J Bacteriol Virol.  2020 Dec;50(4):235-245. 10.4167/jbv.2020.50.4.235.

Effect of Photodynamic Therapy Enhanced by Methylene Blue on Drug-resistant Mycobacterium smegmatis

• Jeong B
• Kim JK
• Bae T
• Park I

• KMID: 2512143
• DOI: http://doi.org/10.4167/jbv.2020.50.4.235

Abstract

Tuberculosis (TB) is an old disease caused by Mycobacterium tuberculosis. Although it has been known for humans for thousands of years, the treatment of this disease still requires a lengthy therapy with multiple antibiotics. Also, the emergence of multidrug-resistant strains made it more difficult to treat TB, calling for a novel treatment approach. In Photodynamic therapy (PDT), a photosensitizer, such as methylene blue (MB), is irradiated by a laser, generating reactive oxygen species and killing microorganisms. Here, using M. smegmatis as a model mycobacterium, we examined the utility of PDT in TB treatment. The photosensitizer MB alone showed weak antimicrobial activity; however, when irradiated by a laser, it efficiently killed M. smegmatis (> 97% killing with 30 mg/ml MB and 54 J/cm² irradiation). Surprisingly, PDT showed more efficient killing activity toward drug-resistant strains of M. smegmatis than the drug-sensitive wild-type strain. In PDT, when the irradiation step alone (Intermittent PDT) or the entire PDT process was repeated (Repeated PDT), the bactericidal activity was significantly enhanced. Since PDT can be applied locally in a short period of time and kills mycobacterium irrespective of its antibiotic resistance status, we conclude that PDT can be a viable option for TB treatment.

Keyword

Tuberculosis; Mycobacterium smegmatis; Drug resistance; Photodynamic therapy; Methylene blue

Figure

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  Fig. 1 Method of intermittent PDT and repeated PDT. (a) represents intermittent PDT. M. smegmatis strains were irradiated with 18 J/cm2 light dose after incubation with 5 μg /ml of methylene blue (MB), incubated again for 2 hours without MB exposure, and then irradiated at the same light dose (18 J/cm2). (b) represents repeated PDT. M. smegmatis strains were irradiated with 18 J/cm2 light dose after incubation with 5 μg /ml of MB and re-incubated with MB for 2 h and re-irradiated at 18 J/cm2 dose of laser light.

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  Fig. 2 The antimicrobial activity of methylene blue (MB) on Mycobacterium smegmatis. The results are mean ± SD from three independent experiments.

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  Fig. 3 The bactericidal effect of photodynamic therapy with methylene blue on Mycobacterium smegmatis. M. smegmatis was incubated in various concentrations of methylene blue (0, 5, 30 μg/ml) and irradiated with different light doses (0, 3.6, 18, 54 J/cm2). Data were presented as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to the corresponding controls (student’s t-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001).

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  Fig. 4 Effects of photodynamic therapy with methylene blue on drug-resistant Mycobacterium smegmatis. Drug-resistant M. smegmatis strains were incubated in various concentrations of methylene blue (0, 5, 30 μg/ml) and irradiated at different light doses (0, 3.6, 18, 54 J/cm2). (a-d) represents ciprofloxacin resistant-, moxifloxacin resistant-, multi drug-resistant (MDR)-, fluoroquinolone-resistant MDR-M. smegmatis strain, respectively. Data were presented as Mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to the corresponding controls (student’s t-test: *, p < 0.05; **, p < 0.01; ***, p <0.001).

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  Fig. 5 The survival rate of Mycobacterium smegmatis by intermittent PDT and repeated PDT. (a-e) represents a wild type-, ciprofloxacin resistant-, moxifloxacin resistant, multi drug-resistant (MDR)-, fluoroquinolone-resistant MDR-M. smegmatis strain, respectively. For comparison of intermittent PDT and repeated PDT, M. smegmatis strains were irradiated for a continuous double time period of those PDT, resulting in 36 J/cm2 of irradiation dose. Control: no photosensitizer and laser light, PDT 18 J/cm2: continuous PDT at the dose of 18 J/cm2, PDT 36 J/cm2: continuous PDT at the dose of 36 J/cm2, I-PDT: intermittent PDT, R-PDT: repeated PDT, I-PDT control: M. smegmatis strains were treated by PDT at the dose of 18 J/cm2 and incubated for 2 hours without methylene blue (MB) exposure and irradiation. R-PDT control: M. smegmatis strains were treated by PDT at the dose of 18 J/cm2 and incubated with MB for 2 hours, but not irradiation. Data were presented as mean ± SD of three independent experiments.

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