Visualization and elimination of polymicrobial biofilms by a combination of ALA-carvacrol-blue light

Chronic wound infections caused by multidrug-resistant (MDR) bacteria are one of the serious threats to public health due to limited therapeutic options and lengthy care. This investigation combines 5-aminolevulinic acid (ALA), blue light (BL), and phytochemical carvacrol, named ABC cocktail or trio-therapy, to efficiently eliminate wound-related MDR pathogens. Both planktonic cells and biofilms of blue light-refractory Escherichia (E.) coli and Klebsiella (K.) pneumoniae succumbed to the trio-therapy partly due to porphyrin accumulations following ALA incubation. ALA either alone or alongside carvacrol could vigorously trigger bursts of reactive oxygen species (ROS) upon blue light irradiation in K. pneumoniae, but not in mammalian cells. The robust antimicrobial activity was extended to polymicrobial biofilms composed of five MDR pathogens (Staphylococcus aureus, E. coli, K. pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) in vitro and in vivo. Strikingly, polymicrobial biofilm in mouse wounds became readily visible in the presence of ALA owing to the increasing generation of porphyrins that exhibited bright red fluorescence in response to blue light. Thus, ALA not only enhances killing efficacy, but also helps to pinpoint the infections for guiding debridement, precise phototherapy, and timely assessment of treatment effectiveness. Featuring a broadened antimicrobial spectrum and advantages of bacterial/biofilm imaging, the trio-therapy can be used either alone or adjunctive to other wound management modalities to effectively combat MDR bacteria in wounds.