Temporal dynamics of the soil resistome and microbiome irrigated with treated wastewater containing clarithromycin

Clarithromycin, a common antibiotic found in domestic wastewater, persists even after treatment and can transfer to soils when treated wastewater (TWW) is used for irrigation. This residual antibiotic may exert selection pressure, promoting the spread of antibiotic resistance. While Predicted No Effect Concentrations (PNECs) are used in liquid media to predict resistance risks, PNEC values for soils, especially for clarithromycin, are lacking. Thus, this study aimed to assess clarithromycin's fate and its concentration threshold affecting soil microbial communities and macrolide resistance genes. The study used a soil microcosm approach with TWW containing clarithromycin at concentrations of 0, 0.01, 0.1, 0.5, and 1 mg/kg_{dry soil} over a three-month period. Results showed clarithromycin persisted with limited degradation, likely due to strong adsorption to soil particles. Two transformation products were identified: decladinose-CLA (abiotic degradation) and phosphate-CLA (bacterial phosphotransferase activity). Soil bacterial communities were more influenced by TWW than by clarithromycin itself, as its antimicrobial effect was reduced due to adsorption. While clarithromycin did not significantly affect the abundance of resistance genes like intl1, mphA, and ereA, concentrations above 0.01 mg/kg increased the ermB gene abundance during the first week. The mefA gene (macrolide efflux pump) showed a hormetic effect: low doses (<0.1 mg/kg) increased gene abundance, while higher doses (>0.5 mg/kg) inhibited gene transfer or the bacteria carrying it. This study performed under controlled conditions provided insights into antibiotic resistance dynamics in soils exposed to clarithromycin, highlighting key concentration thresholds influencing resistance spread in soils.