The mechanistic role of natural antimicrobials in preventing Staphylococcus aureus invasion of MAC-T cells using an in vitro mastitis model.

BACKGROUND: Starting primarily as an inflammation of the mammary gland, mastitis is frequently driven by infectious agents such as Staphylococcus aureus. Mastitis has a large economic impact globally, which includes diagnostic, treatment, and the production costs not to mention the potential milk contamination with antimicrobial residues. Currently, mastitis prevention and cure depends on intramammary infusion of antimicrobials, yet, their overuse risks engendering resistant pathogens, posing further threats to livestock. METHODS: In our study we aimed to investigate, in vitro, using bovine mammary epithelial cells (MAC-T), the efficacy of the AuraShield an antimicrobial mixture (As) in preventing S. aureus attachment, internalisation, and inflammation. The antimicrobial mixture (As) included: 5% maltodextrin, 1% sodium chloride, 42% citric acid, 18% sodium citrate, 10% silica, 12% malic acid, 9% citrus extract and 3% olive extract (w/w). RESULTS AND DISCUSSION: Herein we show that As can significantly reduce both adherence and invasion of MAC-T cells by S. aureus, with no impact on cell viability at all concentrations tested (0.1, 0.2, 0.5, 1%) compared with untreated controls. The anti-apoptotic effect of As was achieved by significantly reducing cellular caspase 1, 3 and 8 activities in the infected MAC-T cells. All As concentrations were proven to be subinhibitory, suggesting that Ac can reduce S. aureus virulence without bacterial killing and that the effect could be dual including a host modulation effect. In this context, we show that As can reduce the expression of S. aureus clumping factor (ClfB) and block its interaction with the host Annexin A2 (AnxA2), resulting in decreased bacterial adherence in infection of MAC-T cells. Moreover, the ability of As to block AnxA2 had a significant decreasing effect on the levels of pro inflammatory cytokine released upon S. aureus interaction with MAC-T cells. CONCLUSION: The results presented in this study indicate that mixtures of natural antimicrobials could potentially be considered an efficient alternative to antibiotics in treating S. aureus induced mastitis.

Natural Antimicrobials Promote the Anti-Oxidative Inhibition of COX-2 Mediated Inflammatory Response in Primary Oral Cells Infected with Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis.

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-1ß 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 H2O2 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 H2O2 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 H2O2. 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.

The Antioxidant Effect of Natural Antimicrobials in Shrimp Primary Intestinal Cells Infected with Nematopsis messor.

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.

The Prebiotic Effect of an Organic Acid Mixture on Faecalibacterium prausnitzii Metabolism and Its Anti-Pathogenic Role against Vibrio parahaemolyticus in Shrimp.

Increasing the abundance of probiotic bacteria in the gut requires either direct dietary supplementation or the inclusion of feed additives able to support the growth of beneficial commensal bacteria. In crustaceans, the increased presence of probiotic-like bacteria in the gut, including of Faecalibacterium prausnitzii (F. prausnitzii), will guarantee a positive health status and a gut environment that will ensure enhanced performance. The aim of this study was to investigate if a mixture of organic acids, AuraAqua (Aq) can stimulate the growth and the anti-pathogenic efficacy of F. prausnitzii through a combination of in vitro and ex vivo models. The results showed that 0.5% Aq was able to improve the growth rate of F. prausnitzii in vitro and in an ex vivo shrimp gut model. Moreover, we were able to demonstrate that Aq increases butyrate production and cellulose degradation in culture or in the shrimp gut model. The growth-stimulating effect of Aq also led to an improved and anti-pathogenic effect against Vibrio parahaemolyticus in a co-culture experiment with shrimp gut primary epithelial cells (SGP). In conclusion, our work demonstrates that Aq can stimulate the growth of F. prausnitzii, increase the production of short-chain fatty acid (SCFA) butyrate, improve substrate digestion, and prevent V. parahaemolyticus invasion of SGP cells.

Essential Fatty Acids as Biomedicines in Cardiac Health.

The destructive impact of cardiovascular diseases on health, including heart failure, peripheral artery disease, atherosclerosis, stroke, and other cardiac pathological conditions, positions these health conditions as leading causes of increased global mortality rates, thereby impacting the human quality of life. The considerable changes in modern lifestyles, including the increase in food intake and the change in eating habits, will unavoidably lead to an unbalanced consumption of essential fatty acids, with a direct effect on cardiovascular health problems. In the last decade, essential fatty acids have become the main focus of scientific research in medical fields aiming to establish their impact for preventing cardiovascular diseases and the associated risk factors. Specifically, polyunsaturated fatty acids (PUFA), such as omega 3 fatty acids, and monounsaturated fatty acids from various sources are mentioned in the literature as having a cardio-protective role, due to various biological mechanisms that are still to be clarified. This review aims to describe the major biological mechanisms of how diets rich in essential fatty acids, or simply essential fatty acid administration, could have anti-inflammatory, vasodilatory, anti-arrhythmic, antithrombotic, antioxidant, and anti-atherogenic effects. This review describes findings originating from clinical studies in which dietary sources of FAs were tested for their role in mitigating the impact of heart disorders in human health.

The Antimicrobial Effect of a Commercial Mixture of Natural Antimicrobials Against Escherichia coli O157:H7.

Ruminants are important reservoirs of E. coli O157:H7 and are considered as the major source of most foodborne outbreaks (e.g., 2017 outbreak in Germany, 2014 and 2016 outbreaks in United States, all linked to beef products). A promising strategy to reduce E. coli O157 is using antimicrobials to reduce the pathogen levels and/or virulence within the animal gastrointestinal tract and thus foodborne disease. The aim of the study was to determine the efficacy of a commercial mixture of natural antimicrobials against E. coli O157. The minimum inhibitory concentration and minimum bactericidal concentration of the antimicrobial were quantitatively determined and found to be 0.5% and 0.75% (v/v) of the natural antimicrobial, respectively. Microbial growth kinetics was also used to determine the effect of the antimicrobial on the pathogen. The natural antimicrobial affected the cell membrane of E. coli O157, as demonstrated by the increase in relative electric conductivity and increase in protein and nucleic acid release. The antimicrobial was also able to significantly reduce the concentration on E. coli O157 in a model rumen system. Biofilm assays showed that subinhibitory concentrations of the antimicrobial significantly reduced the E. coli 0157 biofilm forming capacity without influencing pathogen growth. In addition, the natural antimicrobial was able to reduce motility and exopolysaccharide production. Subinhibitory concentrations of the antimicrobial had no effect on AI-2 production. These findings suggest that the natural antimicrobial exerts an antimicrobial effect against E. coli O157 in vitro and in a model rumen system and could be potentially used to control this pathogen in the animal gut. The results also indicate that subinhibitory concentrations of the antimicrobial effectively reduce biofilm formation, motility, and exopolysaccharide production.

Effect of mineral-enriched diet and medicinal herbs on Fe, Mn, Zn, and Cu uptake in chicken.

BACKGROUND: The goal of our study was to evaluate the effects of different medicinal herbs rich in polyphenol (Lemon balm, Sage, St. John's wort and Small-flowered Willowherb) used as dietary supplements on bioaccumulation of some essential metals (Fe, Mn, Zn and Cu) in different chicken meats (liver, legs and breast). RESULTS: In different type of chicken meats (liver, legs and breast) from chickens fed with diets enriched in minerals and medicinal herbs, beneficial metals (Fe, Mn, Zn and Cu) were analysed by flame atomic absorption spectrometry. Fe is the predominant metal in liver and Zn is the predominant metal in legs and breast chicken meats. The addition of metal salts in the feed influences the accumulations of all metals in the liver, legs and breast chicken meat with specific difference to the type of metal and meat. The greatest influences were observed in legs meat for Fe and Mn. Under the influence of polyphenol-rich medicinal herbs, accumulation of metals in the liver, legs and breast chicken meat presents specific differences for each medicinal herb, to the control group that received a diet supplemented with metal salts only. Great influence on all metal accumulation factors was observed in diet enriched with sage, which had significantly positive effect for all type of chicken meats. CONCLUSIONS: Under the influence of medicinal herbs rich in different type of polyphenol, accumulation of metals in the liver, legs and breast chicken meat presents significant differences from the group that received a diet supplemented only with metal salts. Each medicinal herb from diet had a specific influence on the accumulation of metals and generally moderate or poor correlations were observed between total phenols and accumulation of metals. This may be due to antagonism between metal ions and presence of other chelating agents (amino acids and protein) from feeding diets which can act as competitor for complexation of metals and influence accumulation of metals in chicken meat.Graphical abstract.