Search

Antibiotic resistance is a major 21st century One Health (humans, animals, environment) challenge whose spread limits options to treat bacterial infections. There is growing interest in monitoring water environments, including surface water and wastewater, which have been identified as key recipients, pathways, and sources of antibiotic resistant bacteria (ARB). Aquatic environments also facilitate the transmission and amplification of ARB. Enterococcus spp. often carry clinically-important antibiotic resistance genes and are of interest as environmental monitoring targets. Enterococcus spp. are Gram-positive bacteria that are typically of fecal origin; however, they are also found in relevant environmental niches, with various species and strains that are opportunistic human pathogens. Although the value of environmental monitoring of antibiotic-resistant Enterococcus has been recognized by both national and international organizations, lack of procedural standardization has hindered generation of comparable data needed to implement integrated surveillance programs. Here we provide a comprehensive methodological review to assess the techniques used for the culturing and characterization of antibiotic-resistant Enterococcus across water matrices for the purpose of environmental monitoring. We analyzed 117 peer-reviewed articles from 33 countries across six continents. The goal of this review is to provide a critical analysis of (i) the various methods applied globally for isolation, confirmation, and speciation of Enterococcus isolates, (ii) the different methods for profiling antibiotic resistance among enterococci, and (iii) the current prevalence of resistance to clinically-relevant antibiotics among Enterococcus spp. isolated from various environments. Finally, we provide advice regarding a path forward for standardizing culturing of Enterococcus spp. for the purpose of antibiotic resistance monitoring in wastewater and wastewater-influenced waters within a global surveillance framework. Published version

As climate change exacerbates water scarcity and alters available water and fertilizer resources, it is vital that take appropriate measures to ensure sustainable treatment of water, wastewater, and other waste streams that are protective of public health and support recovery and reuse of water and nutrients. The overarching theme of this dissertation is the advancement of next-generation DNA sequencing (NGS) and computational tools for achieving these goals. A suite of relevant fecal and environmental opportunistic pathogens are examined using both culture-based and NGS-based methods. Of particular concern to this research was not only the attenuation and inactivation of pathogens, but also ensuring that optimal treatment approaches reduce antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Key systems that were the focus of this effort included nutrient reuse (wastewater-derived biosolids and cattle-derived manure), water reuse, and drinking water systems disrupted by a major hurricane. A field study was carried out to survey a suite of pathogens from source-to tap in six small drinking water systems in Puerto Rico six months after Hurricane Maria. The study revealed that pathogenic Leptospira DNA was detected in all systems that were reliant on surface water. On the other hand, Salmonella spp. was detected in surface and groundwater sources and some distribution system waters both by culture and PCR. The study provided comparison of molecular-, microscopic-, and culture-based analysis for pathogen detection and highlighted the need for disaster preparedness for small water systems, including back-up power supply and access to chlorination as soon as possible after a natural disaster. A second field-study examined wastewater derived solids across an international transect of wastewater treatment plants in order to gain insight into the range of ARG concentrations encountered. It was found that, while total ARGs did not vary between treatment or continent of origin, clinically-relevant ARGs (i.e., ARGs encoding resistance to important classes of antibiotics used in humans) were significantly higher in solids derived from Asian wastewater treatment plants. Estimated loading rates of ARGs to soil under a scenario of land application were determined, highlighting in all cases that they are orders of magnitude higher than in the aqueous effluent. Livestock manure, derived from control cattle and cattle undergoing typical antibiotic treatment, and corresponding composts were also evaluated as common soil amendments in a separate study. In this study, the amendments were applied to two soil types in a greenhouse setting, in order to compare the resulting carriage of ARGs on a root (radish) versus leafy (lettuce) vegetable. Remarkably, radishes were found to harbor the highest relative abundance of total ARGs enumerated by metagenomics, even higher than corresponding soils or manures. Although the total microbial load will be lower on a harvested vegetable, the results suggest that the vegetable surface environment can differentially favor the survival of ARBs. The role of wastewater and water reuse treatment processes in reducing ARB and ARGs was also investigated at field-scale. Two independent wastewater treatment plants both substantially reduced total ARG relative and absolute abundance through biological treatment and settling according to metagenomic analysis. The subsequent water reuse treatment train of one system produced water for non- potable purposes and found further reduction in ARGs after chlorination, but a five hundred percent increase in the relative abundance of ARGs in the subsequent distribution system. In the second plant, which employed a membrane-free ozone-biologically-activated carbon-granular activated carbon treatment train for indirect potable reuse, there were notable increases in total ARG relative abundance following ozonation and chlorination. However, these numbers attenuated below background aquifer levels before recharge. Culture-based analysis of these systems targeting resistant ESKAPE pathogens (Escherichia coil, Staphylococcus aureus, Klebsiella spp., Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus spp.) indicated similar trends as the metagenomic ARG analysis for both systems, but was challenged by sub-optimal media for wastewater samples and low confirmation rates, limiting statistical analysis. In order to advance the application of NGS, molecular, and associated bioinformatic tools for monitoring pathogens and antibiotic resistance in environmental systems, newly emerging methods and field standards for antibiotic resistance assessment were also evaluated. Hybrid assembly, the assembly for both short and long metagenomic sequencing reads, were assessed with an in silico framework in order to determine which available assemblers produced the most accurate and long contigs. Hybrid assembly was found to produce longer and more accurate assemblies at all coverages by reducing error as compared to short read assembly, though the outputs differed in composition from long read assembly. Where it is possible, it is beneficial to sequence using both long- and short-read NGS technologies and employ hybrid assembly, but further validation is recommended. Genome resolved metagenomics has also emerged as a strategy to recover individual bacterial genomes from the mixed metagenomic samples though this is often not well validated. In order to address this, genomes were assembled from reclaimed water systems and were compared against whole-genome sequences of antibiotic resistant E.coli isolates. Metagenome-derived genomes were found to produce similar profiles in wastewater treatment plant influents. A final theme to this dissertation addresses the need to standardize targets, methodologies, and reporting of antibiotic resistance in the environment. A systematic literature review was conducted on assays for the enumeration of key ARGs across aquatic environments and recommendations are summarized for the production of comparable data. In sum, this dissertation advances knowledge about the occurrence of pathogens, ARB, and ARGs across aquatic and agricultural systems and across several countries. Advances are made in the application of NGS tools for environmental monitoring of antibiotic resistance and other targets and a path forward is recommended for continued improvement as both DNA sequencing technologies and computational methodologies continue to rapidly advance. Doctor of Philosophy Understanding bacteria in our engineered systems is critical to ensuring drinking water, recycled water, and manure-derived soil amendments are safe for downstream applications. As novel approaches for assessing bacteria are developed, standardized methods and evaluations much be developed to ensure that sound conclusions are made that can appropriately inform policy and practice for the protection of public health. This dissertation focuses on combining bacterial culture and DNA sequencing methods for the study of pathogens (i.e., disease-causing organisms) and antibiotic resistance (i.e., ability of some bacteria to survive antibiotic treatments) in agricultural manure management, water reuse, and drinking water systems. Additionally, this work sought to advance emergent metagenomic analysis tools, which provides a new and potentially powerful pathogen and antibiotic resistance monitoring approach through direct extraction and sequencing of DNA from environmental samples. Antibiotic resistance is a global health challenge and it has been widely recognized that wastewater and agriculture are key control points. When antibiotics are ingested by people or livestock, they select for resistant bacteria in the gut. Mitigation efforts are needed, particularly at wastewater treatment plants and on farms, to ensure that excreted antibiotics and resistant bacteria do not further propagate and pose a risk. However, additional challenges such as climate change have spurred the need for more efficient use of our water and nutrient resources. In this work I examined how nutrient and water reuse treatment methods affect antibiotic resistant bacteria and antibiotic resistance genes using DNA sequencing as well as culture-based methods. In order to assess agricultural practices, a systems approach was conducted at the greenhouse scale to identify key control points to stem the spread of antibiotic resistance when vegetables are grown in soils amended with cattle-derived manure fertilizers. Along the food production chain, vegetables (i.e., radish and lettuce) were found to harbor higher proportions of bacteria carrying antibiotic resistance genes, although the estimated numbers of these bacteria were lower. Solids from an international transect of wastewater treatment plants (Sweden, Switzerland, USA, India, Hong Kong, Phillippenes) were examined because they are also foten used as soil amendments. DNA sequencing of these solids revealed that total measured antibiotic resistance genes did not vary between treatment or continent of origin. Calculations were made to determine the range of total hypothetical outputs of ARGs if the biosolids are land applied. Wastewater reuse systems were also examined using culture and metagenomic DNA analysis so that living pathogens could be compared alongside the total (dead and alive) antibiotic resistance genes. While standard wastewater and subsequent water reuse treatments were found to reduce the absolute numbers of antibiotic resistance genes and bacteria in a treatment plant producing water for non-potable reuse (i.e., irrigation), increases in culturable resistant pathogens and antibiotic resistance genes were apparent in the distribution system (i.e., in the pipes conveying treated water to the point of use). Similar reductions in antibiotic resistant bacteria and resistance genes were also seen in a plant using more advanced treatment (ozonation paired with biofiltration) to produce water suitable for indirect potable reuse via aquifer recharge, but there were indications that ozone and chlorine can increase the proportion of antibiotic resistant bacteria. Finally, genomes were recovered from the metagenomic sequencing analysis and were compared to sequenced culture isolates to validate the capabilities of metagenomic analysis to re-assemble genomes at the strain level, which is often required for pathogen confirmation. Pathogens were also assessed in disrupted drinking water systems in Puerto Rico after Hurricane Maria. Small scale systems that were disrupted by the storm were sampled to identify if pathogens were measurable six months after the hurricane. This work revealed that genes attributed to pathogenic Leptospira were detected in all surface water reliant systems while Salmonella spp. were detected by culture and DNA methods, but only in the source surface and groundwaters, not in the distribution systems delivering water to from the treatment site to the tap. This research also contributed to the advancement of big data analysis pipelines as well as to the standardization of methods to ensure that data produced across studies are comparable. Hybrid assembly, an emergent method that combines both short and long metagenomic DNA sequences generated by different technologies to more accurately recover genomes, was found to improve reliability and accuracy of algorithms aimed at reassembling DNA fragments. Antibiotic resistance is a global challenge, but without standardized methodologies for environmental monitoring, it will be difficult to compare measurements across countries and treatment processes in order to identify effective mitigation strategies. A critical literature review was conducted on assays for the enumeration of key antibiotic resistance genes across aquatic environments so that comparable data can be generated. This will be critical to tap into the tremendous volumes of antibiotic resistance monitoring data being generated around the globe to help identify trends and inform solutions. Collectively, this dissertation advances knowledge about the occurrence of pathogens, antibiotic resistant bacteria and antibiotic resistance genes across aquatic and agricultural systems while also critically evaluating emerging methods for the detection of antibiotic resistance in the environment.

An integrated understanding of factors influencing the occurrence, distribution, and fate of antibiotic resistance genes (ARGs) in vegetable production systems is needed to inform the design and development of strategies for mitigating the potential for antibiotic resistance propagation in the food chain. The goal of the present study was to holistically track antibiotic resistance and associated microbiomes at three distinct pre-harvest control points in an agroecosystem in order to identify the potential impacts of key agricultural management strategies. Samples were collected over the course of a single growing season (67 days) from field-scale plots amended with various organic and inorganic amendments at agronomic rates. Dairy-derived manure and compost amendment samples (n = 14), soil samples (n = 27), and lettuce samples (n = 12) were analyzed via shotgun metagenomics to assess multiple pre-harvest factors as hypothetical control points that shape lettuce resistomes. Pre-harvest factors of interest included manure collection during/post antibiotic use, manure composting, and soil amended with organic (stockpiled manure/compost) versus chemical fertilizer. Microbial community resistome and taxonomic compositions were unique from amendment to soil to lettuce surface according to dissimilarity analysis. The highest resistome alpha diversity (i.e., unique ARGs, n = 642) was detected in amendment samples prior to soil application, while the composted manure had the lowest total ARG relative abundance (i.e., 16S rRNA gene-normalized). Regardless of amendment type, soils acted as an apparent ecological buffer, i.e., soil resistome and taxonomic profiles returned to background conditions 67 d-post amendment application. Effects of amendment conditions surprisingly re-emerged in lettuce phyllosphere resistomes, with the highest total ARG relative abundances recovered on the surface of lettuce plants grown in organically-fertilized soils (i.e., compost- and manure-amended soils). Co-occurrence analysis identified 55 unique ARGs found both in the soil amendments and on lettuce surfaces. Among these, arnA and pmrF were the most abundant ARGs co-occurring with mobile genetic elements (MGE). Other prominent ARG-MGE co-occurrences throughout this pre-harvest lettuce production chain included: TetM to transposon (Clostridiodies difficile) in the manure amendment and TriC to plasmid (Ralstonia solanacearum) on the lettuce surfaces. This suggests that, even with imposing manure management and post-amendment wait periods in agricultural systems, ARGs originating from manure can still be found on crop surfaces. This study demonstrates a comprehensive approach to identifying key control points for the propagation of ARGs in vegetable production systems, identifying potential ARG-MGE combinations that could inform future surveillance. The findings suggest that additional pre-harvest and potentially post-harvest interventions may be warranted to minimize risk of propagating antibiotic resistance in the food chain. Published version

Cattle are commonly administered antibiotics, resulting in excretion of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs). The aim of this study was to determine if the use of dairy manure collected during antibiotic administration influences the bacterial microbiota of lettuce and radishes, including carriage of ARB and ARGs, when applied as a soil amendment and if composting mitigates the effects. Lettuce and radishes were grown in field-plots amended with raw manure from antibiotic-treated (cephapirin, pirlimycin) cows, composted manure from antibiotic-treated cows, composted manure from antibiotic-free cows, or an inorganic chemical fertilizer (control; 12 plots, n = 3). Surficial vegetable bacteria and antibiotic resistomes (i.e., total ARGs) were characterized using heterotrophic plate counts (HPCs) on antibiotic-containing media, 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction (qPCR), and shot-gun metagenomics. The different manure and compost amendments did not result in significant changes to the surficial vegetable bacteria at the phylum level; however, some minor changes at the class and family level were observed. Beta-diversities of the ARGs detected by shotgun metagenomic sequencing were distinctly different between vegetable type (R = 0.30, p = 0.04), with small separations between the resistomes associated with amendment type in unrarefied analysis (R = 0.27, p = 0.02), but not rarefied analysis, of the data. Network analysis highlighted that multi-drug ARG classes commonly co-occurred with plasmid-associated genes and could be a driver of co-and cross-selection of ARGs in the different conditions. Carriage of sul1 and tet(W) ARGs on vegetables quantified by qPCR were strong indicators of manure-based amendment relative to chemical fertilizer, with some reduction incurred via composting (p < 0.05). Also, increased HPCs resistant/tolerant to clindamycin, a class of antibiotics administered to cattle, were on lettuce grown in biological soil amendments relative to chemical fertilizer (p < 0.05). This study demonstrates that amending soil with rawmanure collected fromdairy cows during antibiotic administration may affect the composition of microbiota and resistomes associated with vegetable surfaces. Composting may be an important strategy to reduce some ARGs on fresh produce, but differences in the resistomes of lettuce and radishes suggest the extent of soil contact should be considered. This work was supported by the USDA NIFA-AFRI 2014-05280/2015-68003-23050 awards and the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture.

Wastewater-based testing (WBT) for SARS-CoV-2 has rapidly expanded over the past three years due to its ability to provide a comprehensive measurement of disease prevalence independent of clinical testing. The development and simultaneous application of the field blurred the boundary between measuring biomarkers for research activities and for pursuit of public health goals, both areas with well-established ethical frameworks. Currently, WBT practitioners do not employ a standardized ethical review process (or associated data management safeguards), introducing the potential for adverse outcomes for WBT professionals and community members. To address this deficiency, an interdisciplinary group developed a framework for a structured ethical review of WBT. The workshop employed a consensus approach to create this framework as a set of 11-questions derived from primarily public health guidance because of the common exemption of wastewater samples to human subject research considerations. This study retrospectively applied the set of questions to peer- reviewed published reports on SARS-CoV-2 monitoring campaigns covering the emergent phase of the pandemic from March 2020 to February 2022 (n=53). Overall, 43% of the responses to the questions were unable to be assessed because of lack of reported information. It is therefore hypothesized that a systematic framework would at a minimum improve the communication of key ethical considerations for the application of WBT. Consistent application of a standardized ethical review will also assist in developing an engaged practice of critically applying and updating approaches and techniques to reflect the concerns held by both those practicing and being monitored by WBT supported campaigns., Synopsis: Development of a structured ethical review facilitates retrospective analysis of published studies and drafted scenarios in the context of wastewater-based testing.