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Wastewater treatment plants (WWTPs) are important sources of antibiotic resistance genes (ARGs). Increasing attention has been paid to extracellular ARGs in cell-free form due to their horizontal gene transfer via transformation. However, the fate of the adsorbed form of extracellular ARGs that exist in extracellular polymeric substances (EPS) of activated sludge in WWTP remains largely unknown. Herein, seven EPS-associated ARGs along the anaerobic-anoxic-aerobic (AAO) process were quantified using quantitative polymerase chain reaction. Results show that the absolute abundances of EPS-associated ARGs were 0.69-4.52 logs higher than those of cell-free ARGs. There was no significant difference in the abundances of EPS-associated ARGs along the AAO process. Among these target genes, the abundances of EPS-associated sul genes were higher than those of EPS-associated tet and bla genes. Proteobacteria and Bacteroidetes were identified as the major secretors of EPS-associated ARGs, and they may play an important role in the proliferation of extracellular ARGs. Moreover, the transformation efficiencies of EPS-associated ARGs were 3.55-4.65 logs higher than those of cell-free ARGs, indicating that EPS-associated ARGs have higher environmental risks. These findings have advanced our understanding of EPS-associated ARGs and are useful for the control and risk assessment of ARGs in WWTPs., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antibiotic resistance has become a global public health problem. Recently, various environmental pollutants have been reported to induce the proliferation of antibiotic resistance. However, the impact of multiple pollutants (e.g., heavy metals and antibiotics), which more frequently occur in practical environments, is poorly understood. Herein, one widely distributed heavy metal (Ag + ) and one frequently detected antibiotic (tetracycline) were chosen to investigate their coexisting effect on the proliferation of antibiotic resistance in the activated sludge system. Results show that the co-occurrence of Ag + and tetracycline at environmentally relevant concentrations exhibited no distinct inhibition in reactor performances. However, they inhibited the respiratory activity by 42%, destroyed the membrane structure by 218%, and increased membrane permeability by 29% compared with the blank control bioreactor. Moreover, the relative abundances of target antibiotic resistance genes (ARGs) (e.g., tetA, bla TEM-1 , and sulII) in effluent after exposure of coexisting Ag + and tetracycline were increased by 92-1983% compared with those in control reactor, which were 1.1-4.3 folds higher than the sum of the sole ones. These were possibly attributed to the enrichments of antibiotic-resistant bacteria. The results would illumine the coexisting effect of heavy metals and antibiotics on the dissemination of ARGs in activated sludge system., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antibiotic resistance has been regarded as a global concern and biological wastewater treatment plants (WWTPs) are ideal hotbeds for the emergence and propagation of antibiotic resistance genes (ARGs). Extracellular polymeric substances (EPS), one of the primary components of activated sludge, might affect the distribution of extracellular ARGs in supernatant and EPS matrix, and thus alter their uptake potential by microbial cells. Herein, the presence and significance of EPS-associated ARGs in activated sludge from four WWTPs were assessed. Seven typical ARGs (sulI, sulII, bla TEM-1 , tetA, tetO, tetQ, tetW) and class I integron (intI1) in EPS-associated, cell-free, and intracellular DNA were quantified. Results show that the absolute abundances of EPS-associated, cell-free, and intracellular ARGs were 5.90 × 10 6 -6.45 × 10 9 , 5.53 × 10 4 -4.58 × 10 6 , and 2.68 × 10 8 -1.79 × 10 11 copies/g-volatile suspended solids, respectively. The absolute abundances of EPS-associated ARGs were 0.2-4.6 orders of magnitude higher than those of the corresponding cell-free ARGs. Considering the higher DNA contents in EPS, the transformation abilities of EPS-associated ARGs were 3.3-236.3 folds higher than those of cell-free ARGs. Therefore, EPS-associated ARGs are an important source of extracellular ARGs, and it may play a crucial role in horizontal gene transfer via transformation in WWTPs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aerobic granular sludge with larger size and more compact spherical structure generally shows excellent performance in antibiotic removal, yet little is known about the long-term effect of environmentally-relevant concentration (μg/L) of antibiotics on the proliferation of antibiotic resistance genes (ARGs) and microbial community in aerobic granules. Herein, a sequencing batch reactor (SBR) was set up with dosing different concentrations (0-500 μg/L) of tetracycline to investigate its influences on microbial communities and ARG levels in aerobic granular sludge. Results show that the bioreactor could effectively remove chemical oxygen demand (COD), nitrogen, and tetracycline during the long-term operation. The quantitative polymerase chain reaction (qPCR) analysis shows that tetracycline at μg/L level could greatly enhance the absolute and relative abundances of tetA, sulII, and bla TEM-1 in the effluent and aerobic granules, indicating tetracycline could serve as a selection pressure on the development of ARGs corresponding to different types of antibiotics in aerobic granules. Pearson's correlation analysis also implies that sulII and bla TEM-1 were correlated strongly with tetA. Moreover, the presence of tetracycline altered the microbial communities and diversity of the effluent and aerobic granules in the bioreactor. These findings would advance our understanding of the proliferation and development of ARGs in aerobic granules under tetracycline pressure and serve as a foundation to guide the application of aerobic granular sludge for treatment of antibiotic-containing wastewater., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)