Antibiotic resistance gene propagation: in situ rates and networks of horizontal gene transfer in wastewater

对抗抗菌素耐药性的全球健康危机的一个主要挑战是，抗生素耐药性基因(ARGs)可以通过一种称为水平基因转移(HGT)的过程在细菌之间共享。ARG存在于废水中，对回收废水可能传递的ARG健康影响知之甚少。关于环境细菌导致ARG HGT的条件知之甚少，例如用于处理废水的细菌和临床感染中发现的病原菌。该项目的目标是破译病原体如何从水和废水系统中存在的环境细菌中获得ARGs，并了解ARGs如何在水和废水微生物群落中繁殖。这项研究将开发生物传感器来监测ARGs的HGT，以了解污水处理厂的简单操作参数如何影响ARGs的减少或扩散。如果成功，该项目的结果将确定抗生素耐药性在环境中转移的方法，并确定在水处理过程中阻止这种转移的方法，保护公众健康和国家的供水。了解环境中发现的微生物群落中水平基因转移(HGT)的控制将赋予人们前所未有的能力来管理日益增长的抗生素耐药性威胁。虽然有各种技术可以获得通过HGT获得抗生素耐药性基因(ARGs)的细菌的静态快照，但这些现有的方法不能提供关于经历不断变化的环境的复杂社区内基因流动路径和速度的动态信息。这些方法也不容易区分活的细菌和死亡的细菌。将利用两种新出现的工具来获得这一信息：(1)报告原位结合事件的气体报告生物传感器；(2)用于确定混合群落中ARGs宿主范围的高通量、独立于培养的方法。这些工具将应用于处理生活污水的生物反应器，以更好地了解操作控制如何影响ARG繁殖率和寄主范围。这项研究的目标是(1)开发生物传感器，通过偶联一种产生稀有挥发性气体的酶的合成来原位报告HGT；(2)在实验室规模的废水生物反应器中使用这些工具来监测不同反应器条件下整个群落的HGT速率；以及(3)表征不同生物反应器操作条件下工程质粒和一套与环境相关的ARG的宿主范围。这项研究的结果将促进对废水处理和水再利用系统中ARG的HGT管理机制的了解，这些机制将为保护人类健康的管理战略提供信息。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.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.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.