Systematic Analysis of Antibiotic Resistance Genes (ARGs) in Water and Environmental System
The global research community is aware of the prevalence and ubiquity of antibiotic resistant genes (ARGs) which is increasing over time due to human activities (Allen et al. 2010; Baquero, Martínez, and Cantón 2008; Bergeron et al. 2016; Di Cesare et al. 2015; Huang et al. 2019; Knapp et al. 2010; Pruden et al. 2006; Rizzo et al. 2013; Tacconelli et al. 2018). The main issue of emerging antibiotic resistance (AR) is causing casual infections untreatable and can cause high socioeconomic costs as well as health care burden (CDC 2019; L. G. Li et al. 2020). According to the World Health Organization (WHO), we lack global surveillance, and this poses threat to not only human health but also has risks of system overloading, failure, or indirect hazards(Sanitation Safety Planning, Greywater and Excreta. 2016; WHO 2014). Studies have shown that ARGs can be present even in a pristine environment like the sea bed of the deep ocean (B. Chen et al. 2013). Others have suggested most of the antibiotic resistance and metal resistances are coming from environmental stress in the urban area (Medeiros et al. 2016). My dissertation focuses on targeting ARGs abundance, distribution, and their fate in the water environment impacted by human activities. It consists of four sections: 1) Introduction on antibiotic resistance (AR) and methods of quantitative ARG analysis 2) Method comparison and optimization of qPCR and ddPCR methods for ARGs detection 3) ARGs and pathogen quantification in the water system under the influence of anthropogenic activities, 4) ARG tracking and metagenomic study of microbiome under the effects of Flue Gas Desulfurization (FGD) bioreactors The first chapter focuses on the background of Antibiotic Resistance and describes the method development and optimization of ARG quantification using quantitative polymerase chain reaction (qPCR). DNA is extracted from an environmental sample that potentially has targeted genes and was screened for the presence of ARGs by PCR and gel electrophoresis. Once the target is identified, target genes were cloned into competent cells. The cells were cultured on antimicrobial plates, and once survived passing the blue/white screening, its grown culture DNA can serve as a standard for qPCR analysis. Standards were developed through this method were further used in the studies as described in subsequent chapters. The second chapter focused on the following criteria for comparing both technologies, 1) efficiency, 2) range of detection and limitations under different disciplines and gene targets, (3) optimization, and (4) status on antibiotic resistance genes (ARGs) analysis in the literature. Then droplet digital polymerase chain reaction (ddPCR) is developed and optimized to detect ARGs in environmental samples and compare quantification and detection performances along with qPCR. The performance of ddPCR to qPCR was compared on ARGs (tetA, ereA) standard and samples, and the correlation between lab results and trend of ARGs reported in the literature was discussed. The third chapter describes our study that shows how the antibiotic resistance genes abundance varies in three different water systems across the U.S. with various types of water bodies such as lakes, rivers, underground recharge, and wetlands, particularly with discharge from the wastewater treatment plants (WWTP). The objective of the study is to 1) find effects of WWTP effluents on ARGs abundance in the water system, 2) investigate how ARGs concentration changes throughout different types of water bodies, and 3) find connections of other anthropogenic sources of ARGs inflow into the water system. The WWTP effluents were the major contributor of increased ARG concentration for most of the ARG analyzed except for qnrB. We found that specific types of water systems have a different distribution of ARGs: underground recharge contaminated with qnrA compared to others, the lake was detected to have higher sul3 and qnrB genes compared to others. This study has concluded and is at the writing phase for a journal paper submission The fourth chapter describes the study on the microbiome of the Flue Gas Desulfurization (FGD) bioreactors, for their genetic functions on contaminant uptake from the FGD wastewater into biomass and the expression of ARGs across all sites. FGD is a process to remove toxic volatile substances from the coal combustion process of power plants, and this process wastewater as a byproduct. The wash water of the scrubbing tower is sourced from a nearby water source such as a river or lake, and this makes the presence of ARGs to be expected, because of the known ubiquity of ARGs even in pristine water. The wastewater created from the process also contains high concentrations of heavy metals including Se, Cd, Cr, Hg, As, Pb, Ni, Cu, etc. along with sulfates, chlorides, nitrates (Ma et al. 2019; Martini and Vadbunker 2016; W. Zhang et al. 2019). In the treatment of the contaminated FGD wastewater, two-stage bioreactors which can biologically harness sulfates and other heavy metals were applied with sludge recycling with little or no waste. The microbiome that is present in the bioreactors showed various functional pathways to harness contaminants, as well as antibiotic resistance of different mechanisms. The co-occurrence of ARGs and metal resistance genes (MRGs) has been previously reported (Xiaomin Wang et al. 2021), and the result from this chapter helps understanding and containment of ARGs under stressed environments.