ABSTRACT

Background and Aim: Bovine metritis is an important postpartum disease that compromises reproductive performance, milk production, and profitability in dairy herds. Increasing antimicrobial resistance among uterine pathogens highlights the need for alternative antimicrobial strategies. Carvacrol and thymol, major phenolic monoterpenes in oregano essential oil, have broad antibacterial activity; however, evidence of their activity against diverse clinical isolates associated with bovine metritis remains limited. This study evaluated the in vitro antibacterial activity of purified carvacrol and thymol against clinical bacterial isolates associated with bovine metritis.

Materials and Methods: Forty-three clinical bacterial isolates recovered from bovine metritis cases were evaluated, comprising four Staphylococcus aureus, six coagulase-negative staphylococci, ten Streptococcus spp., ten Trueperella pyogenes, ten Escherichia coli, and three Klebsiella pneumoniae isolates. Five American Type Culture Collection reference strains were included exclusively for quality control and excluded from statistical analyses. Antibacterial activity was evaluated using disk diffusion and broth microdilution assays to determine inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs). Ceftiofur and ethanol served as positive and negative controls, respectively. The clinical isolate was considered the experimental unit, and assays were performed in technical triplicate.

Results: Carvacrol and thymol inhibited all bacterial groups evaluated. Gram-positive isolates exhibited significantly larger inhibition zones and lower MIC and MBC values than Gram-negative isolates (p < 0.05). Mean MIC values ranged from 0.0062 ± 0.0004 to 0.0065 ± 0.0004 mg/mL for Gram-positive isolates and from 0.0101 ± 0.0019 to 0.0143 ± 0.0042 mg/mL for Gram-negative isolates. Corresponding mean MBC values ranged from 0.0143 ± 0.0010 to 0.0166 ± 0.0012 mg/mL and from 0.0209 ± 0.0040 to 0.0292 ± 0.0084 mg/mL, respectively. Coagulase-negative staphylococci, Streptococcus spp., and T. pyogenes showed high susceptibility, whereas K. pneumoniae was comparatively less susceptible.

Conclusion: Purified carvacrol and thymol demonstrated antibacterial activity against clinically relevant bacteria associated with bovine metritis, particularly Gram-positive isolates. These findings support further investigation of these naturally derived antimicrobial candidates. Cytotoxicity, antibiofilm activity, pharmaceutical formulation, pharmacokinetics, and controlled in vivo efficacy should be evaluated before clinical application in dairy cattle.

Keywords: antimicrobial alternatives, bovine metritis, carvacrol, clinical bacterial isolates, minimum bactericidal concentration, minimum inhibitory concentration, phenolic monoterpenes, thymol.