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Background  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).    Results  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 (H 2 O 2 ) 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).    Conclusion  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

gut pathogens

Mixtures of natural antimicrobials can reduce Campylobacter jejuni, Salmonella enterica and Clostridium perfringens infections and cellular inflammatory response in MDCK cells