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

Igori Balta,Nicolae Corcionivoschi,Phittawat Sopharat,Gratiela Gradisteanu-Pircalabioru,Carmel Kelly,Todd Callaway,Ozan Gundogdu,Adela Marcu,Patrick Ward,Lavinia Stef,Myriam Deshaies,Mark Linton,Ioan Pet

doi:10.1186/s13099-021-00433-5

Abstract

BackgroundThe 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).ResultsMinimum 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).ConclusionThe 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

Highlights

The classification of natural antimicrobials as potential antibiotic replacements is still hampered by the absence of clear biological mechanisms behind their mode of action
The present study investigated the possible role of natural antimicrobials and their anti-pathogenic effect against C. jejuni RC039, S. enterica SE 10/72 and C. perfringens ATCC® 13124 in canine derived Madin–Darby Canine Kidney cells (MDCK)
Establishing the sub‐inhibitory concentrations First we aimed to establish the anti-pathogenic effect of a natural antimicrobial mixture (Auraguard) and to determine the sub-inhibitory concentrations for C. jejuni, S. enterica and C. perfringens

Summary

Introduction

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 under‐ lying 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). Infections caused by foodborne zoonotic agents, such as Campylobacter spp., Salmonella spp., and Clostridium spp., are associated with high morbidity rates worldwide in both humans and animals [3]. Poultry meat is considered the main source of Campylobacter for human infections, the presence of this bacterium in dogs and cats increases significant public health concerns as pets can potentially be considered a transmission vector [5]. Clostridium perfringens is mainly associated with acute haemorrhagic diarrhoea syndrome (AHDS) in dogs [9]. In cases of severe haemorrhagic canine gastroenteritis,a severe necrotising inflammation of the intestinal tract epithelium can occur which can lead to rapid mortality [10]

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