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Wastewater is a common pathway for the spread of antibiotic resistance (AR) genes and bacteria into the environment. Biological treatment can mitigate this path, but horizontal gene transfer (HGT) between bacteria also occurs in such processes, although the influence of bioreactor habitat and ecology on HGT frequency is not well understood. Here, we quantified how oxidation-reduction (redox) conditions impact the fate of a Green fluorescent protein (Gfp)-tagged AR plasmid (pRP4- gfp ) within an E. coli host (EcoFJ1) in the liquid phase and biofilms in bioreactors. Replicate reactors treating domestic wastewater were operated under stable aerobic (+195 ± 25 mV), anoxic (-15 ± 50 mV), and anaerobic (-195 ± 15 mV) conditions, and flow cytometry and selective plating were used to quantify donor strain, EcoFJ1(pRP4- gfp ), and putative transconjugants over time. Plasmid pRP4- gfp -bearing cells disappeared rapidly in aerobic ecosystems (∼2.0 log reduction after 72 h), especially in the liquid phase. In contrast, EcoFJ1(pRP4- gfp ) and putative transconjugants persisted much longer in anaerobic biofilms (∼1.0 log reduction, after 72 h). Plasmid transfer frequencies were also higher under anaerobic conditions. In parallel, protozoan abundances were over 20 times higher in aerobic reactors relative to anaerobic reactors, and protozoa numbers significantly inversely correlated with pRP4- gfp signals across all reactors ( p &lt; 0.05). Taken together, observed HGT frequency and plasmid retention are impacted by habitat conditions and trophic effects, especially oxygen conditions and apparent predation. New aerobic bioreactor designs are needed, ideally employing passive aeration to save energy, to minimize resistance HGT in biological wastewater treatment processes.

Impact of Redox Conditions on Antibiotic Resistance Conjugative Gene Transfer Frequency and Plasmid Fate in Wastewater Ecosystems