a Photochemotherapy Group, Department of Biological Sciences, University of Central Lancashire, Preston PR1 2HE, UK b Public Health Laboratory, Royal Preston Hospital, Preston PR2 4HT, UK
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Abstract |
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Introduction |
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Recently, we reported the antibacterial activity of a range of non-antibiotic cationic heterocyclics which showed increased activity upon low-power illumination.3,4 This photobactericidal activity was apparent against a range of pathogenic bacteria including epidemic strains of MRSA.4 The present study is an extension of this work to clinically relevant strains of vancomycin-resistant E. faecalis and E. faecium using the photosensitizer methylene blue and its two methylated derivatives.
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Materials and methods |
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Methylene blue and 1,9-dimethyl methylene blue were purchased from Aldrich (Gillingham, UK) and were recrystallized from methanol containing hydrochloric acid to yield the hydrochloride salts. The preparation of 1-methyl methylene blue was as reported previously.5
Light source
An Exal light box, giving a light fluence of 1.7 mW/cm2 was used, the fluence being measured with a Skye SKP 200 light meter (Skye Instruments Ltd, UK). A 1 h illumination period using this source thus gave a total light dose of 6.3 J/cm2. The output of the light source included a strong band at 650 nm as has been reported previously.4
Bacterial cell culture
Enterococcus spp.strains used were a standard, vancomycin-sensitive E. faecalis NCTC 775; a vancomycin-resistant VanB-strain of E. faecalis, 214801 and two vancomycin-resistant VanA strains of E. faecium, 1630 and 1658 (Public Health Laboratory Service, Preston UK). These were grown aerobically in Brain Heart Infusion Broth (Difco Laboratories, UK) at 37°C overnight.
Bacterial cultures were grown to an optical density equivalent to McFarland's Standard No. 3 (109 cfu/mL), and diluted to an approximate concentration of 106 cfu/mL. This was confirmed as 106 cfu/mLusing a surface count on horse blood agar.6 To establish approximate bactericidal concentrations, an initial range of doubling dilutions (01000µM) of each phenothiazinium was placed in 270µL aliquots in flat-bottomed microtitre plates and 30µL of bacterial culture added to each well. The microtitre trays were then incubated for 18 h at 37°C, aerobically in the dark. From each well showing inhibition of growth, 1µL was subcultured on 5% (v/v) defibrinated horse blood agar. These plates were incubated for 18 h at 37°C, aerobically. Using this procedure with closer concentration ranges, minimum lethal drug concentrations (MLCs) were determined as the lowest concentration at which bacterial growth was not detected. The four organisms were tested against each of the photosensitizers in triplicate and each experiment was replicated three times.
To investigate the photosensitizing effects of the phenothiaziniums, the above procedure was duplicated but after the addition of 30µL of bacterial culture to each well, a 1 h illumination of the cultures, both with and without photosensitizers, was carried out (total light dose = 6.3 J/cm2). The microtitre trays were then incubated for 18 h at 37°C, aerobically in the dark and each well showing inhibition of growth was subcultured as above in order to determine the MLC for each compound.
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Results and discussion |
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While the use of photobactericidal agents against systemic disease is likely to be problematic,
the disinfection of sites of drug-resistant infection such as burn wounds or colonized breathing
tubes, drains, catheters, etc. offers some potential. Phenothiaziniums exhibit low mammalian
toxicity, e.g. methylene blue is used routinely as a marker dye in surgery as a 1% aqueous
solution (27 mM),9 and in recent photovirucidal
work, dimethyl methylene blue was highly effective without collateral damage to red blood cells.8 In addition the closely related cationic dye crystal violet
has been used topically against MRSA.10
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Notes |
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References |
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2 . Bostic, G. D., Perri, M. B., Thal, L. A. & Zervos, M. J. (1998). Comparative in vitro and bactericidal activity of oxazolidinone antibiotics against multidrug-resistant enterococci. Diagnostic Microbiology and Infectious Disease 30, 10912.[ISI][Medline]
3 . Wainwright, M., Phoenix, D. A., Marland, J., Wareing, D. R. & Bolton, F. J. (1997). In-vitro photobactericidal activity of aminoacridines. Journal of Antimicrobial Chemotherapy 40, 5879.[Abstract]
4 . Wainwright, M., Phoenix, D. A., Laycock, S. L., Wareing, D. R. & Wright, P. A. (1998). Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus. FEMS Microbiology Letters 160, 17781.[ISI][Medline]
5 . Wainwright, M., Phoenix, D. A., Rice, L., Burrow, S. M. & Waring, J. (1997). Increased cytotoxicity and phototoxicity in the methylene blue series viachromophore methylation. Journal of Photochemistry and Photobiology, B: Biology 40, 2339.[ISI]
6 . Miles, A. A., Misra, S. S. & Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene 38, 73249.
7 . Pal, M. K. & Ghosh, T. C. (1990). Induction of metachromasia and circular dichroism in the dye 1,9-dimethyl methylene blue by S. aureus wall teichoic acid. Indian Journal of Biochemistry and Biophysics 27, 1768.[ISI][Medline]
8 . Wagner, S. J., Skripchenko, A., Robinette, D., Foley, J. W. & Cincotta, L. (1998). Factors affecting virus photinactivation by a series of phenothiazine dyes. Photochemistry and Photobiology 67, 3439.[ISI][Medline]
9 . Creagh, T. A., Gleeson, M., Travis, D., Grainger, R., McDermott, T. E. & Butler, M. R. (1995). Is there a role for in vivo methylene blue staining in the prediction of bladder tumour recurrence? British Journal of Urology 75, 4779.[ISI][Medline]
10 . Saji, M., Taguchi, S., Uchiyama, K., Osono, E., Hayama, N. & Ohkuni, H. (1995). Efficacy of gentian violet in the eradication of methicillin-resistant Staphylococcus aureus from skin-lesions. Journal of Hospital Infection 31, 2258.[ISI][Medline]
Received 6 May 1999; returned 9 August 1999; revised 18 August 1999; accepted 1 September 1999