Characterization of Antibiotic Resistance in Enterobacteriaceae From Agricultural Manure and Soil in Portugal

Intensive livestock production uses antibiotics to treat and prevent infectious diseases. Antibiotics are expelled unaltered in animal manure, which is often used as a fertilizer. This practice spreads antibiotic residues and antibiotic-resistant bacteria and their genes into the soil. These resistance determinants affect soil microbial communities, raising concerns about the potential environmental and public health risks. This study aims to characterize soil contaminated with antibiotic resistant bacteria and the putative role played by manure as a vector for these resistance determinants. Six poultry and dairy cattle farms in the central region of Portugal were sampled. Ampicillin-resistant Enterobacteriaceae isolates from manure and manured-soil samples were tested for their resistance profile to 13 antibiotics and the presence of tetracycline and sulfonamide resistance genes. High multidrug resistance rates (>70%) were observed in both manure and soil samples. This resistance was higher in the poultry samples; manured-soil isolates were more resistant to cefoxitin (91.7%), sulfamethoxazole/ trimethoprim (79.2%), chloramphenicol (79.2%), and to a lesser extent tetracycline (12.5%). Manure isolates have high resistance frequency to tetracycline (73.9%), amoxicillin with clavulanic acid (66.0%), chloramphenicol (65.3%), and sulfamethoxazole with trimethoprim (36.5%). Manure isolates revealed high frequency of tet(A), sul1, and sul3 genes. The multiple antibiotic resistance index reveals a putative risk of soil contamination due to antibiotic exposure. The results obtained are important for soil management regarding resistance determinants spread through agricultural practices. To our knowledge, this is the first study of tetracycline and sulfonamide resistance in Portuguese manure and soil.