Unravelling a vicious circle: animal feed marketed in Costa Rica contains irregular concentrations of tetracyclines and abundant oxytetracycline-resistant Gram-positive bacteria

Diverse tetracyclines are used to prevent and control bacterial infections in livestock and farmed fish. These drugs are administered through the diet, but farmers seldom check whether feed contains antibiotic-resistant bacteria that may colonise their crops or transfer their resistance traits to species of veterinary relevance. To examine whether antibiotic dosage defines the abundance of antibiotic-resistant bacteria in animal feed, we determined the concentration of parental compounds and epimers of oxytetracycline (OTC), doxycycline, tetracycline and chlortetracycline, as well as the abundance and resistance level of OTC-resistant bacteria in samples of fish (n = 21), poultry (n = 21), swine (n = 21), and shrimp feed (n = 21) marketed in Costa Rica. Fish feed contained the highest amounts of tetracyclines (119–8365 mg kg⁻¹) and the largest proportion of bacteria resistant to 10 μg ml⁻¹ (1.8–92.4%) or 100 μg ml⁻¹ of OTC (12.5–63.8%). Poultry (78–438 mg kg⁻¹) and swine (41–1076 mg kg⁻¹) feed had intermediate concentrations of tetracyclines and OTC-resistant bacteria (0.2–66% and 0.3–49%, respectively), whereas shrimp feed showed the lowest amounts of tetracyclines (21.5–50.3 mg kg⁻¹), no OTC and no culturable OTC-resistant bacteria. In line with these results, the MIC₅₀ of OTC for 150 isolates from fish and poultry feed was > 256 µg ml⁻¹, while that of 150 bacteria isolated from swine feed was 192 µg ml⁻¹. Phenotypic tests, fatty acid profiles and proteotypic analyses by matrix-assisted laser desorption/ionisation-time of flight mass-spectroscopy revealed that most OTC-resistant isolates were Gram-positive bacteria of low G+C% content from the genera Staphylococcus and Bacillus. Clear correlations between OTC dosage and feed colonisation with OTC-resistant bacteria were seen in medicated feed for fish (r = 0.179–0.651). Nonetheless, some unmedicated feed for fish, swine and poultry contained large populations of OTC-resistant bacteria, suggesting that raw materials and manufacturing processes may also influence carriage of OTC-resistant bacteria in animal feed.