Tetracyclines, sulphonamides, and quinolones are families of antimicrobials (AMs) widely used in the poultry industry and can excrete up to 90% of AMs administrated, which accumulate in poultry litter. Worryingly, poultry litter is widely used as an agriculture fertilizer, contributing to the spread AMs residues in the environment. The aim of this research was to develop a method that could simultaneously identify and quantify three AMs families in poultry litter by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Samples of AMs free poultry litter were used to validate the method according to 657/2002/EC and VICH GL49. Results indicate that limit of detection (LOD) ranged from 8.95 to 20.86 μg kg⁻¹, while limits of quantitation (LOQ) values were between 26.85 and 62.58 µg kg⁻¹ of tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, enrofloxacin, ciprofloxacin, flumequine, sulfachloropyridazine, and sulfadiazine. Recoveries obtained ranged from 93 to 108%. The analysis of field samples obtained from seven commercial poultry flocks confirmed the adequacy of the method since it detected means concentrations ranging from 20 to 10,364 μg kg⁻¹. This provides us an accurate and reliable tool to monitor AMs residues in poultry litter and control its use as agricultural fertilizer.

Antimicrobials are currently used in poultry for disease treatment. However, their excretion in bird feces may contaminate the environment. Considering this, the objective of this work was to quantify antimicrobials residues concentrations in therapeutically treated broiler chicken droppings throughout the post-treatment period. For this aim a multiresidue method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was validated. Forty-eight male broiler chickens were distributed and treated with commercial formulations of 5 different antimicrobials. Results showed that oxytetracycline and 4-epi-oxytetracycline, presented the highest concentrations during all sampling period, detecting concentrations of 1471.41 µg kg⁻¹ at the last sampling point (day 22 post-treatment). Florfenicol, tylosin, enrofloxacin, and ciprofloxacin were eliminated and detected in treated chicken droppings until d 18 post-treatment. Sulfachloropyridazine decrease gradually during post-treatment period until day 30. Results demonstrate that studied antimicrobials in treated chicken droppings were eliminated for prolonged periods, therefore becoming a significant route of residues dissemination into the environment.

Oxytetracycline (OTC) is widely used in broiler chickens. During and after treatment a fraction of OTC is excreted in its original form and as its epimer, 4-epi-OTC in droppings. To address the transfer of OTC into the environment, we evaluated the dissemination of OTC and 4-epi-OTC from treated birds to the environment and sentinels, through the simultaneous analysis of broiler droppings and litter. Male broiler chickens were bred in controlled conditions. One group was treated by orogastric tube with 80 mg kg⁻¹ of OTC and two groups received no treatment (sentinels). OTC+4-epi-OTC were analyzed and detected by a HPLC-MS/MS post the end of treatment. The highest concentrations of OTC+4-epi-OTC were detected in the droppings of treated birds 14-days following the end of treatment (2244.66 µg kg⁻¹), and one day following the end of treatment in the litter (22,741.68 µg kg⁻¹). Traces of OTC+4-epi-OTC were detected in the sentinels’ droppings and litter (<12.2 µg kg⁻¹). OTC+4-epi-OTC can be transferred from treated birds to the environment and to other untreated birds. The presence and persistence of OTC+4-epi-OTC in litter could contribute to the selection of resistant bacteria in the environment, increasing the potential hazard to public and animal health.

Lincomycin is the first antimicrobial agent described for the lincosamide class and it is commonly used for the treatment of infectious enteric and respiratory diseases in poultry. Maximum residue limits (MRLs) in edible tissues have been established for this antimicrobial, however, no regulation has been proposed yet for by-products that are not intended for direct human consumption. Feathers are a by-product from poultry farming that might be used as an ingredient for diets fed to other farm animal species. The presence of antimicrobial residues in them is not monitored in spite of the fact that several studies have proved that they can persist in feathers. Currently though, no evidence has been presented regarding the behaviour of lincomycin in this matrix. Hence, this work intended to assess the depletion of lincomycin residues in feathers of birds treated with therapeutic doses and compare them with those detected in muscle and liver samples. Samples were collected for several days after ceasing treatment from a group of broiler chickens treated with a 25% lincomycin formulation. Methanol and Florisil® columns were used to extract and retain the analyte, and samples were analysed using a triple quadrupole mass spectrometer (API 5500, AB SCIEX™). On day 1 after ceasing treatment, average concentrations of lincomycin detected in feather samples reached up to 8582 μg kg⁻¹ and by day 16, these had only declined by 63%, to an average of 3138 μg kg⁻¹. Lincomycin residues were detected in feathers at every sampling point, even after they were not detectable in edible tissues. Depletion time was 98 days for feathers, considering the LOQ established for the methodology as cut-off value for the calculations. Data showed that lincomycin is highly persistent in feathers, which may result in this matrix becoming a re-entry route for its residues into the food chain.

Oxytetracycline (OTC) is administered in the poultry industry for the treatment of digestive and respiratory diseases. The use of OTC may contribute to the selection of resistant bacteria in the gastrointestinal tract of birds or in the environment. To determine the effect of OTC on the selection of resistant Escherichia coli strains post-treatment, bacteria were isolated from droppings and litter sampled from untreated and treated birds. Bacterial susceptibility to tetracyclines was determined by the Kirby–Bauer test. A total of 187 resistant isolates were analyzed for the presence of tet(A), (B), (C), (D), (E), and (M) genes by PCR. Fifty-four strains were analyzed by PFGE for subtyping. The proportion of tetracycline-resistant E. coli strains isolated was 42.88%. The susceptibility of the strains was treatment-dependent. A high clonal diversity was observed, with the tet(A) gene being the most prevalent, followed by tet(C). Even at therapeutic doses, there is selection pressure on resistant E. coli strains. The most prevalent resistance genes were tet(A) and tet(C), which could suggest that one of the main mechanisms of resistance of E. coli to tetracyclines is through active efflux pumps.

Objective: We set out to evaluate the persistence of sulfachloropyridazine, oxytetracycline, and enrofloxacin in broiler chicken litter following therapeutic oral treatment and its association with the isolation of Escherichia coli resistant to these antimicrobials. Methods: Forty broiler chickens were raised under controlled conditions and divided into three experimental groups, each treated with a different antimicrobial, in addition to an untreated/control group. Litter samples were collected post treatment, analyzed by UPLC-MS/MS, and processed for the isolation of E. coli. The antimicrobial susceptibility of E. coli was assessed using the Kirby–Bauer disk diffusion method. Results: Chemical analysis detected concentrations of antimicrobials throughout post treatment, reaching maxima of 42,910.14 μg kg⁻¹, 92,712 μg kg⁻¹, and 9567 μg kg⁻¹ for sulfachloropyridazine, oxytetracycline plus 4-epi-oxytetracycline, and enrofloxacin plus ciprofloxacin, respectively. It was estimated that the concentrations of sulfachloropyridazine, oxytetracycline, and enrofloxacin would persist in broiler litter for 61, 244, and 514 days, respectively. A very strong association was observed between the presence of antimicrobial residues and the antimicrobial resistance of E. coli (p-value < 0.0001, and Cramer’s coefficient of 0.47), and an independence between the level of residue concentration and susceptibility (p-value 0.5262). Conclusions: The persistence of antimicrobial residues contributes to the selection of resistant bacteria, regardless of persistent antimicrobial concentrations. These findings highlight the need for stricter regulations on poultry litter management, including residue thresholds and resistance monitoring, to minimize environmental and public health risks. Proper treatment of poultry litter is essential to ensure its sustainable and safe re-use in agricultural systems.