Effects on extracellular and intracellular antibiotic resistance genes and their potential hosts in activated sludge under stress of high concentrations of antibiotics

Abstract

Antibiotic resistance genes (ARGs) including intracellular ARGs (iARGs) and extracellular ARGs (eARGs) are harboured in wastewater treatment plants. This study investigated the effects of short-term and long-term cyclic stress on i/eARGs and microbial communities under high concentrations of sulfamethoxazole (SMX) and trimethoprim (TMP). Secretion of extracellular polymeric substances (EPS) was inhibited under both antibiotic stress modes. Under short-term stress of SMX, the absolute abundance of i-sul1 and i-sul2, which belong to the sulfonamide resistance genes, increased, while i-aac, i-mefA and i-intl-1 were also enriched. In contrast, short-term TMP stress had a limited effect on iARGs, though the absolute abundance of dfrA12 and dfrG increased. Under long-term cyclic stress of SMX, the greatest increase in absolute abundance of i/eARG was observed during the first cyclic stress stage. Under both stresses, there are complex interactions between iARGs and eARGs, with mobile genetic elements (MGEs) promoting certain subtypes associated with them. Both stresses notably altered the intracellular microbial community composition, with abundant, intermediate, and rare taxa responding differently. Abundant taxa (e.g. Brevundimonas) were main potential hosts for iARGs, and intermediate taxa (e.g. Methylocystis) and rare taxa (e.g. Paracoccus) were main releasers for eARGs. These findings provide valuable insights into the dynamics of iARGs and eARGs under high antibiotic stress, advancing our understanding of ARG behavior in wastewater treatment systems.