Impact of Long-Term Manure and Sewage Sludge Application to Soil as Organic Fertilizer on the Incidence of Pathogenic Microorganisms and Antibiotic Resistance Genes

The reuse of stabilized (under thermophilic conditions) sewage sludge and manure on agricultural soils is a common practice. The aim of this study was to evaluate the risks associated with their repeated applications on the spread of pathogenic bacteria and antibiotic resistance genes (ARGs) that encode resistance to tetracycline (tetA and tetW), sulphonamide (sul1 and sul2), erythromycin (ermB), vancomycin (vanA) and integron genetic element (intI1). The trial fields has been regularly fertilized every 3rd year since 1996 with manure (MF; 330 kg N/ha) and sewage sludge (SF; 330 kg N/ha and SF3; 990 kg N/ha). Unfertilized soil (CF) served as a control. Samples were collected at different time points: (i) right before fertilization (which was also 3 years after the last fertilization), (ii) 5 months after fertilization, and (iii) 11 months after fertilization. The relative abundance of amplicon sequence variants (ASVs) assigned to potentially pathogenic bacteria was low (0.3% and 0.25% in sludge and manure, respectively), and no association with the application of these fertilizers was found. On the other hand, our data indicate that an increased relative abundance of the ARGs sul1 and tetW was significantly associated with these fertilizer applications, and sul1 was increased in all treatments regardless of the time. It is suggested that sul1 should be monitored in organically fertilized soils to prevent its spread and possible further accumulation in crops.