This study evaluated whether co-administering piperine, a known efflux pump inhibitor, with amoxicillin improves antibacterial efficacy against a metal-tolerant, antibiotic-resistant Escherichia coli O125 strain in broiler chickens. The investigation combined in vitro susceptibility testing, toxicity and antioxidant assessment of piperine, an in vivo infection model with treated groups, microbiological and molecular endpoints, validated drug assays, compartmental pharmacokinetic analysis, and residue depletion assessment to address both efficacy and regulatory safety.
Minimum inhibitory concentrations (MICs) determined in vitro were 32 µg/mL for amoxicillin and 5 µg/mL for piperine. The combination exhibited synergism with a fractional inhibitory concentration (FIC) index of 0.2518, indicating that the two agents interacted to lower the concentration required to inhibit bacterial growth in vitro.
Acute and subacute toxicity evaluations were performed in accordance with ARRIVE 2.0 reporting and OECD Test Guideline 223. Dietary piperine given at 100 mg/kg was reported as well tolerated in the experimental animals. Additionally, piperine improved antioxidant status markers, suggesting a potential ancillary protective effect against oxidative stress in treated birds.
The in vivo study used 225 day-old broiler chicks allocated into five groups: negative control (uninfected), positive control (infected untreated), and three infected groups treated orally for five days with either amoxicillin alone, piperine alone, or the amoxicillin–piperine combination. The infecting organism was a resistant E. coli O125 isolate. Serum and tissues were sampled at multiple time points for microbiological, molecular, chemical, and pharmacokinetic analyses.
Co-administration of piperine with amoxicillin reduced mean E. coli counts in tissues to 2.2 log10 CFU/g. Analysis of efflux-associated gene expression showed downregulation on the order of 0.4–0.5 fold change in the combination-treated group compared with relevant controls. These findings support that piperine acted as a resistance-modulating agent, consistent with efflux pump inhibition contributing to the improved antibacterial effect.
Drug concentration measurements in serum and tissues were performed using a validated high-performance liquid chromatography (HPLC) method. Compartmental analysis found that amoxicillin followed a two-compartment pharmacokinetic model, whereas piperine fit a one-compartment model. When co-administered, piperine significantly increased amoxicillin maximum plasma concentration (Cmax) and area under the concentration–time curve (AUC), and it decreased apparent clearance. These changes indicate enhanced systemic exposure to amoxicillin and prolonged tissue persistence when given with piperine.
Residue depletion analyses showed extended persistence of amoxicillin in tissues when administered with piperine. From a regulatory perspective, the combination required an extended withdrawal period of 5 days post-cessation to comply with established amoxicillin maximum residue limits (MRLs), compared with a 3-day withdrawal period when amoxicillin was used alone. This finding highlights a trade-off between improved therapeutic exposure and the need to adjust withdrawal intervals to ensure food safety.
Tissue assays detected low concentrations of zinc, copper, and cadmium across sampled tissues. The study reported the presence of these metals but indicated concentrations were low; detailed values were presented in the full dataset and are not restated here beyond that characterization.
Toxicity and animal experiments complied with ARRIVE 2.0 recommendations and OECD test guidance. The experimental protocol was approved by the Institutional Animal Care and Use Committee (ARC AHRI, registration number ARC AHRI, 6725). Serum and tissue drug assays were performed using validated HPLC methodology. The authors declared no competing interests.
In this broiler model of resistant, metal-tolerant E. coli infection, dietary piperine at 100 mg/kg enhanced the antibacterial efficacy of amoxicillin through both efflux pump inhibition (evidenced by reduced bacterial counts and downregulated efflux gene expression) and pharmacokinetic interactions (increased amoxicillin Cmax and AUC, decreased clearance, and longer tissue persistence). However, co-administration prolonged amoxicillin residues in tissues, necessitating a longer withdrawal period (5 days) to meet amoxicillin MRLs compared with amoxicillin alone (3 days). Low tissue levels of zinc, copper, and cadmium were also detected.
These results indicate piperine can potentiate amoxicillin activity against resistant E. coli in broilers but require consideration of toxicity testing, residue depletion, and adjusted withdrawal intervals before application in production settings.