Antimicrobial resistance is a threat to public health that requires our immediate attention. With increasing numbers of microbes that are becoming resistant to routinely used antimicrobials, it is vital that we look to other, non-traditional therapies for the treatment of infections. Antimicrobial blue light (aBL) is an innovative approach that has demonstrated efficacy for the inactivation of an array of microbial pathogens. In the present study, we investigated the potential for resistance development to aBL in Gram-negative pathogenic bacteria by carrying out multiple aBL exposures on bacteria. In the first aBL exposure, clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and uropathogenic Escherichia coli [107 colony forming units/mL (CFU/mL)] were irradiated in phosphate-buffered saline with aBL at 405 nm until a >99.99% reduction in bacterial viability was achieved. Irradiation was then repeated for each bacterial species over 20 cycles of aBL exposure. The potential for resistance development to aBL was also investigated in vivo, in superficial mouse wounds infected with a bioluminescent strain of P. aeruginosa (PAO1; 108 CFU) and irradiated with a sub-curative radiant exposures of 108 or 216 J/cm2 aBL over 5 cycles of treatment (over 5 days) prior to bacterial isolation from the animal tissue. PAO1 isolated from infected tissue were treated with aBL at 216 J/cm2, in vitro, in parallel with unexposed PAO1 or PAO1 isolates from mouse wound infections not treated with aBL. No statistically significant correlation was found between the aBL-susceptibility of bacteria in vitro and the number of cycles of aBL exposure any bacterial species (P ≥ 0.26). In addition, serial exposure of infected mouse wounds to aBL did not result in any change in the susceptibility to aBL of PAO1 (P = 0.97). In conclusion, it is unlikely that sequential exposure to aBL will result in aBL-resistance in Gram-negative bacteria. Also, multiple aBL treatments may potentially be administered to an infected wound without resistance development becoming a concern.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria. An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process. Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells. In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

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