Effect of Silver Nanoparticles and Antibiotics on Antibiotic Resistance Genes in Anaerobic Digestion

Abstract

Water resource recovery facilities have been described as creating breeding ground conditions for the selection, transfer, and dissemination of antibiotic resistance genes (ARGs) among various bacteria. The objective of this study was to determine the effect of direct addition of antibiotic and silver nanoparticles (Ag NPs, or nanosilver) on the occurrence of ARGs in thermophilic anaerobic digesters. Test thermophilic digesters were amended with environmentally-relevant concentrations of Ag NP (0.01, 0.1, and 1.0 mg-Ag/L; corresponding to ≈ 0.7, 7.0, and 70 mg-Ag/kg total solids) and sulfamethoxazole (SMX) that span susceptible to resistant classifications (1, 5, and 50 mg/L) as potential selection pressures for ARGs. Tetracycline (tet(O), tet(W)) and sulfonamide (sulI, sulII) ARGs and the integrase enzyme gene (intI1) associated with Class 1 integrons were measured in raw sludge, test thermophilic digesters, a control thermophilic digester, and a control mesophilic digester. There was no apparent effect of Ag NPs on thermophilic anaerobic digester performance. The maximum SMX addition (50 mg/L) resulted in accumulation of volatile fatty acids and low pH, alkalinity, and volatile solids reduction. There was no significant difference between ARG gene copy numbers (absolute or normalized to 16S rRNA genes) in amended thermophilic digesters and the control thermophilic digester. Antibiotic resistance gene copy numbers in digested sludge ranged from 10³ to 10⁶ copies per µL (≈ 8 × 10¹ to 8 × 10⁴ copies per lg) of sludge as result of a 1-log reduction of ARGs (2- log reduction for intI1). Quantities of the five ARGs in raw sludge ranged from 10⁴ to 10⁸ copies per lL (≈ 4 × 10² to 4 × 10⁶ per lg) of sludge. Test and control thermophilic digesters (53 °C, 12-day solids retention time [SRT]) consistently reduced but did not eliminate levels of all analyzed genes. The mesophilic digester (37 °C, 20-day SRT) also reduced levels of sulI, sulII, and intI1 genes, but levels of tet(O) and tet(W) were the same or higher than in raw sludge. Antibiotic resistance gene reductions remained constant despite the application of selection pressures, which suggests that digester operating conditions are a strong governing factor of the bacterial community composition and thus the prevalence of ARGs.