Balta, IgoriMarcu, AdelaLinton, W. Mark R.Kelly, Carmel A.Gundogdu, OzanStef, LaviniaPet, IoanWard, PatrickDeshales, Myriamcallaway, ToddSopharat, PhittawatGradisteanu-Pircalabioru, GratielaCorcionivoschi, Nicolae2021-08-092021-08-092021-06-07Balta, I., Marcu, A., Linton, M., Kelly, C., Gundogdu, O., Stef, L., Pet, I., Ward, P., Deshaies, M., Callaway, T., Sopharat, P., Gradisteanu-Pircalabioru, G. and Corcionivoschi, N. (2021) ‘Mixtures of natural antimicrobials can reduce Campylobacter jejuni, Salmonella enterica and Clostridium perfringens infections and cellular inflammatory response in MDCK cells’, Gut Pathogens, 13(1). doi: 10.1186/s13099-021-00433-5.1757-4749https://doi.org/10.1186/s13099-021-00433-5Publication history: Accepted - 1 June 2021; Published online - 7 June 2021Background: 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 ( 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). 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).enOpen Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Campylobacter jejuniSalmonella entericaClostridium perfringensNatural antimicrobialsHydrogen peroxideERK kinaseMDCK cellsVirulenceArticle