Journal of Antimicrobial Chemotherapy (1999) 44, 823-825
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
Photobactericidal activity of methylene blue derivatives against vancomycin-resistant Enterococcus spp.
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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The toxicities and phototoxicities of methylene blue and its two methylated derivatives were measured against one standard and three vancomycin-resistant pathogenic strains of Enterococcus spp. Each of the compounds was bactericidal and the derivatives exhibited photobactericidal activity on illumination at a ‘light’ dose of 6.3 J/cm2 against one or more of the strains. Increased bactericidal and photobactericidal activity in the methylated derivatives is thought to be due to their higher hydrophobicities allowing greater interaction with the bacterial cell wall. In addition, the derivatives exhibited higher inherent photosensitizing efficacies.
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Introduction |
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The increasing problem of drug-resistant microbes is particularly important where there is impaired immune response, either as a consequence of immunosuppressant therapy or an immunodeficient syndrome. Major outbreaks of staphylococcal and enterococcal infection owing to strains resistant to a wide range of chemotherapeutic agents have been reported.1 Whilst vancomycin and the related glycopeptide antibiotic teicoplanin provide an effective option in the treatment of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant strains of Enterococcus faecalis and Enterococcus faecium are now often a cause of nosocomial infection. Glycopeptide resistance in enterococci is due to modification of the cell wall (VanA–VanE strains).1 Currently, although there are several chemotherapeutic alternatives available for the treatment of drug-resistant disease (e.g. oxazolidinones2) the continued search for novel therapies is imperative.
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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Photosensitizers
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 (0–1000µM) 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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Against all strains employed in the current study, methylene blue exhibited little activity and was not photobactericidal (Table I). The methylated methylene blue derivatives were bactericidal against all strains and photobactericidal in all but two cases (Table I). The activity of the phenothiaziniums did not correlate with phenotype, suggesting that sites of action for the photosensitizers were unaffected by differences in cell wall morphology in the VanA or VanB strains employed. Although on this small sample the difference in dark and light MLC rarely exceeded two-fold, the differences were relevant and not due to experimental variation, being completely reproducible.
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Methylene blue and its mono- and dimethylated derivatives were chosen for this study owing to the increases in hydrophobicity and photosensitizing efficacy that result from chromophoric methylation (Table II).5 In addition, methylation inhibits cellular reduction of the phenothiazinium chromophore to the leuco form which is colourless and thus inactive in terms of photosensitization with long wavelength light.5 Against methicillin-resistant strains of S. aureus, methylated derivatives of methylene blue exhibited far greater activity than the parent compound.4
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The increased hydrophobicities (LogP) of the methylated phenothiaziniums (Table II) may have allowed greater partitioning into the cell wall. The strong binding of dimethyl methylene blue by the bacterial cell wall carbohydrate teichoic acid has been reported,7 as have the DNA-binding properties of methylene blue and dimethyl methylene blue.8 However, the generally low levels of photoactivation encountered (Table I) suggest less specific target sites, e.g. cell wall rather than DNA. Given low target specificity, the photobactericidal activity of the phenothiaziniums correlated with measured singlet oxygen efficiencies (Table II).
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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* Corresponding author. Tel: +44-1772-893534; Fax: +44-1772-892929; E-mail: m.wainwright{at}uclan.ac.uk
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Woodford, N. (1998). Glycopeptide-resistant enterococci: a decade of experience. Journal of Medical Microbiology 47, 849–62.
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, 109–12.[Web of Science][Medline]
3
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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, 177–81.[Web of Science][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, 233–9.
6 . Miles, A. A., Misra, S. S. & Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene 38, 732–49.
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, 176–8.[Web of Science][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, 343–9.[Web of Science][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, 477–9.[Web of Science][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, 225–8.[Web of Science][Medline]
Received 6 May 1999; returned 9 August 1999; revised 18 August 1999; accepted 1 September 1999
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