Comparative analysis of intracellular and extracellular antibiotic resistance gene abundance in anaerobic membrane bioreactor effluent

The growing practice of wastewater reuse poses a significant risk to further dissemination of antibiotic resistance due to the abundance of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in wastewater effluents. Anaerobic membrane bioreactors (AnMBRs) are an emerging wastewater treatment technology capable of reducing the total ARGs and ARB load discharged to receiving environments compared to conventional aerobic treatment processes. While size exclusion is effective at retaining ARB and its associated intracellular ARGs, the abundance and fate of extracellular ARGs in an AnMBR effluent have not been examined. This study elucidates the effect of combined antibiotics loading (ampicillin, erythromycin, and sulfamethoxazole) on the abundance of intracellular and extracellular ARGs in an AnMBR effluent over a period of five weeks. Quantification of targeted genes revealed an overall enrichment of intracellular ARGs (iARGs) and depletion of extracellular (exARGs) in response to antibiotics addition, which suggests exARG uptake as a significant mode of horizontal gene transfer in AnMBR effluents. Comparison of the iARG and exARG abundance profiles showed a potential bias for exARG uptake located on small plasmids compared to large plasmids.\n\nImportanceAntibiotic resistance dissemination is facilitated through horizontal gene transfer (HGT) of ARGs. Currently, conjugation is considered to be the dominant mechanism during wastewater treatment. However, recent studies have detected high abundances of exARGs, implying that transformation may play a greater role in dissemination. While previous studies quantified iARGs and exARGs in wastewater treatment facilities, they did not evaluate temporal changes between the two forms. Further, almost no research has differentiated between iARGs and exARGs in anaerobic processes, which are being considered to replace aerobic activated sludge processes. This study specifically investigates the abundance of targeted iARGs and exARGs in AnMBRs in response to antibiotic pressure to quantify potential exchange of ARGs between intracellular and extracellular compartments. Our findings suggest that exARGs located on small plasmids are preferentially taken up by cells under antibiotic pressure compared to large plasmids, which implies heterogenous HGT mechanisms among the plasmid community.