Inhibition of Biofilm Production and Determination of In Vitro Time-Kill Thymus vulgaris L. Essential Oil (TEO) for the Control of Mastitis in Small Ruminants

<i>Staphylococcus aureus</i> and coagulase-negative staphylococci (CNS) are the main causative agents of mastitis in sheep. Their ability to form biofilms in vivo is considered an important virulence factor underlying mastitis outbreaks refractory to antibiotic treatments. Furthermore, pre- and postdipping immersion during milking in iodine substances could determine the presence of residues in milk and therefore represent a health risk factor for consumers. The aim of this study was to evaluate the antibacterial and biofilm inhibitory activity of <i>Thymus vulgaris</i> L. essential oil (TEO) against staphylococci strains isolated from ovine clinical mastitis. In particular, 3 reference strains (<i>S. aureus</i> 25923 and 11623 and <i>S. epidermidis</i> 12228) and 12 clinical isolates (6 <i>S. aureus</i> and 6 CNS) were used. TEO solutions, from a concentration of 1% (<i>v</i>/<i>v</i>) to 1.25% (<i>v</i>/<i>v</i>), corresponding to 9.28-2.32 mg/mL, were obtained after solubilization in 10% dimethyl sulfoxide (DMSO) and used to evaluate the bacterial time-kill compared to that of an iodine-based solution. Antibacterial efficacy was then assessed by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), while biofilm inhibition was assessed by minimum biofilm inhibitory concentration (MBIC) using a spectrophotometer at a wavelength of 570 nm. Additionally, biofilm-associated genes (icaA and icaD) were evaluated in all tested strains by PCR. The tested TEO concentrations were able to significantly and prominently reduce bacterial growth compared to controls, as demonstrated by bacterial time-kills. The MIC value was obtained at a concentration of 0.50% (<i>v</i>/<i>v</i>) for a single coagulation-positive isolate (<i>S. aureus</i> (f)) and at a concentration of 0.25% (<i>v</i>/<i>v</i>) for all other isolates. TEO showed effective bactericidal action with a 99.9% reduction in CFU/mL of all isolates in the MBC test at a concentration of 0.25% (<i>v</i>/<i>v</i>) for most of the tested strains. Furthermore, a marked inhibition in biofilm formation at all tested concentrations was observed, with MBIC value of 0.25%. All <i>S. aureus</i> tested were biofilm-producing strains and positive for icaA and icaD genes, while two CNS biofilm-producing strains were negative for both genes. These preliminary results suggest that TEO could be a promising alternative as an udder disinfectant during milking practices. Although in vivo studies are needed to confirm the efficacy and safety of TEO as an adjuvant in the prevention and treatment of udder infections, TEO could help counteract the emergence of antimicrobial resistance and reduce the potential risk of iodine residues in milk.