In vitro study: methylene blue-based antibacterial photodynamic inactivation of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is one of the most antibiotic-resistant and opportunistic pathogens in immunocompromised and debilitated patients. It is considered the cause of most severe skin infections and is frequently found in hospital burn units. Due to its high antibiotic resistance, eliminating P. aeruginosa from skin infections is quite challenging. Therefore, this study aims to assess the novel in vitro antibacterial activity of methylene blue using a 635-nm diode laser to determine the effective power and energy densities for inhibition of P. aeruginosa. The strain was treated with various concentrations of methylene blue and 635-nm diode laser at powers of 300 mW/cm² and 250 mW/cm². The diode laser's potency in the photo-destruction of methylene blue and its degradation through P. aeruginosa were also evaluated. Colony-forming unit (CFU)/ml, fluorescence spectroscopy, optical density, and confocal microscopy were used to measure the bacterial killing effect. As a result, the significant decrease of P. aeruginosa was 2.15-log₁₀, 2.71-log₁₀, and 3.48-log₁₀ at 60, 75, and 90 J/cm² after excitation of MB for 240, 300, and 360 s at a power of 250 mW/cm², respectively. However, a maximum decrease in CFU was observed by 2.54-log₁₀ at 72 J/cm² and 4.32-log₁₀ at 90 and 108 J/cm² after 300 mW/cm² of irradiation. Fluorescence images confirmed the elimination of bacteria and showed a high degree of photo-destruction compared to treatment with methylene blue and light alone. In conclusion, MB-induced aPDT demonstrated high efficacy, which could be a potential approach against drug-resistant pathogenic bacteria. KEY POINTS: • Combination of methylene blue with 635-nm diode laser for antibacterial activity. • Methylene blue photosensitizer is employed as an alternative to antibiotics. • aPDT showed promising antibacterial activity against Pseudomonas aeruginosa.

Keywords: Antibiotics; Diode laser; Methylene blue; Photosensitizer; Pseudomonas aeruginosa; aPDT.

Fig. 1

Antimicrobial susceptibility of Pseudomonas aeruginosa. A Comparison of antibiogram results with CLSI 2020. B Zone of inhibition measured in mm on MHA-containing plate

Fig. 2

A Photobleaching of methylene blue concentration at a power of 300 mW/cm² for selected irradiation times and dosages. B Optical density of control groups and photodegraded methylene blue at 600 nm. Mean ± standard deviation indicated by errors bar. C Methylene blue degradation before and after incubation with P. aeruginosa. D 635-nm diode laser therapy system developed by NILOP and their application inside a biosafety cabinet. μg/ml indicates methylene blue concentrations

Fig. 3

A Antibacterial activity of methylene blue using 15.625 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, and 500 μg/ml concentrations in the absence of light on P. aeruginosa. B Application of MB alone in well diffusion method on MHA-containing plate. C Application of 635-nm diode laser without methylene blue against P. aeruginosa. s, seconds. μg/ml indicates methylene blue concentrations

Fig. 4

A P. aeruginosa colonies clearly seen under S/ST Stereo Microscope before and after treatment with MB and diode laser. B Photographs of P. aeruginosa colonies on nutrient agar plates after treatment with various concentrations of methylene blue in combination with 635-nm diode laser at a power of 300 mW/cm² for exposures of 60, 120, 180, 240, 300, and 360 s and 18, 36, 54, 72, 90, and 108 J/cm² of dosages. C Indicating a reduction of CFU/ml in percentage. D Log₁₀ reduction of CFU/ml after treatment compared with the control group. The standard errors are very low and are presented as the mean ± standard deviation of each independent sample, which also demonstrates the logarithmic values in log₁₀ CFU/ml reduction. s, seconds. μg/ml indicates methylene blue concentrations

Fig. 5

A Photographs of P. aeruginosa colonies on nutrient agar plates after treatment with various concentrations of methylene blue in combination with 635-nm diode laser at a power of 250 mW/cm² for exposures of 60, 120, 180, 240, 300, and 360 s and 18, 36, 54, 72, 90, and 108 J/cm² of dosages. B Indicating reduction of CFU/ml in percentage. C Log₁₀ reduction of CFU/ml after treatment compared with the control group. The standard errors are very low and are presented as the mean ± standard deviation of each independent sample, which also demonstrates the logarithmic values in log₁₀ CFU/ml reduction. s, seconds. μg/ml indicates methylene blue concentrations

Fig. 6

Optical density of P. aeruginosa was measured at a wavelength of 600 nm before and after treatment with aPDT at powers A 300 mW/cm² and B 250 mW/cm². The standard errors are very low and are presented as the mean ± standard deviation of each independent sample. μg/ml indicates methylene blue concentrations

Fig. 7

Spectra of fluorescence spectroscopy were recorded at excitation wavelength 270 nm and emission wavelength 285 to 525 nm after treatment with various concentrations of methylene blue for specific time exposure at powers A 300 mW/cm² (18, 36, 54, 72, 90, and 109 J/cm²) and B 250 mW/cm² (15, 30, 45, 60, 75, and 90 J/cm²). C Linear relationship/calibration between fluorescence intensity at 336 nm and D 436 nm and exposure time to determine the gradual decrease and increase in fluorescence intensity after treatment

Fig. 8

Confocal laser scanning microscopy (CLSM) images of P. aeruginosa. The dense green lawn, fluorescent colony-like structures represent the live cells, while the non-fluorescent empty places in the lawn indicate the inhibition of bacterial load after treatment with aPDT at powers of 300 mW/cm² and 250 mW/cm². The circles in the last treated samples indicate few low-fluorescent live colonies after treatment, while only four colonies remain live at 108 J/cm² as compared to 90 J/cm². s, seconds