First in vitro characterization of three oxytetracycline degradation products against avian pathogenic Escherichia coli isolates: antibacterial activity, formation in gizzard, and resistance gene profiling

Although oxytetracycline (OTC) has been widely used in poultry since 1950, comprehensive studies characterizing its degradation products for potential therapeutic applications have not been conducted. The aims of the present study were to evaluate the antibacterial activity of three OTC degradation products (4-α-epi-OTC [4-EOTC], α-apo-OTC, and β-apo-OTC) individually and in combination with OTC, to identify and assess the genetic determinants conferring antimicrobial resistance (AMR) in avian pathogenic Escherichia coli (APEC) strains exposed to tetracyclines and OTC degradation products, and to assess OTC degradation in a simulated poultry gizzard. The microdilution method was used to determine the minimal inhibitory concentration (MIC) of OTC, chlortetracycline, tetracycline, 4-EOTC, α-apo-OTC, and β-apo-OTC against 15 APEC strains. The fractional inhibitory concentration index (FICI) was calculated to assess the combined effect of OTC with its degradation products. A conjugation assay was conducted to determine whether resistance to OTC and its active degradation products is mediated by the identified tetracycline resistance genes. Results showed that while α-apo-OTC and β-apo-OTC lacked detectable antibacterial activity, 4-EOTC retained inhibitory activity against all tested APEC strains. Notably, a synergistic effect was observed between OTC and 4-EOTC (FICI ≤ 0.5). Preliminary evidence indicated that E. coli employed the same resistance determinant ( tetA ) against both OTC and its degradation products, while confirming that OTC remained stable under simulated gizzard conditions. The identification of antibacterial activity in 4-EOTC represents a key finding of this study and warrants further investigation to define its activity spectrum and explore its potential therapeutic applications. IMPORTANCE Tetracyclines are among the most extensively used antimicrobials in veterinary medicine, with oxytetracycline (OTC) serving as a cornerstone compound of this family. While several studies have reported that OTC undergoes degradation in the environment, the biological roles of the resulting chemical derivatives in poultry remain poorly understood. The present study demonstrates that one major degradation product, 4-EOTC, retains antibacterial activity against avian pathogenic Escherichia coli (APEC) strains and, when combined with OTC, produces a synergistic effect. Importantly, the tetA gene confers resistance in APEC against 4-EOTC as it does against OTC, highlighting the possible contribution of OTC degradation products to the spread of antimicrobial resistance (AMR). Furthermore, OTC was found to remain stable under simulated poultry gizzard conditions, indicating that its degradation likely occurs in downstream compartments of the poultry gastrointestinal tract. Collectively, these findings reveal that OTC degradation products are not biologically inert and open new avenues for the development of next-generation tetracyclines.