.[Medline]

7 Wood SR, Metcalf DM, Devine D, et al. (2006) Erythrosine is a potential photosensitizer for the photodynamic therapy of oral plaque biofilms. J Antimicrob Chemother 57:680–4.[Abstract/Free Full Text]

8 van Geel IP, Oppelaar H, Marijnissen JP, et al. (1996) Influence of fractionation and fluence rate in photodynamic therapy with Photofrin or mTHPC. Radiat Res 145:602–9.[Web of Science][Medline]

9 Tsutsui H, MacRobert AJ, Curnow A, et al. (2002) Optimisation of illumination for photodynamic therapy with mTHPC on normal colon and a transplantable tumour in rats. Lasers Med Sci 17:101–9.[Medline]


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