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

Abstract

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

“…Ajose et al 2023, have recently reviewed ethnoveterinary medicinal plants and ethnoveterinary medicinal products utilised in dairy and livestock farming to combat mastitis, with the most prominent plants as Becium obovatum , Malva parviflora , Brucea antidysenterica , Acorus calamus L., Triticum sp, Arachis hypogeam , Peganum harmala , Citrus limon , Withania somnifera and others [ 8 ]. Among plant extracts, Sorghum phenol extract [ 5 ], sweet orange peel ( Citrus sinensis ), pomegranate ( Punica granatum ) [ 22 , 23 ], indian screw tree ( Helicteres isora ) [ 24 ], lemon balm ( Melissa officinalis ), a specie of laurel wood ( Knema retusa ) [ 25 ] and olive [ 13 ] extracts demonstrated strong antimicrobial activity against different S. aureus strains including other BM-associated pathogens. After in vitro disc diffusion assays from recent studies, the authors have reported the inhibitory zones against S. aureus significant average zones ranging from 22 to 31 mm [ 24 , 26 ].…”

“…The Knema retusa extract (KRe) showed significant antibacterial effects at observed MIC between 32 and 256 ug/mL and MBC between 64 and 512 ug/mL against S. aureus and S. haemolyticus , inhibiting biofilm formation and bacterial surface adherence [ 25 ]. In an earlier investigation, a natural antimicrobial mixture of organic acids, citrus and olive extracts was assessed for its anti-staphylococcal activity [ 13 ]. This blend manifested a multifaceted impact during the infection process.…”

“…Cells were infected for 6 h of incubation at 37 °C in 5% CO 2 in the presence of 0, 0.1, 0.2, 0.5% and 1% AuraShield (As) for 2–3 h. Post infection the supernatant, containing un-attached bacteria was removed and wells were washed with 1 ml pre-warmed PBS. The cytotoxicity of As was determined as previously described [ 13 ] using the MTT assay (Sigma-Aldrich, Gillingham, England, UK). To quantify the number of cell-associated bacteria, infected monolayers were washed three times with PBS and treated with 0.1% Triton X-100 in PBS at 41.5 °C and 37 °C for 15 min.…”

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

“…The TGP cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Cell Biologics, USA) as described above and maintained in 75cm 2 flasks (Sigma-Aldrich, Arklow, Ireland, SIAL0641) at 37 °C and 5% CO 2 . To test the effect of Aq on the ability of S. agalactiae to infect TGP cells, the assay was performed as previously described [ 17 ]. Briefly, the TGP cells (5.5 × 10 5 /well) were cultured for 24 h until 80–90% confluent in 6-well culture plates at pH 7.2.…”

Emerging Approaches for Mitigating Biofilm-Formation-Associated Infections in Farm, Wild, and Companion Animals

“…Most biofilm-forming bacteria originate in the bacterial plaque formed on the tooth surface. The identification of biological agents and efficient control of biofilm formation and consequently dental plaque are of constant concern in the veterinary practice [ 30 ].…”