Browsing by Author "Garner, Emily"

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    Assessing Vulnerabilities to the Spread of Pathogens and Antibiotic Resistance in Agricultural and Water Systems Using Culture-, Molecular-, and Metagenomic-based Techniques
    Keenum, Ishi M. (Virginia Tech, 2021-09-09)
    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.  
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    Combined effects of composting and antibiotic administration on cattle manure–borne antibiotic resistance genes
    Keenum, Ishi M.; Williams, Robert K.; Ray, Partha P.; Garner, Emily; Knowlton, Katharine F.; Pruden, Amy (2021-04-01)
    Background Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here, we carried out a comprehensive parallel examination of the effects of small-scale (> 55 °C × 3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration (cephapirin/pirlimycin or sulfamethazine/chlortetracycline/tylosin, respectively), versus from untreated cattle, on “resistomes” (total antibiotic resistance genes (ARGs) determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally heated to > 55 °C × 15 days. Results Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition (ANOSIM; R = 0.424, p = 0.001, respectively) in all composts at the small-scale. Reduction in relative abundance (16S rRNA gene normalized) of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions (ANOSIM; R = 0.728, p = 0.001), except when externally heated. Sul1, intI1, beta-lactam ARGs, and plasmid-associated genes increased in all finished composts as compared with the initial condition. External heating more effectively reduced certain clinically relevant ARGs (blaOXA, blaCARB), fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotic condition and slower in the beef manure with antibiotic condition. Conclusions This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, tet(W), and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered.
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    Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016
    Garner, Emily; Brown, Connor L.; Schwake, David Otto; Rhoads, William J.; Arango-Argoty, Gustavo; Zhang, Liqing; Jospin, Guillaume; Coil, David A.; Eisen, Jonathan A.; Edwards, Marc A.; Pruden, Amy (Centers for Disease Control and Prevention, 2019-11)
    During the water crisis in Flint, Michigan, USA (2014–2015), 2 outbreaks of Legionnaires’ disease occurred in Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella pneumophila isolates submitted to a laboratory in Genesee County during the second outbreak with 103 water isolates collected the following year. We documented a genetically diverse range of L. pneumophila strains across clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates, 3 water isolates from a Flint hospital, 1 water isolate from a Flint residence, and the reference Paris strain) had a high degree of similarity (2–1,062 single-nucleotide polymorphisms), all L. pneumophila sequence type 1, serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were widespread in Flint hospital water samples but bore no resemblance to available clinical isolates. L. pneumophila strains in Flint tap water after the outbreaks were diverse and similar to some disease-causing strains.
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    DeepARG: a deep learning approach for predicting antibiotic resistance genes from metagenomic data
    Arango-Argoty, Gustavo; Garner, Emily; Pruden, Amy; Heath, Lenwood S.; Vikesland, Peter J.; Zhang, Liqing (2018-02-01)
    Background Growing concerns about increasing rates of antibiotic resistance call for expanded and comprehensive global monitoring. Advancing methods for monitoring of environmental media (e.g., wastewater, agricultural waste, food, and water) is especially needed for identifying potential resources of novel antibiotic resistance genes (ARGs), hot spots for gene exchange, and as pathways for the spread of ARGs and human exposure. Next-generation sequencing now enables direct access and profiling of the total metagenomic DNA pool, where ARGs are typically identified or predicted based on the “best hits” of sequence searches against existing databases. Unfortunately, this approach produces a high rate of false negatives. To address such limitations, we propose here a deep learning approach, taking into account a dissimilarity matrix created using all known categories of ARGs. Two deep learning models, DeepARG-SS and DeepARG-LS, were constructed for short read sequences and full gene length sequences, respectively. Results Evaluation of the deep learning models over 30 antibiotic resistance categories demonstrates that the DeepARG models can predict ARGs with both high precision (> 0.97) and recall (> 0.90). The models displayed an advantage over the typical best hit approach, yielding consistently lower false negative rates and thus higher overall recall (> 0.9). As more data become available for under-represented ARG categories, the DeepARG models’ performance can be expected to be further enhanced due to the nature of the underlying neural networks. Our newly developed ARG database, DeepARG-DB, encompasses ARGs predicted with a high degree of confidence and extensive manual inspection, greatly expanding current ARG repositories. Conclusions The deep learning models developed here offer more accurate antimicrobial resistance annotation relative to current bioinformatics practice. DeepARG does not require strict cutoffs, which enables identification of a much broader diversity of ARGs. The DeepARG models and database are available as a command line version and as a Web service at http://bench.cs.vt.edu/deeparg.
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    Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes
    Prieto Riquelme, Maria Virginia; Garner, Emily; Gupta, Suraj; Metch, Jake; Zhu, Ni; Blair, Matthew F.; Arango-Argoty, Gustavo; Maile-Moskowitz, Ayella; Li, An-dong; Flach, Carl-Fredrik; Aga, Diana S.; Nambi, Indumathi M.; Larsson, D. G. Joakim; Bürgmann, Helmut; Zhang, Tong; Pruden, Amy; Vikesland, Peter J. (ACS, 2022-06-27)
    Wastewater-based surveillance (WBS) for disease monitoring is highly promising but requires consistent methodologies that incorporate predetermined objectives, targets, and metrics. Herein, we describe a comprehensive metagenomics-based approach for global surveillance of antibiotic resistance in sewage that enables assessment of 1) which antibiotic resistance genes (ARGs) are shared across regions/communities; 2) which ARGs are discriminatory; and 3) factors associated with overall trends in ARGs, such as antibiotic concentrations. Across an internationally sourced transect of sewage samples collected using a centralized, standardized protocol, ARG relative abundances (16S rRNA gene-normalized) were highest in Hong Kong and India and lowest in Sweden and Switzerland, reflecting national policy, measured antibiotic concentrations, and metal resistance genes. Asian versus European/US resistomes were distinct, with macrolide-lincosamide-streptogramin, phenicol, quinolone, and tetracycline versus multidrug resistance ARGs being discriminatory, respectively. Regional trends in measured antibiotic concentrations differed from trends expected from public sales data. This could reflect unaccounted uses, captured only by the WBS approach. If properly benchmarked, antibiotic WBS might complement public sales and consumption statistics in the future. The WBS approach defined herein demonstrates multisite comparability and sensitivity to local/regional factors.
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    Evaluation of Metagenomic-Enabled Antibiotic Resistance Surveillance at a Conventional Wastewater Treatment Plant
    Majeed, Haniyyah J.; Riquelme, Maria V.; Davis, Benjamin C.; Gupta, Suraj; Angeles, Luisa F.; Aga, Diana S.; Garner, Emily; Pruden, Amy; Vikesland, Peter J. (Frontiers, 2021-05-13)
    Wastewater treatment plants (WWTPs) receive a confluence of sewage containing antimicrobials, antibiotic resistant bacteria, antibiotic resistance genes (ARGs), and pathogens and thus are a key point of interest for antibiotic resistance surveillance. WWTP monitoring has the potential to inform with respect to the antibiotic resistance status of the community served as well as the potential for ARGs to escape treatment. However, there is lack of agreement regarding suitable sampling frequencies and monitoring targets to facilitate comparison within and among individual WWTPs. The objective of this study was to comprehensively evaluate patterns in metagenomic-derived indicators of antibiotic resistance through various stages of treatment at a conventional WWTP for the purpose of informing local monitoring approaches that are also informative for global comparison. Relative abundance of total ARGs decreased by ∼50% from the influent to the effluent, with each sampling location defined by a unique resistome (i.e., total ARG) composition. However, 90% of the ARGs found in the effluent were also detected in the influent, while the effluent ARG-pathogen taxonomic linkage patterns identified in assembled metagenomes were more similar to patterns in regional clinical surveillance data than the patterns identified in the influent. Analysis of core and discriminatory resistomes and general ARG trends across the eight sampling events (i.e., tendency to be removed, increase, decrease, or be found in the effluent only), along with quantification of ARGs of clinical concern, aided in identifying candidate ARGs for surveillance. Relative resistome risk characterization further provided a comprehensive metric for predicting the relative mobility of ARGs and likelihood of being carried in pathogens and can help to prioritize where to focus future monitoring and mitigation. Most antibiotics that were subject to regional resistance testing were also found in the WWTP, with the total antibiotic load decreasing by ∼40–50%, but no strong correlations were found between antibiotics and corresponding ARGs. Overall, this study provides insight into how metagenomic data can be collected and analyzed for surveillance of antibiotic resistance at WWTPs, suggesting that effluent is a beneficial monitoring point with relevance both to the local clinical condition and for assessing efficacy of wastewater treatment in reducing risk of disseminating antibiotic resistance.
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    A Framework for Standardized Monitoring of Antibiotic Resistance in Aquatic Environments and Application to Wastewater, Recycled Water, Surface Water, and Private Wells
    Liguori, Krista Margaretta (Virginia Tech, 2023-07-10)
    Antimicrobial resistance (AMR) is a One-Health (human, animal, environment) challenge that requires collaborative, interdisciplinary action. Comparable surveillance data are needed to effectively inform policy interventions aimed at preventing the spread of AMR. Environmental monitoring lags behind that of other One Health sectors and is in need of agreed upon targets and standardized methods. A challenge is that there are numerous microorganisms, antibiotic resistance genes (ARGs), and mobile genetic elements and corresponding methods that have been proposed. In this dissertation, a framework for AMR monitoring of aquatic environments was developed through a combination of literature review and stakeholder input, via surveys and a workshop. Through this process, three targets were selected for standardization: the sulfonamide resistance gene (sul1), the class 1 integron integrase gene (intI1), and cefotaxime-resistant Escherichia coli. Quantitative polymerase chain reaction (qPCR)- and culture-based protocols were developed and pilot tested in two independent laboratories on a set of six water matrices: wastewater, recycled water, and surface water from six different wastewater utilities engaging in water reuse located in five states across the USA. The impact of wastewater treatment and advanced water treatment processes was examined in terms of removal of these targets. Finally, qPCR and culture methods were used to examine the relationship between sul1, intI1, E. coli, and fecal indicators in private household wells across four states in the Southern USA that were identified as susceptible to storm events. The overall findings provide a useful baseline occurrence of the proposed AMR monitoring indicators across a range of water types and protocols that are accessible to water utilities.
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    A human exposome framework for guiding risk management and holistic assessment of recycled water quality
    Garner, Emily; Zhu, Ni; Strom, Laurel; Edwards, Marc A.; Pruden, Amy (Royal Society of Chemistry, 2016-04-12)
    Challenges associated with water scarcity and increasing water demand are leading many cities around the globe to consider water reuse as a step towards water sustainability. Recycled water may be used in a spectrum of applications, from irrigation or industrial use to direct potable reuse, and thus presents a challenge to regulators as not all applications require the same level of treatment. We propose that traditional drinking water standards identifying “safe” water quality are insufficient for recycled water and that using the “human exposome” as a framework to guide development of a risk management strategy offers a holistic means by which to base decisions impacting water quality. A successful and comprehensive plan for water reuse must consider 1) health impacts associated with both acute and chronic exposures, 2) all routes of exposure by which individuals may encounter recycled water, and 3) water quality at the true point of use after storage and transport through pipe networks, rather than at the point of treatment. Based on these principles we explore key chemical differences between recycled and traditional potable water, implications for distribution systems with respect to design and operation, occurrence of chronic contaminants, and the presence of emerging and often underappreciated microbial contaminants. The unique nature of recycled water has the potential to provide rapid regrowth conditions for certain microbial contaminants in these systems, which must be considered to achieve safe water quality at the point of use.
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    Metagenomic profiling of historic Colorado Front Range flood impact on distribution of riverine antibiotic resistance genes
    Garner, Emily; Wallace, Joshua S.; Arango-Argoty, Gustavo; Wilkinson, Caitlin; Fahrenfeld, Nicole; Heath, Lenwood S.; Zhang, Liqing; Arabi, Mazdak; Aga, Diana S.; Pruden, Amy (Nature Publishing Group, 2016-12-05)
    Record-breaking floods in September 2013 caused massive damage to homes and infrastructure across the Colorado Front Range and heavily impacted the Cache La Poudre River watershed. Given the unique nature of this watershed as a test-bed for tracking environmental pathways of antibiotic resistance gene (ARG) dissemination, we sought to determine the impact of extreme flooding on ARG reservoirs in river water and sediment. We utilized high-throughput DNA sequencing to obtain metagenomic profiles of ARGs before and after flooding, and investigated 23 antibiotics and 14 metals as putative selective agents during post-flood recovery. With 277 ARG subtypes identified across samples, total bulk water ARGs decreased following the flood but recovered to near pre-flood abundances by ten months post-flood at both a pristine site and at a site historically heavily influenced by wastewater treatment plants and animal feeding operations. Network analysis of de novo assembled sequencing reads into 52,556 scaffolds identified ARGs likely located on mobile genetic elements, with up to 11 ARGs per plasmid-associated scaffold. Bulk water bacterial phylogeny correlated with ARG profiles while sediment phylogeny varied along the river’s anthropogenic gradient. This rare flood afforded the opportunity to gain deeper insight into factors influencing the spread of ARGs in watersheds.
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    Metagenomics-Based Environmental Monitoring of Antibiotic Resistance: Towards Standardization
    Davis, Benjamin Cole (Virginia Tech, 2022-06-13)
    Antibiotic resistance (AR) is a critical and looming threat to human health that requires action across the One Health continuum (humans, animals, environment). Coordinated surveillance within the environmental sector is largely underdeveloped in current National Action Plans to combat the spread of AR, and a lack of effective study approaches and standard analytical methods have led to a dearth of impactful environmental monitoring data on the prevalence and risk of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments. In this dissertation, integrated surveillance approaches of surface water and wastewater systems are demonstrated, and efforts are made towards standardizing both metagenomic- and culture-based techniques for globally comparable environmental monitoring. A field study of differentially-impacted watersheds on the island of Puerto Rico post-Hurricane Maria demonstrated the effectiveness of metagenomics in defining direct impact of anthropogenic stress and human fecal contamination on the proliferation of ARGs in riverine systems. The contribution of treated wastewater effluents to the dissemination of highly mobile and clinically-relevant ARGs and their connection to local clinical settings was also revealed. At the international scale, a transect of conventional activated sludge wastewater treatment plants (WWTPs), representing both US/European and Asian regions, were found to significantly attenuate ARG abundance through the removal of total bacterial load and human fecal indicators, regardless of influent ARG compositions. Strong structural symmetry between microbiome and ARG compositions through successional treatment stages suggested that horizontal gene transfer plays a relatively minor role in actively shaping resistomes during treatment. Risk assessment models, however, indicated high-priority plasmid-borne ARGs in final treated effluents discharged around the world, indicating potentially increased transmission risks in downstream environments. Advancements were also made toward standardizing methods for the generation of globally representative and comparable metagenomic- and culture-based AR monitoring data via two comprehensive and critical literature reviews. The first review provides guidance in next-generation sequencing (NGS) studies of environmental AR, proposing a framework for experimental controls, adequate sequencing depths, appropriate use of public databases, and the derivation of datatypes that are conducive for risk assessment. The second review focuses on antibiotic-resistant Enterococcus spp. as robust monitoring targets and an attractive alternative to more widely adopted Gram-negative organisms, while proposing workflows that generate universally equivalent datatypes. Finally, quantitative metagenomic (qMeta) techniques were benchmarked using internal reference standards for high-throughput quantification of ARGs with statistical reproducibility.
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    Occurrence and Control of Microbial Contaminants of Emerging Concern through the Urban Water Cycle: Molecular Profiling of Opportunistic Pathogens and Antibiotic Resistance
    Garner, Emily (Virginia Tech, 2018-03-26)
    In an era of pervasive water stress caused by population growth, urbanization, drought, and climate change, limiting the dissemination of microbial contaminants of emerging concern (MCECs) is of the utmost importance for the protection of public health. In this dissertation, two important subsets of MCECs, opportunistic pathogens (OP) and antibiotic resistant genes (ARG), are studied across several compartments of the urban water cycle, including surface water, stormwater, wastewater, recycled water, and potable water. Collectively, this dissertation advances knowledge about the occurrence of OPs and ARGs across these water systems and highlights trends that may be of value in developing management strategies for limiting their regrowth and transmission. Field studies of two surface water catchments impacted by stormwater runoff demonstrated the prevalence of ARGs in urban stormwater compared to pristine, unimpacted sites, or to days when no precipitation was recorded. The role of wastewater reuse in transmitting OPs and ARGs was also investigated. Traditional tertiary wastewater treatment plants producing water for non-potable use were found to be largely ineffective at removing ARGs, but plants using advanced oxidation processes or ozonation paired with biofiltration to produce direct potable reuse water were highly effective at removing ARGs. Non-potable reclaimed water consistently had greater quantities of sul1, a sulfonamide ARG, and Legionella and Mycobacterium, two OPs of significant public health concern, present than corresponding potable systems. Limited regrowth of OPs and ARGs did occur in simulated premise (i.e., building) plumbing systems operated with direct potable reuse waters, but regrowth was comparable to that observed in systems fed with potable water derived from surface or groundwater. Advancements were also made in understanding the role of several hypothesized driving forces shaping the antibiotic resistome in natural and engineered water systems: selection by antimicrobials and other compounds, horizontal gene transfer, and microbial community composition. Finally, whole-genome and metagenomic characterization were applied together towards profiling L. pneumophila in clinical and water samples collected from Flint, Michigan, where an economically-motivated switch to an alternative water source created conditions favorable for growth of this organism and likely triggered one of the largest Legionnaires' Disease outbreaks in U.S. history.
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    Tenets of a holistic approach to drinking water-associated pathogen research, management, and communication
    Proctor, Caitlin; Garner, Emily; Hamilton, Kerry A.; Ashbolt, Nicholas J.; Caverly, Lindsay J.; Falkinham, Joseph O. III; Haas, Charles N.; Prevost, Michele; Prevots, D. Rebecca; Pruden, Amy; Raskin, Lutgarde; Stout, Janet; Haig, Sarah-Jane (Pergamon-Elsevier Science, 2022-03-01)
    In recent years, drinking water-associated pathogens that can cause infections in immunocompromised or otherwise susceptible individuals (henceforth referred to as DWPI), sometimes referred to as opportunistic pathogens or opportunistic premise plumbing pathogens, have received considerable attention. DWPI research has largely been conducted by experts focusing on specific microorganisms or within silos of expertise. The resulting mitigation approaches optimized for a single microorganism may have unintended consequences and trade-offs for other DWPI or other interests (e.g., energy costs and conservation). For example, the ecological and epidemiological issues characteristic of Legionella pneumophila diverge from those relevant for Mycobacterium avium and other nontuberculous mycobacteria. Recent advances in understanding DWPI as part of a complex microbial ecosystem inhabiting drinking water systems continues to reveal additional challenges: namely, how can all microorganisms of concern be managed simultaneously? In order to protect public health, we must take a more holistic approach in all aspects of the field, including basic research, monitoring methods, risk-based mitigation techniques, and policy. A holistic approach will (i) target multiple microorganisms simultaneously, (ii) involve experts across several disciplines, and (iii) communicate results across disciplines and more broadly, proactively addressing source water-to-customer system management.
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    Towards a harmonized method for the global reconnaissance of multi-class antimicrobials and other pharmaceuticals in wastewater and receiving surface waters
    Singh, Randolph R.; Angeles, Luisa F.; Butryn, Deena M.; Metch, Jacob W.; Garner, Emily; Vikesland, Peter J.; Aga, Diana S. (Elsevier, 2019-01-17)
    Antimicrobial resistance is a worldwide problem that is both pressing and challenging due to the rate at which it is spreading, and the lack of understanding of the mechanisms that link human, animal and environmental sources contributing to its proliferation. One knowledge gap that requires immediate attention is the significance of antimicrobial residues and other pharmaceuticals that are being discharged from wastewater treatment plants (WWTPs) on the dissemination of antimicrobial resistance in the environment. In this work we provide an approach to develop a harmonized analytical method for 8 classes of antimicrobials and other pharmaceuticals that can be used for global monitoring in wastewater and receiving waters. Analysis of these trace organic chemicals in the influent and effluent wastewater, and in the respective upstream and downstream receiving waters from different countries across the globe is not trivial. Here, we demonstrated that sample preparation using solid-phase extraction (SPE) not only provides a convenient and cost-effective shipping of samples, but also adds stability to the analytes during international shipping. It is important that SPE cartridges are maintained at cold temperature during shipment if the duration is longer than 7 days because a significant decrease in recoveries were observed after 7 days in the cartridges stored at room temperature, especially for sulfonamides and tetracyclines. To compensate for sample degradation during shipment, and matrix effects in liquid chromatography/mass spectrometry, the use of stable isotope labeled compounds should be employed when available and affordable. The importance of applying a defined tolerance for the ion ratios (Q/q) that have been optimized for wastewater and surface water is discussed. The tolerance range was set to be the mean Q/q of the analyte standard at various concentrations ±40% for the influent, and ±30% for the effluent, upstream, and downstream samples; for tetracyclines and quinolones, however, the tolerance range was ±80% in order to minimize false negative and false positive detection. The optimized procedures were employed to reveal differences in antimicrobial and pharmaceutical concentrations in influent, effluent, and surface water samples from Hong Kong, India, Philippines, Sweden, Switzerland, and United States. The antimicrobials with the highest concentrations in influent and effluent samples were ciprofloxacin (48,103 ng/L, Hong Kong WWTP 1) and clarithromycin (5178 ng/L, India WWTP 2), respectively. On the other hand, diclofenac (108,000 ng/L, Sweden WWTP 2), caffeine (67,000 ng/L, India WWTP 1), and acetaminophen (28,000 ng/L, India WWTP 1) were the highest detected pharmaceuticals in the receiving surface water samples. Hong Kong showed the highest total antimicrobial concentrations that included macrolides, quinolones, and sulfonamides with concentrations reaching 60,000 ng/L levels in the influent. Antidepressants were predominant in Sweden, Switzerland, and the United States. © 2019
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    Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus
    Brown, Connor L.; Garner, Emily; Jospin, Guillaume; Coil, David A.; Schwake, David Otto; Eisen, Jonathan A.; Mukhopadhyay, Biswarup; Pruden, Amy (PLoS, 2020-01-01)
    Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1–3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence.