Chemical Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.
Molecular Bacteriology & Chemical Genetics Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.
Appl Environ Microbiol. 2019 Feb 6;85(4). doi: 10.1128/AEM.02487-18. Print 2019 Feb 15.
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.
抗生素的不适当和过度使用极大地促成了与食物污染相关的细菌物种对抗生素耐药性的发展。天然产物的联合使用可以是一种有效的替代障碍策略，以灭活食源性病原体。在这里，我们探索了精油inalool 和维生素 C 与铜（LVC）结合的促氧化剂特性，使用三维（3D）棋盘微量稀释测定法对抗食源性病原体和血清型 Typhi。MIC 方面的抗菌活性表明，与单独成分的作用相比，三联组合具有协同作用。通过活菌/死菌染色测定证实了三联组合的杀菌作用。用末端脱氧核苷酸转移酶介导的 dUTP-生物素缺口末端标记测定法和扫描电子显微镜成像进行的活性氧（ROS）测量强烈表明，ROS 产生的增加是 LVC 组合（芳樟醇[1.298 mM]、维生素 C[8 mM]、铜[16.3 μM]）增强抗菌效力的潜在机制。此外，氧化应激调节剂突变体（、、和突变体）对 LVC 的超敏反应证实了 ROS 参与细胞死亡。活菌/死菌染色和细胞形态变化表明，氧化应激不会将细胞转化为存活但非可培养状态（VBNC）；相反，杀伤与细胞内和细胞外氧化爆发有关。此外，LVC 组合对人体细胞没有毒性，同时它有效地将酸性果汁中的病原体水平降低了 3 到 4 个对数 CFU/ml，而不会对感官特性产生不利影响。这项研究为组合抗菌治疗开辟了新的前景。需要开发有效的抗菌疗法来减轻食品系统中的细菌病原体。我们使用 3D 棋盘测定法确定了安全的协同组合食品级成分：维生素 C、铜和精油芳樟醇。单独使用这些成分时，必须添加大量才能发挥其抗菌作用，这会导致不想要的感官特性。该三联组合可以以低浓度（芳樟醇 1.298 mM；维生素 C 8 mM；铜 16.3 μM）异常抑制食源性病原体如和血清型 Typhi，并且在酸性饮料中显示出强大的微生物抑制作用。我们发现由于 Fenton 化学产生的活性氧，氧化应激调节剂缺失突变体（、、和突变体）的敏感性增加。这项研究的结果表明，可能可以使用植物源抗菌剂以协同组合来控制微生物污染物。
