Exploring Thymus vulgaris Extract as a Phytotherapeutic Agent: A Multifaceted Approach to tackle Avian Colibacillosis...

Avian colibacillosis remains a major threat to poultry production and food security, whereas its antibiotic-based control accelerates antimicrobial resistance. This study investigated the antibacterial potential of the hydroethanolic extract of Thymus vulgaris, alone and in combination with antibiotics, against avian pathogenic Escherichia coli (APEC). The phytochemical composition of the extract was analyzed by HPLC, while its antibacterial activity was assessed using agar diffusion and minimum inhibitory concentration (MIC) assays. Antibiotic-extract interactions were evaluated by the checkerboard method, and mechanisms of action were explored through assays targeting membrane permeability, proton pump inhibition, catalase inhibition, oxidative stress, and biofilm formation. Its antioxidant, anti-inflammatory, and cytotoxic activities were also evaluated. HPLC analysis revealed 16 phenolic compounds, including rosmarinic, ferulic, and salicylic acids, as well as flavonoids such as quercetin and luteolin, with an extraction yield of 15% ± 0.55. The extract displayed significant antibacterial activity (MIC: 5.46-10.93 mg/ml, p < 0.05). In synergy tests, the extract enhanced ampicillin efficacy 4-8-fold and showed additive effects with ciprofloxacin and tetracycline (2-4-fold). Mechanistic assays demonstrated disruption of bacterial membranes (32.66% electrolyte leakage, p < 0.05), inhibition of H⁺-ATPase proton pumps (p < 0.05), catalase activity reduction by 79.2% (p < 0.05), >50% decrease in cell hydrophobicity, and inhibition of biofilm formation (49.07-72.47%), alongside eradication of mature biofilms (41.89-64.67%) (p < 0.05). Beyond antimicrobial effects, the extract exhibited notable antioxidant capacity. In DPPH radical scavenging, the extract showed an IC₅₀ of 9.76 ± 0.228 μg/ml (p < 0.05), while in the TAC assay, it reached 467.25 ± 1.889 μg/ml (p < 0.05), and it reduced ampicillin-induced lipid peroxidation by 42.85% (p < 0.05). Anti-inflammatory testing revealed inhibition of protein denaturation (4.95-52.48% at 15-2000 µg/ml, p < 0.05), although weaker than aspirin. Hemolysis assays confirmed the extract was non-hemolytic at concentrations up to 11 mg/ml (p < 0.05). In conclusion, Thymus vulgaris extract demonstrated safe, multi-target bioactivity, supporting its potential as a promising alternative to combat avian colibacillosis and antimicrobial resistance.