A Combination of Linalool, Vitamin C, and Copper Synergistically Triggers Reactive Oxygen Species and DNA Damage and Inhibits subsp. Serovar Typhi and .

Inappropriate and disproportionate use of antibiotics is contributing immensely to the development of antibiotic resistance in bacterial species associated with food contamination. The use of natural products in combination can be a potent alternative hurdle strategy to inactivate foodborne pathogens. Here, we explored the pro-oxidant properties of essential oil inalool and itamin C in combination with opper (LVC) in combating the foodborne pathogens and subsp. serovar Typhi using a three-dimensional (3D) checkerboard microdilution assay. Antibacterial activity in terms of the MIC revealed that the triple combination exerted a synergistic effect compared to the effects of the individual constituents. The bactericidal effect of the triple combination was confirmed by a live/dead staining assay. Reactive oxygen species (ROS) measurements with the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay and scanning electron microscopy imaging strongly suggested that the increase in ROS production is the underlying mechanism of the enhanced antibacterial potency of the LVC combination (linalool [1.298 mM], vitamin C [8 mM], copper [16.3 μM]). In addition, the hypersensitivity of oxidative stress regulator mutants (, , , and mutants) toward LVC corroborated the involvement of ROS in cell death. Live/dead staining and changes in cellular morphology revealed that oxidative stress did not transform the cells into the viable but nonculturable (VBNC) state; rather, killing was associated with intracellular and extracellular oxidative burst. Furthermore, the LVC combination did not display toxicity to human cells, while it effectively reduced the pathogen levels in acidic fruit juices by 3 to 4 log CFU/ml without adversely altering the organoleptic properties. This study opens a new outlook for combinatorial antimicrobial therapy. There is a need to develop effective antibacterial therapies for mitigating bacterial pathogens in food systems. We used a 3D checkerboard assay to ascertain a safe synergistic combination of food-grade components: vitamin C, copper, and the essential oil linalool. Individually, these constituents have to be added in large amounts to exert their antibacterial effect, which leads to unwanted organoleptic properties. The triple combination could exceptionally inhibit foodborne Gram-negative pathogens like and subsp. serovar Typhi at low concentrations (linalool, 1.298 mM; vitamin C, 8 mM; copper, 16.3 μM) and displayed potent microbial inhibition in acidic beverages. We found increased susceptibility in deletion mutants of oxidative stress regulators (, , , and mutants) due to ROS generation by Fenton's chemistry. The results of this study show that it may be possible to use plant-based antimicrobials in synergistic combinations to control microbial contaminants.