Blends of Organic Acids Are Weaponizing the Host iNOS and Nitric Oxide to Reduce Infection of Piscirickettsia salmonis in vitro.

For the last 30 years, Piscirickettsia salmonis has caused major economic losses to the aquaculture industry as the aetiological agent for the piscirickettsiosis disease. Replacing the current interventions, based on antibiotics, with natural alternatives (e.g., organic acids) represents a priority. With this study, we aimed to better understand their biological mechanism of action in an in vitro model of infection with salmon epithelial cells (CHSE-214). Our first observation revealed that at the sub-inhibitory concentration of 0.5%, the organic acid blend (Aq) protected epithelial cell integrity and significantly reduced P. salmonis invasion. The MIC was established at 1% Aq and the MBC at 2% against P. salmonis. The sub-inhibitory concentration significantly increased the expression of the antimicrobial peptides Cath2 and Hepcidin1, and stimulated the activity of the innate immune effector iNOS. The increase in iNOS activity also led to higher levels of nitric oxide (NO) being released in the extracellular space. The exposure of P. salmonis to the endogenous NO caused an increase in bacterial lipid peroxidation levels, a damaging effect which can ultimately reduce the pathogen's ability to attach or multiply intracellularly. We also demonstrate that the increased NO release by the host CHSE-214 cells is a consequence of direct exposure to Aq and is not dependent on P. salmonis infection. Additionally, the presence of Aq during P. salmonis infection of CHSE-214 cells significantly mitigated the expression of the pro-inflammatory cytokines IL-1ß, IL-8, IL-12, and IFNγ. Taken together, these results indicate that, unlike antibiotics, natural antimicrobials can weaponize the iNOS pathway and secreted nitric oxide to reduce infection and inflammation in a Piscirickettsia salmonis in vitro model of infection.

Organic acids mitigate Streptococcus agalactiae virulence in Tilapia fish gut primary cells and in a gut infection model.

BACKGROUND: Streptococcus agalactiae, a Gram-positive bacterium, has emerged as an important pathogen for the aquaculture industry worldwide, due to its increased induced mortality rates in cultured fish. Developing interventions to cure or prevent infections based on natural alternatives to antibiotics has become a priority, however, given the absence of scientific evidence regarding their mode of action progress has been slow. METHODS: In this study we aimed to investigate the effect of a mixture of organic acids (natural antimicrobials), AuraAqua (Aq), on the virulence of S. agalactiae using Tilapia gut primary epithelial cells and an in vitro Tilapia gut culture model. Our results show that Aq was able to reduce significantly, in vitro, the S. agalactiae levels of infection in Tilapia gut primary epithelial cells (TGP) when the MIC concentration of 0.125% was tested. RESULTS AND DISCUSSION: At bacterial level, Aq was able to downregulate bacterial capsule polysaccharide (CPS) gene expression, capC, resulting in a significant decrease in bacterial surface capsule production. The decrease in CPS production was also associated with a reduction in the pro-inflammatory IFNγ, IL1ß, TNFα, SOD and CAT gene expression and H2O2 production in the presence of 0.125% Aq (P < 0.0001). The antimicrobial mixture also reduced the levels of S. agalactiae infection in an in vitro gut culture model and significantly reduced the IFNγ, IL1ß, TNFα, SOD, CAT gene expression and H2O2 production in infected tissue. Moreover, genes involved in Tilapia resistance to S. agalactiae induced disease, MCP-8 and Duo-1, were also downregulated by Aq, as a consequence of reduced bacterial levels of infection. CONCLUSION: Conclusively, our study shows that mixtures of organic acids can be considered as potential alternative treatments to antibiotics and prevent S. agalactiae infection and inflammation in the Tilapia fish digestive tract.

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 Block the Host NF-κB Pathway and Reduce Enterocytozoon hepatopenaei Infection Both In Vitro and In Vivo.

The objective of this work was to investigate, for the first time, the antioxidant effect of a mixture of natural antimicrobials in an Enterocytozoon hepatopenaei (EHP) shrimp-gut model of infection and the biological mechanisms involved in their way of action. The study approach included investigations, firstly, in vitro, on shrimp-gut primary (SGP) epithelial cells and in vivo by using EHP-challenged shrimp. Our results show that exposure of EHP spores to 0.1%, 0.5%, 1%, and 2% AuraAqua (Aq) significantly reduced spore activity at all concentrations but was more pronounced after exposure to 0.5% Aq. The Aq was able to reduce EHP infection of SGP cells regardless of cells being pretreated or cocultured during infection with Aq. The survivability of SGP cells infected with EHP spores was significantly increased in both scenarios; however, a more noticeable effect was observed when the infected cells were pre-exposed to Aq. Our data show that infection of SGP cells by EHP activates the host NADPH oxidases and the release of H2O2 produced. When Aq was used during infection, a significant reduction in H2O2 was observed concomitant with a significant increase in the levels of CAT and SOD enzymes. Moreover, in the presence of 0.5% Aq, the overproduction of CAT and SOD was correlated with the inactivation of the NF-κB pathway, which, otherwise, as we show, is activated upon EHP infection of SGP cells. In a challenge test, Aq was able to significantly reduce mortality in EHP-infected shrimp and increase the levels of CAT and SOD in the gut tissue. Conclusively, these results show, for the first time, that a mixture of natural antimicrobials (Aq) can reduce the EHP-spore activity, improve the survival rates of primary gut-shrimp epithelial cells and reduce the oxidative damage caused by EHP infection. Moreover, we show that Aq was able to stop the H2O2 activation of the NF-κB pathway of Crustins, Penaeidins, and the lysozyme, and the CAT and SOD activity both in vitro and in a shrimp challenge test.

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 Effect Citrox BCL on Legionella pneumophila Mechanisms of Biofilm Formation, Oxidative Stress and Virulence.

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 tatB and tatC gene expressions. Moreover, inhibition of pvcA and pvcB 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.

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.

The in vitro and ex vivo effect of Auranta 3001 in preventing Cryptosporidium hominis and Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium is a major cause of diarrhea worldwide in both humans and farm animals with no completely effective treatment available at present. In this study, we assessed the inhibitory effect of different concentrations of Auranta 3001 (0.1, 0.5 and 1%), a novel natural feed supplement, on C. hominis and C. parvum invasion of human ileocecal adenocarcinoma (HCT-8), bovine primary cells and C. parvum invasion of HCT-8, bovine primary cells and bovine intestinal biopsies. The effect of the feed supplement on the production of pro-inflammatory cytokines IL-8 and INF-γ, the anti-inflammatory cytokine IL-10, the expression of CpSUB1 protease gene during infection was also assessed by quantitative PCR (q-PCR). Transepithelial electrical resistance (TEER) was employed to measure the integrity of tight junction dynamics of the culture models. RESULTS: Pre-treatment of intestinal cells or oocysts with the Auranta 3001 significantly reduced the invasiveness of C. hominis and C. parvum against HCT-8 and bovine primary cells in a dose dependent manner. The most pronounced reduction in the invasiveness of both parasites was observed when Auranta 3001 was present during infection. Levels of IL-8 were significantly reduced in both HCT-8 and bovine primary cells, while the levels of INF-γ and IL-10 showed opposite trends in the two cell lines during infection in the presence of Auranta 3001. CpSUB1 gene protease expression, which mediates infection, was significantly reduced suggesting that this enzyme is a possible target of Auranta 3001. CONCLUSIONS: Although, C. hominis and C. parvum use different invasion mechanisms to infect cells, the novel feed additive can significantly attenuate the entry of Cryptosporidium in HCT-8 cells, primary bovine cells and bovine intestinal biopsies and thus provide an alternative method to control cryptosporidiosis.