Recipient Organization
GEORGE WASHINGTON UNIVERSITY
2121 EYE STREET NW SUITE 601
WASHINGTON,DC 20052
Performing Department
(N/A)
Non Technical Summary
While agriculture production urges a growing demand of freshwater, wastewater reuse is recognized as a promising strategy to supply water for irrigation and promote water sustainability. However, a serious public health concern on wastewater reuse is the potential transmission of pathogens, especially antibiotic-resistant bacteria (ARB) from reused treated wastewater to food. Environmental bacteria may acquire antibiotic resistance through horizontal gene transfer of antibiotic resistance genes (ARGs). Wastewater carries a large number of ARGs, and the high bacterial density and diversity environments in biological wastewater treatment processes can facilitate the transfer of ARGs among bacteria. While three major pathways (including natural transformation, transduction, and conjugation) were believed to horizontally transfer ARGs among bacteria, vesiduction (horizontal gene transfer mechanism via vesicle-associated ARGs released from bacteria) has been recently recognized as an emerging but overlooked route to transfer genes horizontally. The vesicle-associated ARGs can be extensively released from bacteria, stable and persistent in environment, and escape from or resist to water treatment processes. However, our knowledge on transfer and fate of these vesicle-associated ARGs in wastewater treatment and reuse is not sufficient to develop control strategies. In the proposed research, we aim to i) understand the horizontal transfer of ARGs mediated by membrane vesicles in water matrices relevant to agriculture water reuse, ii) elucidate the persistence and removal of vesicle-associated ARGs in key steps of wastewater treatment and reclamation, and iii) determine the occurrence, abundance, and removal of vesicle-associated ARGs in each step in municipal water reclamation and recycling facilities. First, we will conduct lab tests to evaluate the horizontal transfer abilities of vesicle-associated ARGs to selected bacterial species that are environmentally and clinically relevant, and we will also explore the mechanisms/factors controlling this transfer in water matrices. Next, bench-top tests will be performed to quantitatively investigate the damage/degradation of vesicle-associated ARGs through characterizing DNA, proteins, lipids, vesicle sizes, and vesicle integrity/morphology under exposure to simulated wastewater treatment conditions. Finally, real wastewater and reclaimed water will be collected in water reclamation and recycling facilities, and the metagenomic and quantitative polymerase chain reaction (qPCR) analysis will be conducted to understand the occurrence, abundance, and removal of vesicle-associated ARGs.This study will advance our knowledge of the fate, transfer, and risks of ARGs in agricultural water reuse, provide insights into the control of antibiotic-resistant pathogens in agricultural water reuse, and shed light on the management of water reuse for agricultural irrigation.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
The goal of this research is to advance our knowledge on transfer, fate, and occurrence of vesicle-associated antibiotic resistance genes (ARGs) in wastewater treatment processes for agriculture water reuse, hence providing insights into the control of ARGs and antibiotic-resistant bacteria (ARB). In particular, this proposed research aims to i) understand the horizontal transfer of ARGs mediated by membrane vesicles in water matrices relevant to agriculture water reuse, ii) elucidate the persistence and removal of vesicle-associated ARGs in key steps of wastewater treatment and reclamation, and iii) determine the occurrence, abundance, and removal of vesicle-associated ARGs in each step in municpla water reclamation and recycling facilities.
Project Methods
First, we will conduct bench-top experiments to determine the horizontal transfer frequencies of vesicle-associated ARGs to different bacterial species and explore the mechanisms/factors controlling this transfer in water matrices. In particular, we will include both Gram-negative and Gram-positive as the donor and recipient strains, especially the environmentally and clinically relevant pathogenic strains, for which the acquisition of antibiotic resistance is concerning. Then the transfer frequency of vesicle-associated ARGs will be examined by incubating the isolated vesicles from one donor strain with one recipient strain in water matrices relevant to wastewater treatment and reuse. The transfer frequency of vesicle-associated ARGs will also be compared with other gene transfer routes, including transformation and conjugation. In addition, the influence of nutrient levels, temperature, and other environmental stressors on the transfer frequency will also be examined.Next, bench-top tests will be performed to quantitatively investigate the persistence and removal of vesicle-associated ARGs in wastewater treatment and reuse processes. Especially, the degradation of vesicle-associated ARGs through characterizing DNA, proteins, lipids, vesicle sizes, and vesicle integrity/morphology in wastewater and reclaimed water matrices will be examined. In addition, the removal efficiency of vesicle-associated ARGs will be evaluated through filtration processes (sand filtration, activated carbon filtration, microfiltration, and ultrafiltration) and disinfection processes (chlorine, chloramine, and UV). The degradation kinetics and the horizontal transfer frequencies of the ARGs exposed to the disinfection processes will also be investigated.Finally, the occurrence, abundance, and removal of vesicle-associated ARGs in real wastewater treatment and reuse processes will be examined. In particular, the wastewater after each step of treatment and the reclaimed water will be collected from several wastewater treatment and recycling facilities. Then the vesicles will be isolated by filtration and ultracentrifuge. Next, metagenomic analysis will be conducted to determine the occurrence and diversity of vesicle-associated ARGs. Then the most commonly occurred and clinically relevant ARGs will be selected, and their removal efficiencies after each treatment process will be examined through quantitative polymerase chain reaction (qPCR) analysis. In addition, the occurrence and removal of vesicle-associated ARGs will also be compared with total ARGs in wastewater treatment and reuse processes.