Antimicrobial Blue Light Reduces Human-Wound Pathogens' Resistance to Tetracycline-Class Antibiotics in Biofilms

Abstract

Biofilms contribute to chronic infections and the development of antimicrobial resistance (AMR). We are developing an antimicrobial blue light (aBL) device to reduce bacterial bioburden in wounds and decrease reliance on systemic antibiotics. aBL induces the generation of reactive oxygen species (ROS) through photoexcitation of endogenous chromophores, causing bacterial damage and death. This study explores the combination of tetracyclines (TCs) with aBL for the treatment of biofilm infections in vitro. Tetracyclines (TCs), including second-generation minocycline (MC), doxycycline (DOCT), and third-generation agents omadacycline (OM) and tigecycline (TG), were evaluated for their ability to enhance bactericidal effects and ROS production during aBL treatment of abiotic biofilm. TCs were tested under dark conditions and with varying aBL light parameters against biofilms of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (PA), and Escherichia coli (E. coli). Results showed that TCs alone were ineffective against these biofilm cultures. However, when combined with aBL either before or after TC treatment, significant enhancement of microbicidal activity was observed. When the aBL is added before the TCs, there was equivalent bactericidal effect, indicating that TCs primary action against biofilms were not as photosensitizers. These findings suggest that aBL can significantly enhance the antimicrobial activity of TCs, potentially offering a new effective approach to treating biofilm-associated infections and combating AMR when aBL is applicable.

Keywords: antimicrobial blue light; antimicrobial resistance; biofilms; phototherapy; tetracyclines; wound infection.

Figure 1

Chemical structure of (A) four tetracyclines: minocycline, doxycycline, omadacycline, and tigecycline. (B) Correlation between molar absorption coefficient and wavelength for each tetracycline in PBS and emission spectra of the blue light source (purple).

Figure 1

Chemical structure of (A) four tetracyclines: minocycline, doxycycline, omadacycline, and tigecycline. (B) Correlation between molar absorption coefficient and wavelength for each tetracycline in PBS and emission spectra of the blue light source (purple).

Figure 2

Measurement of reactive oxygen species levels using DCF-DA probe in MRSA planktonic culture treated with 10 µg/mL of TCs in combination with blue light.

Figure 3

Measurement of reactive oxygen species levels using DCF-DA probe in MRSA biofilm treated with 10 µg/mL of TCs in combination with blue light.

Figure 4

Measurement of reactive oxygen species by flow cytometer using dihydrorhodamine (DHR123) as a ROS probe in MRSA planktonic culture treated with 10 µg/mL of TCs in combination with blue light.

Figure 5

Bar graph illustrating the log10 colony-forming unit (CFU/g) reduction of 48 h of bacterial biofilms in MRSA after treatment with different conditions, procedure 1 (TCs incubation followed by aBL) and procedure 2 (aBL application followed by TCs incubation). The differences between untreated or treated biofilms were analyzed with a one-way ANOVA followed by Tukey’s multiple comparison tests: ns, not significant. The data for antibiotics alone in the dark are shown in Table 1.

Figure 6

Comparation of post-effects of TC combined with aBL on MRSA biofilm at various time points—0 h, 24 h, and 48 h—both with and without TCs during these intervals. (A). Different time point incubation of TC without aBL (DARK). (B). Post-effect of TCs + aBL, followed by incubation with BHI only after 24 h and 48 h. (C). Post-effect of TCs + aBL, followed by incubation with TCs after 24 h and 48 h. The differences between aBL alone or aBL + TCs biofilms were analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test: ns, not significant, #### p < 0.0001, ## p < 0.01, # p < 0.1, related to aBL.