Temporal dynamic of soil microbial communities and antibiotic resistance markers exposed to increasing concentrations of sulfamethoxazole

The reuse of treated wastewater (TWW) for irrigation is widely applied to alleviate pressure on freshwater resources. However, TWW contains antibiotics that once in soils, can exert selective pressure, promoting the emergence and spread of antimicrobial resistance (AMR) in the environment. Current environmental risk assessments for antibiotic residues rely on indicators such as Predicted No Effect Concentrations (PNECs), usually determined in liquid media. These PNECs aim to predict antibiotic concentrations that may promote resistance in the environment. Given the complexity of soil matrices, few studies have established PNEC values for soil, which likely differ significantly from aquatic environments. To address this gap, we developed a simplified experimental model using soil microcosms irrigated with TWW and the antibiotic sulfamethoxazole (SMX) to estimate threshold concentrations favouring resistance transfer or/and emergence within the soil microbiome. We identified SMX concentrations between 0.01 and 0.1 mg/kgdry soil that likely increased the abundance of sulfonamide resistance genes in soil. A time window of 1 to 7 days post-exposure showed a temporary rise in sul1 and intl1 gene abundance (over 1 log/soil 16S rDNA), the appearance of SMX transformation products, and an increase in some Rhodocyclaceae. After 1.5 months of incubation and complete SMX transformation, the relative abundance of sul1 and intl1 remained about 0.5 log higher than in SMX-free controls and soils with SMX levels below 0.1 mg/kg dry soil. A persistent transformation product, 4-N-glucuronide-SMX, was also observed. Here, the estimated PNEC for SMX in soil, between 0.01 and 0.1 mg/kg, exceeds typical SMX concentrations found in soils exposed to TWW. This may suggest low impact on resistance selection for this compound in the context of TWW exposure. However further studies on other soils, water, and antibiotics need to be conducted to expand our knowledge on soil PNECs.