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Staphylococcus aureus , Streptococcus pyogenes and Enterococcus faecalis can colonize the tooth root canals, adhere to dentin walls, and frequently cause periodontitis in dogs. Bacterial periodontal diseases are common in domesticated pets, causing severe oral cavity inflammation and a strong immune response. This study investigates the antioxidant effect of a natural antimicrobial mixture (Auraguard-Ag) on the ability of S. aureus , S. pyogenes and E. faecalis to infect primary canine oral epithelial cells as well as its impact on their virulence factors. Our data show that a concentration of 0.25% Ag is sufficient to inhibit the growth of all three pathogens, whereas a concentration of 0.5% will become bactericidal. The sub-inhibitory concentration of 0.125% Ag reveals that the antimicrobial mixture can significantly reduce biofilm formation and exopolysaccharide production. The impact on these virulence factors was further translated into a significantly reduced ability to infect primary canine oral epithelial cells and restore epithelial tight junctions, with no impact on the epithelial cell viability. The post-infection inflammatory cytokines (IL-1β and IL-8) and the COX-2 mediator were also reduced both in mRNA and protein expression levels. The oxidative burst, detected upon infection, was also decreased in the presence of Ag, as our results show a significant decrease in H 2 O 2 released by the infected cells. We show that inhibition of either NADPH or ERK activity will result in a downregulation of COX-2 expression and lower levels of H 2 O 2 in infected cells. Conclusively, our study shows that natural antimicrobials reduce pro-inflammatory events, post infection, through an antioxidative mechanism that involves the downregulation of the COX-2 mediator via the inactivation of ERK in the absence of H 2 O 2 . As a result, they significantly reduce the risk of secondary bacterial infections and host oxidative stress caused by Staphylococcus aureus , Streptococcus pyogenes and Enterococcus faecalis accumulation in biofilms in an in vitro canine oral infection model.

Legionella pneumophila is responsible for causing Legionnaires' disease and Pontiac fever, also known as legionellosis. The aim of this study was to investigate the mechanistic effect of a mixture of natural antimicrobials (Citrox BCL) in preventing L. pneumophila biofilm formation and reducing its in vitro virulence. The minimum inhibitory concentrations were detected at 0.06%, and the MBC was established at 0.125%. Based on the growth curve profile, the sub-inhibitory concentration of 0.02% was further used to study the mechanistic implications in the absence of a cytotoxic effect on A549 cells. At 24 h post-infection, Citrox BCL reduced ( p = 0.005) the intracellular growth of L. pneumophila when the A549 cells or the bacteria were pre-treated with 0.02% Citrox BCL. This result was replicated when Citrox BCL was added during the 24 h infection assay leading to a reduction in intracellular growth ( p = 0.003). Herein we show that at the sub-inhibitory concentration of 0.02%, Citrox CBL lowers the ROS levels in infected A549 cells and causes a 45% reduction in L. pneumophila EPS production, a reduction associated with the decline in biofilm formation. Overall, our results corroborate the low c-di-GMP production with the decrease in biofilm formation and low EPS levels. The low EPS levels seemed to be caused by the downregulation of the tat B and tat C gene expressions. Moreover, inhibition of pvc A and pvc B gene expressions, leading to lower siderophore levels, suggests that Citrox BCL reduces the ability of L. pneumophila to sequester iron and reduce biofilm formation through iron starvation.

Nematopsis messor infections severely impact on shrimp’s health with devastating economic consequences on shrimp farming. In a shrimp primary intestinal cells (SGP) model of infection, a sub-inhibitory concentration (0.5%) of natural antimicrobials (Aq) was able to reduce the ability of N. messor to infect (p < 0.0001). To prevent N. messor infection of SGP cells, Aq inhibits host actin polymerization and restores tight junction integrity (TEER) and the expression of Zo-1 and occluding. The oxidative burst, caused by N. messor infection, is attenuated by Aq through the inhibition of NADPH-produced H2O2. Simultaneous to the reduction in H2O2 released, the activity of catalase (CAT) and superoxide dismutase (SOD) were also significantly increase (p < 0.0001). The antimicrobial mixture inactivates the ERK signal transduction pathway by tyrosine dephosphorylation and reduces the expression of DCR2, ALF-A, and ALF-C antimicrobial peptides. The observed in vitro results were also translated in vivo, whereby the use of a shrimp challenge test, we show that in N. messor infected shrimp the mortality rate was 68% compared to the Aq-treated group where the mortality rate was maintained at 14%. The significant increase in CAT and SOD activity in treated and infected shrimp suggested an in vivo antioxidant role for Aq. In conclusion, our study shows that Aq can efficiently reduce N. messor colonization of shrimp’s intestinal cells in vitro and in vivo and the oxidative induced cellular damage, repairs epithelial integrity, and enhances gut immunity.