A major challenge to combating the global health crisis of antibacterial resistance is that antibiotic resistance genes (ARGs) can be shared among bacteria through a process called horizontal gene transfer (HGT). ARGs are present in wastewater, and there is little understanding of ARG health impacts potentially conveyed by recycling wastewater. Little is known regarding the conditions that result in HGT of ARGs from environmental bacteria such as those used to treat wastewater and pathogenic bacteria found in clinical infections. The goals of this project are to decipher how pathogens acquire ARGs from environmental bacteria present in water and wastewater systems and to understand how ARGs are propagated in water and wastewater microbial communities. This research will develop biosensors to monitor HGT of ARGs to understand how simple operational parameters in a wastewater treatment plant impact the reduction or proliferation of ARGs. If successful, the results of this project will identify methods of antibiotic resistance transfer in the environment and identify ways to halt this transfer during water treatment processes, protecting public health and the Nation's water supply.<br/><br/>Understanding the controls over horizontal gene transfer (HGT) in microbial communities found in the environment would impart an unprecedented ability to manage the growing threat of antibiotic resistance. While a variety of technologies are available for obtaining static snapshots of bacteria that have acquired antibiotic resistance genes (ARGs) through HGT, these existing approaches do not provide dynamic information on the pathways and rates of gene flow within complex communities that experience a changing environment. These approaches also cannot easily differentiate between living and dead bacteria. Two emerging tools will be leveraged to obtain this information: (1) gas-reporting biosensors that report on in situ conjugation events; and (2) a high-throughput, culture-independent method for determining the host-range of ARGs in a mixed community. These tools will be applied in bioreactors treating domestic wastewater to better understand how operational controls impact ARG propagation rates and host range. The objectives of this research are to (1) develop biosensors that report on HGT in situ by coupling the synthesis of an enzyme that produces a rare volatile gas to broad-range plasmid transfer; (2) use these tools in bench-scale wastewater bioreactors to monitor HGT rates across a community under various reactor conditions; and (3) characterize the host range of the engineered plasmids and of a suite of environmentally-relevant ARGs under different bioreactor operational conditions. The results of this research will advance the knowledge of the mechanisms that govern HGT of ARGs in wastewater treatment and water reuse systems that will inform management strategies to protect human health.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.