The classification of natural antimicrobials as potential antibiotic replacements is still hampered by the absence of clear biological mechanisms behind their mode of action. This study investigated the mechanisms underlying the anti-bacterial effect of a mixture of natural antimicrobials (maltodextrin, citric acid, sodium citrate, malic acid, citrus extract and olive extract) against Campylobacter jejuni RC039, Salmonella enterica SE 10/72 and Clostridium perfringens ATCC® 13124 invasion of Madin–Darby Canine Kidney cells (MDCK). Minimum sub-inhibitory concentrations were determined for Campylobacter jejuni (0.25%), Salmonella enterica (0.50%) and Clostridium perfringens (0.50%) required for the in vitro infection assays with MDCK cells. The antimicrobial mixture significantly reduced the virulence of all three pathogens towards MDCK cells and restored the integrity of cellular tight junctions through increased transepithelial resistance (TEER) and higher expression levels of ZO-1 (zonula occludens 1) and occludin. This study also identified the ERK (external regulated kinase) signalling pathway as a key mechanism in blocking the pro-inflammatory cytokine production (IL-1β, IL-6, IL-8, TNF-α) in infected cells. The reduction in hydrogen peroxide (H2O2) production and release by infected MDCK cells, in the presence of the antimicrobial mixture, was also associated with less tetrathionate formed by oxidation of thiosulphate (p < 0.0001). The present study describes for the first time that mixtures of natural antimicrobials can prevent the formation of substrates used by bacterial pathogens to grow and survive in anaerobic environments (e.g. tetrathionate). Moreover, we provide further insights into pathogen invasion mechanisms through restoration of cellular structures and describe their ability to block the ERK–MAPK kinase pathway responsible for inflammatory cytokine release

中文翻译：

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天然抗微生物剂的混合物可以减少空肠弯曲杆菌、肠沙门氏菌和产气荚膜梭菌的感染和MDCK细胞的细胞炎症反应。

由于其作用模式背后缺乏明确的生物学机制，将天然抗菌剂归类为潜在的抗生素替代品仍然受到阻碍。本研究调查了天然抗菌剂混合物（麦芽糊精、柠檬酸、柠檬酸钠、苹果酸、柑橘提取物和橄榄提取物）对空肠弯曲杆菌 RC039、肠道沙门氏菌 SE 10/72 和产气荚膜梭菌 ATCC 的抗菌作用机制® 13124 入侵 Madin-Darby 犬肾细胞 (MDCK)。确定了 MDCK 细胞体外感染测定所需的空肠弯曲杆菌 (0.25%)、肠沙门氏菌 (0.50%) 和产气荚膜梭菌 (0.50%) 的最低亚抑制浓度。抗菌混合物显着降低了所有三种病原体对 MDCK 细胞的毒力，并通过增加跨上皮抗性 (TEER) 和 ZO-1（封闭小带 1）和 occludin 的更高表达水平来恢复细胞紧密连接的完整性。该研究还确定了 ERK（外部调节激酶）信号通路是阻断受感染细胞中促炎细胞因子产生（IL-1β、IL-6、IL-8、TNF-α）的关键机制。在抗菌混合物存在的情况下，受感染的 MDCK 细胞产生和释放过氧化氢 (H2O2) 的减少也与硫代硫酸盐氧化形成的连四硫酸盐减少有关 (p < 0.0001)。本研究首次描述了天然抗微生物剂的混合物可以防止细菌病原体用于在厌氧环境中生长和存活的底物（例如连四硫酸盐）的形成。此外，我们通过恢复细胞结构提供了对病原体入侵机制的进一步见解，并描述了它们阻断负责炎症细胞因子释放的 ERK-MAPK 激酶途径的能力