<i>In Vitro</i> Evaluation of the Antibacterial Effect of Photodynamic Therapy with Methylene Blue

Catão, Maria Helena Chaves de Vasconcelos; Batista, Ana Luzia Araújo

doi:10.1590/pboci.2020.073

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ORIGINAL ARTICLE • Pesqui. Bras. Odontopediatria Clín. Integr. 20 • 2020 • https://doi.org/10.1590/pboci.2020.073 copy

In Vitro Evaluation of the Antibacterial Effect of Photodynamic Therapy with Methylene Blue

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Objective:

To evaluate, in vitro, the effect of photodynamic therapy (PDT) compared to laser therapy and the use of a photosensitizer alone.

Material and Methods:

The following therapies were used: PDT, laser therapy and photosensitizer alone. For PDT, methylene blue (MB) at different concentrations and red laser InGaAlP 660nm were used. For the use of low-power laser (LPL) alone, red laser InGaAlP 660 nm and infrared laser AsGaAl, 830 nm, both in continuous emission were used. Standard ATCC strains of Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) species were used. The antibacterial effect of PDT was quantified by the diameter of the inhibition halos.

Results:

PDT (LPL 660 nm, 320 J/cm²) with MB at concentration of 50 µg/mL showed antibacterial efficacy only when tested against S. aureus and E. coli strains, as well as with the isolated use of MB at the same concentration. Using LPL alone, whether red or infrared, with different dosimetry, no antibacterial effect was observed. In none of the therapeutic modalities used, P. aeruginosa inactivation was observed.

Conclusion:

Antibacterial effects of PDT (LPL 660 nm + MB 50 µg/mL) were observed for S. aureus and E. coli, as well as with the isolated use of MB (50 µg/mL). For P. aeruginosa, no antibacterial effect with any of the protocols recommended in the study was observed.

Keywords:
Microbiology; Bacteria; Low-Level Light Therapy; Photochemotherapy

Microorganisms	PDT (methylene blue [µg/mL] / Diameter of Growth Inhibition Halos (mm)
Microorganisms	50 µg/mL	25 µg/mL	12.5 µg/mL	6.25 µg/mL	3.125 µg/mL	1.5625 µg/mL
Staphylococcus Aureus (ATCC 25923)	12	0	0	0	0	0
Escherichia Coli (ATCC 25922)	12	0	0	0	0	0
Pseudomonas aeruginosa (ATCC 27857)	0	0	0	0	0	0

Microorganisms	Concentration	Mean Halo	N	SD	Minimum Halo	Maximum Halo	p-value¹ 1 Mann-Whitney U test at 5% significance level; ,² 2 For the application of the Mann-Whitney U test, the methylene blue concentration was divided into two groups, one less than or equal to 25 µg/mL and the other equal to 50 µg/mL.
Pseudomonas aeruginosa	1.5625 µg/mL	0.0	2	0.00	0	0	1.000
	3.125 µg/mL	0.0	2	0.00	0	0
	6.25 µg/mL	0.0	2	0.00	0	0
	12.5 µg/mL	0.0	2	0.00	0	0
	25 µg/mL	0.0	2	0.00	0	0
	50 µg/mL	0.0	2	0.00	0	0
	Total	0.0	12	0.00	0	0
Staphylococcus Aureus	1.5625 µg/mL	0.0	2	0.00	0	0	0.001
	3.125 µg/mL	0.0	2	0.00	0	0
	6.25 µg/mL	0.0	2	0.00	0	0
	12.5 µg/mL	0.0	2	0.00	0	0
	25 µg/mL	0.0	2	0.00	0	0
	50 µg/mL	12.00	2	0.00	12	12
	Total	2.00	12	4.671	0	12
Escherichia Coli	1.5625 µg/mL	0.0	2	0.00	0	0	0.001
	3.125 µg/mL	0.0	2	0.00	0	0
	6.25 µg/mL	0.0	2	0.00	0	0
	12.5 µg/mL	0.0	2	0.00	0	0
	25 µg/mL	0.0	2	0.00	0	0
	50 µg/mL	12.00	2	0.00	12	12
	Total	2.00	12	4.671	0	12
Total	1.5625 µg/mL	0.0	6	0.00	0	0
	3.125 µg/mL	0.0	6	0.00	0	0
	6.25 µg/mL	0.0	6	0.00	0	0	-
	12.5 µg/mL	0.0	6	0.00	0	0
	25 µg/mL	0.0	6	0.00	0	0
	50 µg/mL	8.00	6	6.197	0	12
	Total	1.33	36	3.825	0	12

Microorganisms	Wave Length	N
Pseudomonas aeruginosa	(830 nm) Infrared	8
	(660 nm) Red	8
	Total	16
Staphylococcus aureus	(830 nm) Infrared	8
	(660 nm) Red	8
	Total	16
Eschericha coli	(830 nm) Infrared	8
	(660 nm) Red	8
	Total	16
Total	(830 nm) Infrared	24
	(660 nm) Red	24
	Total	48

Microorganisms	Concentration	Mean Halo	N	SD	Minimum Halo	Maximum Halo	p-value^* * Mann-Whitney U test at 5% significance level.
Pseudomonas aeruginosa	1.5625 µg/mL	0.0	2	0.00	0	0	1.000
	3.125 µg/mL	0.0	2	0.00	0	0
	6.25 µg/mL	0.0	2	0.00	0	0
	12.5 µg/mL	0.0	2	0.00	0	0
	25 µg/mL	0.0	2	0.00	0	0
	50 µg/mL	0.0	2	0.00	0	0
	Total	0.0	12	0.00	0	0
Staphylococcus aureus	1.5625 µg/mL	0.0	2	0.00	0	0	0.001
	3.125 µg/mL	0.0	2	0.00	0	0
	6.25 µg/mL	0.0	2	0.00	0	0
	12.5 µg/mL	0.0	2	0.00	0	0
	25 µg/mL	0.0	2	0.00	0	0
	50 µg/mL	12.00	2	0.00	12	12
	Total	2.00	12	4.671	0	12
Escherichia Coli	1.5625 µg/mL	0.0	2	0.00	0	0	0.001
	3.125 µg/mL	0.0	2	0.00	0	0
	6.25 µg/mL	0.0	2	0.00	0	0
	12.5 µg/mL	0.0	2	0.00	0	0
	25 µg/mL	0.0	2	0.00	0	0
	50 µg/mL	12.00	2	0.00	12	12
	Total	2.00	12	4.671	0	12
Total	1.5625 µg/mL	0.0	6	0.00	0	0
	3.125 µg/mL	0.0	6	0.00	0	0
	6.25 µg/mL	0.0	6	0.00	0	0
	12.5 µg/mL	0.0	6	0.00	0	0
	25 µg/mL	0.0	6	0.00	0	0
	50 µg/mL	8.00	6	6.197	0	12

Microorganisms	Concentration	Mean Halo	N	SD	Minimum Halo	Maximum Halo	p-value^* * Mann-Whitney U test at 5% significance level.
Pseudomonas aeruginosa	1.5625 µg/mL	0.00	2	0.000	0	0	0.436
	3.125 µg/mL	0.00	2	0.000	0	0
	6.25 µg/mL	0.00	2	0.000	0	0
	12.5 µg/mL	0.00	2	0.000	0	0
	25 µg/mL	0.00	2	0.000	0	0
	50 µg/mL	0.00	2	0.000	0	0
	Total	0.00	12	0.000	0	0
Staphylococcus aureus	1.5625 µg/mL	0.00	2	0.000	0	0	0.001
	3.125 µg/mL	0.00	2	0.000	0	0
	6.25 µg/mL	0.00	2	0.000	0	0
	12.5 µg/mL	0.00	2	0.000	0	0
	25 µg/mL	0.00	2	0.000	0	0
	50 µg/mL	12.00	2	0.000	12	12
	Total	2.00	12	4.671	0	12
Eschericha coli	1.5625 µg/mL	0.00	2	0.000	0	0	0.001
	3.125 µg/mL	0.00	2	0.000	0	0
	6.25 µg/mL	0.00	2	0.000	0	0
	12.5 µg/mL	0.00	2	0.000	0	0
	25 µg/mL	0.00	2	0.000	0	0
	50 µg/mL	12.00	2	0.000	12	12
	Total	2.00	12	4.671	0	12
Total	1.5625 µg/mL	0.00	6	0.000	0	0
	3.125 µg/mL	0.00	6	0.000	0	0
	6.25 µg/mL	0.00	6	0.000	0	0
	12.5 µg/mL	0.00	6	0.000	0	0
	25 µg/mL	0.00	6	0.000	0	0
	50 µg/mL	8.00	6	6.197	0	12
	Total	1.33	36	3.825	0	12

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[1] Author to whom correspondence should be addressed: Maria Helena Chaves de Vasconcelos Catão, Rua Alfredo Farias Pimentel, n 61, Sandra Cavalcanti, Campina Grande, PB, Brazil. 50.410-675. Phone: +55 83 991345863. E-mail: mhelenact@zipmail.com.br.