VTechWorks Repository :: Browsing by Author "Pruden, Amy"

Browsing by Author "Pruden, Amy"

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Abundance of Antibiotic Resistance Genes in Feces Following Prophylactic and Therapeutic Intramammary Antibiotic Infusion in Dairy Cattle
Willing, Brittany Faith (Virginia Tech, 2013-12-04)
Prophylactic and therapeutic antibiotic treatments have the potential to increase excretion of antibiotic resistance genes (ARGs) by dairy cattle through selection pressure on the gut microbiome. The objective of these studies was to evaluate the effect of cephapirin benzathine administered prophylactically at the end of lactation and pirlimycin hydrochloride administered therapeutically during a clinical mastitis infection on the abundance and relative abundance of ARGs in dairy cow feces. For prophylactic treatment using cephapirin benzathine, nineteen end-of-lactation cows were used. Treatment cows (n = 9) received cephapirin benzathine as an intramammary infusion prior to dry-off, and control cows (n =10) received no antibiotics. All cows received an internal non-antibiotic teat sealant. Fecal grab samples were collected for each cow on d -2 (baseline, used as covariate), d 1, 3, 5, 7, and once per week until d 49. Fecal samples were collected in sterile containers, then freeze-dried and subject to DNA extraction. The abundance of ampC, blaCMY-2, ermB, sul1, tetO, tetW, integrase-specific gene int1, and 16S rRNA were quantified using quantitative polymerase chain reaction (qPCR). The genes ampC and blaCMY-2 encode resistance to ß-lactam antibiotics, ermB to macrolides, sul1 to sulfonamides, tetO and tetW to tetracyclines, and int1 a class-1 integrase gene that facilitates horizontal transfer of ARGs across bacteria. The 16S rRNA gene was used as a representation of bacterial population. Absolute abundance was defined as number of ARG copies per gram of freeze-dried feces, while relative abundance was defined as ARG copy numbers per copy of 16S rRNA gene, which is indicative of the proportion of bacteria carrying ARGs. Non-normal data were logarithmically transformed and were statistically analyzed using PROC GLIMMIX in SAS 9.2. Abundance and relative abundance of sul1 and blaCMY-2 were below the limit of quantification in most samples and therefore not suitable for statistical comparisons. The int1 gene was not detectable in any sample. There were significant interactions between treatment and day for the abundance and relative abundance of ampC, tetO, and tetW. The abundance and relative abundance of ampC increased with time in control cows while remaining constant in antibiotic treated cows through the dry period. Antibiotics may act to stabilize the gut microbiome in response to diet and housing changes. There was a significant main effect of treatment for ermB with a significantly greater proportion of bacteria carrying ermB in control cows when compared to antibiotic treated cows. The tetracycline resistance genes tetO and tetW behaved similarly with a significant treatment by day interaction for the abundance and relative abundance of both genes. The relative abundance of both tetO and tetW were greater in control cows when compared to antibiotic treated cows on days 3, 5, 7, and 14. The abundance of both tetO and tetW resistance genes increased in antibiotic treated cows from day 1 to 49. There was also a significant increase in tetW relative abundance when comparing day 1 to 49. Administering long-acting antibiotics as intramammary dry treatment changed fecal bacteria composition during the dry period perhaps by stabilizing GI bacteria through dietary and housing changes. However, the use of prophylactic dry cow treatment does not uniformly or predictably lead to changes in fecal ARGs. In a second study, after clinical mastitis detection and identification, 6 lactating dairy cows received therapeutic mastitis treatment (pirlimycin hydrochloride as an intramammary infusion). Fecal grab samples were collected from each cow on d 0, 3, 9, and 12. Collection and analytical methods were as previously described. Abundance and relative abundance of sul1 and blaCMY-2 were again below the limit of quantification and therefore not suitable for statistical comparison. The int1 gene was not detected in any sample. The abundance of 16S rRNA genes decreased with day and relative abundance ermB, tetO, and tetW increased with day. There was no significant effect of day on the relative abundance of ampC or the abundance of ampC, ermB, tetO, and tetW in feces of cows with clinical mastitis. Administering fast-acting antibiotics as therapeutic intramammary mastitis treatment to dairy cows increased the relative abundance (gene copies per 16S rRNA) of selected ARGs but not the total abundance of ARGs in feces. The use of antibiotics for prevention and treatment of bacterial infections does not uniformly or predictably increase ARGs.
Adherence and Biofilm Formation of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus in household plumbing
Mullis, Summer (Virginia Tech, 2012-09-05)
Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and found in drinking water distribution systems and household plumbing. They are opportunistic pathogens of humans, causing lung disease. Their ability to adhere and form biofilm is attributed to a waxy, lipid-rich, cell envelope. This highly hydrophobic envelope also contributes to the characteristic antibiotic-, chlorine-, and disinfectant- resistance of NTM. NTM in household plumbing reside primarily in biofilms and the ability to form biofilm has been linked to virulence. Shedding of cells from biofilm and the subsequent aerosolization of microorganisms through showerheads presents a significant public health risk, particularly to those individuals with associated risk factors. Three species of NTM, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus, were examined for adherence and biofilm formation on surfaces common to household plumbing systems, including glass, copper, stainless steel, polyvinyl chloride, and galvanized steel. All experiments were conducted with sterile, Blacksburg tap water in a CDC Biofilm Reactor. Highest adherence was observed by M. avium on galvanized steel surfaces, reaching 15,100 CFU/cm2 surface at 6 hours incubation at room temperature. After 3 weeks incubation at room temperature, biofilm formation of M. avium was also highest on galvanized steel surfaces, reaching 14,000,000 CFU/cm2 surface. Lowest adherence was observed by M. abscessus on polyvinyl chloride (PVC) surfaces, reaching 40 CFU/cm2. Lowest biofilm formation was observed by M. intracellulare on glass surfaces, reaching 5,900 CFU/cm2. Surfaces, such as galvanized (zinc), on which high adherence and biofilm formation was observed, should be avoided in household plumbing systems of NTM patients and individuals at risk for developing NTM disease. Additionally, surfaces such as copper, harbor fewer NTM and may provide a safer alternative for household plumbing of NTM patients.
Advanced Applications of Raman Spectroscopy for Environmental Analyses
Lahr, Rebecca Halvorson (Virginia Tech, 2014-01-09)
Due to an ever-increasing global population and limited resource availability, there is a constant need for detection of both natural and anthropogenic hazards in water, air, food, and material goods. Traditionally a different instrument would be used to detect each class of contaminant, often after a concentration or separation protocol to extract the analyte from its matrix. Raman spectroscopy is unique in its ability to detect organic or inorganic, airborne or waterborne, and embedded or adsorbed analytes within environmental systems. This ability comes from the inherent abilities of the Raman spectrometer combined with concentration, separation, and signal enhancement provided by drop coating deposition Raman (DCDR) and surface-enhanced Raman spectroscopy (SERS). Herein the capacity of DCDR to differentiate between cyanotoxin variants in aqueous solutions was demonstrated using principal component analysis (PCA) to statistically demonstrate spectral differentiation. A set of rules was outlined based on Raman peak ratios to allow an inexperienced user to determine the toxin variant identity from its Raman spectrum. DCDR was also employed for microcystin-LR (MC-LR) detection in environmental waters at environmentally relevant concentrations, after pre-concentration with solid-phase extraction (SPE). In a cellulose matrix, SERS and normal Raman spectral imaging revealed nanoparticle transport and deposition patterns, illustrating that nanoparticle surface coating dictated the observed transport properties. Both SERS spectral imaging and insight into analyte transport in wax-printed paper microfluidic channels will ultimately be useful for microfluidic paper-based analytical device (𝜇PAD) development. Within algal cells, SERS produced 3D cellular images in the presence of intracellularly biosynthesized gold nanoparticles (AuNP), documenting in detail the molecular vibrations of biomolecules at the AuNP surfaces. Molecules involved in nanoparticle biosynthesis were identified at AuNP surfaces within algal cells, thus aiding in mechanism elucidation. The capabilities of Raman spectroscopy are endless, especially in light of SERS tag design, coordinating detection of analytes that do not inherently produce strong Raman vibrations. The increase in portable Raman spectrometer availability will only facilitate cheaper, more frequent application of Raman spectrometry both in the field and the lab. The tremendous detection power of the Raman spectrometer cannot be ignored.
Advanced Biofilm and Aerobic Granulation Technologies for Water and Wastewater Treatment
Sun, Yewei (Virginia Tech, 2020-04-10)
Attached growth biological processes offer advantages over traditional water purification technologies through high biomass retention, easy sludge-water separation, multiple multispecies synergies in proximity, resilience to shock loading, low space requirements, and reactor operational flexibility. Traditionally, attached growth refers to biofilms that require abiotic carrying media for bacteria to attach and grow on. While biofilms have been broadly applied in wastewater treatment, its potential for potable reuse or stormwater treatment has not been well studied. The treatment trains of pre-ozonation followed by biologically active filtration (ozone- BAF) is an advanced biofilm technology for potable reuse that can generate high-quality potable water at reduced energy and chemical demands by removing pollutant through three different pathways: oxidation, adsorption, and biodegradation. However, these pathways can result in both desirable and undesirable effects, and the mechanism behind it is still unclear. To understand the mechanisms of various pollutant removal, parallel performance comparisons of ozone-BAF treatment trains with spent and regenerated granular activated carbon (GAC), along with a range of pre-oxidant ozone doses were performed. Another common issue of BAF is the headloss buildup during its operation, which has become a significant energy and maintenance burden at many utilities. Thus, a mathematical model was developed to predict BAF headloss buildup in response to organic removal and nitrification. For stormwater treatment, the feasibility of using biofilms for stormwater biological nitrogen removal (BNR) is still largely unknown, as very limited research effort has been dedicated to this aspect. Thus, a mathematical model was developed to evaluate the potential of using BNR techniques for stormwater nitrogen removal. Aerobic granules are an even more advanced attached growth process, which eliminates the need for abiotic carrying media. So far, aerobic granular sludge is only formed in sequential batch reactors but not in a continuous flow system. Therefore, continuous flow aerobic granulation from traditional activated sludge was investigated and, for the first time, successfully achieved in continuous flow plug-flow bioreactors fed with real municipal wastewater. Besides, the role and critical value of an essential operational parameter, feast/famine ratio, for continuous flow aerobic granulation were determined.
Advancing Monitoring and Mitigation of Antibiotic Resistance in Wastewater Treatment Plants and Water Reuse Systems
Majeed, Haniyyah JaRae (Virginia Tech, 2020-10-22)
Wastewater treatment plants (WWTPs) receive a confluence of sewage containing antibiotics, antibiotic resistant bacteria, antibiotic resistance genes (ARGs), and pathogens, thus serving as key point of interest for the surveillance of antibiotic resistance (AR) dissemination. This thesis advances knowledge about the fate of AR indicators throughout treatment and reuse. The field study informs approaches for monitoring AR at a WWTP by characterizing the resistome (i.e., full profile of ARGs) and microbiome across eight sampling events via metagenomic sequencing, complemented by antibiotic data. The WWTP significantly reduced the total load of ARGs and antibiotics, although correlations between ARGs and antibiotics were generally weak. Quantitative polymerase chain reaction was applied to validate the quantitative capacity of metagenomics, whereby we found strong correlations. The influent and effluent to the WWTP were remarkably stable with time, providing further insight into the sampling frequency necessary for adequate surveillance. The laboratory study examined the effects of commonly applied disinfection processes (chlorination, chloramination, and ultraviolet irradiation [UV]) on the inactivation of antibiotic resistant pathogens and corresponding susceptible pathogens in recycled and potable water. Further, we evaluated their regrowth following disinfection by simulating distribution. Acinetobacter baumannii, an environmental opportunistic pathogen, regrew especially well following UV disinfection, although not when a disinfectant residual was present. Enterococcus faecium, a fecal pathogen, did not regrow following any disinfection process. There were no significant differences between water types. The findings of this study emphasize a need to move beyond the framework of assessing treatment efficacy based on the attenuation of fecal pathogens.
Aerosolization of Ebola Virus Surrogates in Wastewater Systems
Lin, Kaisen (Virginia Tech, 2016-09-26)
Recent studies have shown that Ebola virus can persist in wastewater, and the potential for the virus to be aerosolized and pose a risk of inhalation exposure has not been evaluated. We considered this risk for three wastewater systems: toilets, a lab-scale model of an aeration basin, and a lab-scale model of converging sewer pipes. We measured the aerosol size distribution generated by each system, spiked Ebola virus surrogates into each system, and determined the emission rate of viruses into the air. While the number of aerosols released ranged from 105 to 107 per flush from the toilets or per minute from the lab-scale models, the total volume of aerosols generated by these systems was ~10-8 to 10-7 mL per flush or per minute in all cases. The Ebola virus surrogates MS2 and Phi6, spiked into toilets at an initial concentration of 107 PFU mL-1, were not detected in air after flushing. Airborne concentrations of MS2 and Phi6 were ~20 PFU L-1 and ~0.1 PFU L-1, respectively, associated with the aeration basin and sewer models. This corresponds to emission rates of 547 PFU min-1 and 3.8 PFU min-1 of MS2 and Phi6, respectively, for the aeration basin and 79 PFU min-1 and 0.3 PFU min-1 for the sewer model. Since information on the aerosolization of Ebola virus is quite limited, these emission rates can greatly help inform risk assessment of inhalation exposure to Ebola virus.
AgroSeek: a system for computational analysis of environmental metagenomic data and associated metadata
Liang, Xiao; Akers, Kyle; Keenum, Ishi M.; Wind, Lauren L.; Gupta, Suraj; Chen, Chaoqi; Aldaihani, Reem; Pruden, Amy; Zhang, Liqing; Knowlton, Katharine F.; Xia, Kang; Heath, Lenwood S. (2021-03-10)
Background Metagenomics is gaining attention as a powerful tool for identifying how agricultural management practices influence human and animal health, especially in terms of potential to contribute to the spread of antibiotic resistance. However, the ability to compare the distribution and prevalence of antibiotic resistance genes (ARGs) across multiple studies and environments is currently impossible without a complete re-analysis of published datasets. This challenge must be addressed for metagenomics to realize its potential for helping guide effective policy and practice measures relevant to agricultural ecosystems, for example, identifying critical control points for mitigating the spread of antibiotic resistance. Results Here we introduce AgroSeek, a centralized web-based system that provides computational tools for analysis and comparison of metagenomic data sets tailored specifically to researchers and other users in the agricultural sector interested in tracking and mitigating the spread of ARGs. AgroSeek draws from rich, user-provided metagenomic data and metadata to facilitate analysis, comparison, and prediction in a user-friendly fashion. Further, AgroSeek draws from publicly-contributed data sets to provide a point of comparison and context for data analysis. To incorporate metadata into our analysis and comparison procedures, we provide flexible metadata templates, including user-customized metadata attributes to facilitate data sharing, while maintaining the metadata in a comparable fashion for the broader user community and to support large-scale comparative and predictive analysis. Conclusion AgroSeek provides an easy-to-use tool for environmental metagenomic analysis and comparison, based on both gene annotations and associated metadata, with this initial demonstration focusing on control of antibiotic resistance in agricultural ecosystems. Agroseek creates a space for metagenomic data sharing and collaboration to assist policy makers, stakeholders, and the public in decision-making. AgroSeek is publicly-available at https://agroseek.cs.vt.edu/ .
Airborne Transmission of Influenza a Virus in Indoor Environments
Yang, Wan (Virginia Tech, 2012-03-30)
Despite formidable advances in virology and medicine in recent decades, we know remarkably little about the dynamics of the influenza virus in the environment during transmission between hosts. There is still controversy over the relative importance of various transmission routes, and the seasonality of influenza remains unexplained. This work focuses on developing new knowledge about influenza transmission via the airborne route and the virus' inter-host dynamics in droplets and aerosols. We measured airborne concentrations of influenza A viruses (IAVs) and size distributions of their carrier aerosols in a health center, a daycare center, and airplanes. Results indicate that the majority of viruses are associated with aerosols smaller than 2.5 µm and that concentrations are sufficient to induce infection. We further modeled the fate and transport of IAV-laden droplets expelled from a cough into a room, as a function of relative humidity (RH) and droplet size. The model shows that airborne concentrations of infectious IAV vary with RH through its influence on virus inactivation and droplet size, which shrinks due to evaporation. IAVs associated with large droplets are removed mostly by settling, while those associated with aerosols smaller than 5 µm are removed mainly by ventilation and inactivation. To investigate the relationship between RH and influenza transmission further, we measured the viability of IAV in droplets at varying RHs. Results suggest that there exist three regimes defined by RH: physiological conditions (~100% RH) with high viability, concentrated conditions (~50% to ~99% RH) with lower viability, and dry conditions (<~50% RH) with high viability. A droplet's extent of evaporation, which is determined by RH, affects solute concentrations in the droplet, and these appear to influence viability. This research considerably advances the current understanding of the dynamics of the influenza virus while it is airborne and provides an explanation for influenza's seasonality. Increased influenza activity in winter in temperate regions could be due to greater potential for IAV carrier aerosols to remain airborne and higher viability of IAV at low RH. In tropical regions, transmission could be enhanced due to better survival of IAV at extremely high RH.
Antibiotic Resistance Characterization in Human Fecal and Environmental Resistomes using Metagenomics and Machine Learning
Gupta, Suraj (Virginia Tech, 2021-11-03)
Antibiotic resistance is a global threat that can severely imperil public health. To curb the spread of antibiotic resistance, it is imperative that efforts commensurate with a “One Health” approach are undertaken. Given that interconnectivities among ecosystems can serve as conduits for the proliferation and dissemination of antibiotic resistance, it is increasingly being recognized that a robust global environmental surveillance framework is required to promote One Health. The ideal aim would be to develop approaches that inform global distribution of antibiotic resistance, help prioritize monitoring targets, present robust data analysis frameworks to profile resistance, and ultimately help build strategies to curb the dissemination of antibiotic resistance. The work described in this dissertation was aimed at evaluating and developing different data analysis paradigms and their applications in investigating and characterizing antibiotic resistance across different resistomes. The applications presented in Chapter 2 illustrate challenges associated with various environmental data types (especially metagenomics data) and present a path to advance incorporation of data analytics approaches in Environmental Science and Engineering research and applications. Chapter 3 presents a novel approach, ExtrARG, that identifies discriminatory ARGs among resistomes based on factors of interest. The results in Chapter 4 provide insight into the global distribution of ARGs across human fecal and sewage resistomes across different socioeconomics. Chapter 5 demonstrates a data analysis paradigm using machine learning algorithms that helps bridge the gap between information obtained via culturing and metagenomic sequencing. Lastly, the results of Chapter 6 illustrates the contribution of phages to antibiotic resistance. Overall, the findings provide guidance and approaches for profiling antibiotic resistance using metagenomics and machine learning. The results reported further expand the knowledge on the distribution of antibiotic resistance across different resistomes.
Antibiotic resistance gene abundance in feces of calves fed pirlimycin-dosed whole milk
Littier, Heather Melissa (Virginia Tech, 2015-08-31)
Exposure to antibiotics has the potential to increase the incidence and proliferation of antibiotic resistance genes (ARG) in the gut and fecal microbiome. Non-saleable, antibiotic-containing milk from cows treated with antibiotics (waste milk) is commonly fed to dairy calves but the effects of ingestion of antibiotics at an early age on the gut microbiome and the development of ARG in the naive gut are not well understood. Pirlimycin, a lincosamide antibiotic acting against Gram positive bacteria through inhibiting protein synthesis by binding to the 50S ribosome, is commonly used as mastitis therapy. Lincosamides are also considered highly important in human medicine, often used against Staphylococcus aureus and Clostridium difficile infections. Emerging microbial resistance to pirlimycin is of concern for both animal and human health. The objective of this study was to determine the effect of early lincosamide antibiotic exposure on the abundance of ARG in feces of milk-fed calves. Eight female Holstein calves were blocked by age, paired by block, and randomly assigned to pasteurized whole milk (control; n = 4) or milk containing 0.2 mg/L of pirlimycin (treatment; n = 4). Calves were enrolled after receiving two colostrum feedings and were fed 5.68 L of pasteurized whole milk, treatment, or control, divided into two daily feedings, from d 1 to d 50 of age. After weaning calves were fed non-medicated starter grain ad libitum. Fecal samples were collected weekly until 85 d of age and freeze-dried. DNA was extracted using QiaAmp® Fast DNA Stool Mini Kit and qPCR was used to quantify the absolute abundance (gene copies/g of wet feces) and relative abundance (gene copies/copies of 16S rRNA genes) of erm(B), tet(O), tet(W) and 16S rRNA genes. Data was analyzed using PROC GLIMMIX in SAS. Abundance of 16S rRNA genes, tet(O) and tet(W) were not different between control and pirlimycin-fed calves nor were the relative abundance of tet(O) (mean = 0.050 tet(O) copies/16S rRNA genes) or tet(W) (0.561 tet(W) copies/16S rRNA genes). While abundance of erm(B) was higher in pirlimycin-fed calves compared to control calves (6.46 and 6.04 log gene copies/g wet feces; P = 0.04) the relative abundance of erm(B) (0.273 gene copies/16S rRNA genes) in feces of calves was not influenced by treatment. There was an effect of day (P < 0.10) for absolute abundance of tet(O), tet(W), and erm(B) indicating that the levels change with time as the fecal microbiome develops. This study suggests that feeding pirlimycin-containing non-saleable milk to growing calves may increase environmental loading of erm(B), which codes for resistance to highly important macrolide and lincosamide antibiotics. Additional research is needed on effects of feeding waste milk to calves on other fecal ARG and on the post-excretion and post-application fate of these genes.
Antibiotics in Agroecosystems: Introduction to the Special Section
Franklin, Alison M.; Aga, Diana S.; Cytryn, Eddie; Durso, Lisa M.; McLain, Jean E.; Pruden, Amy; Roberts, Marilyn C.; Rothrock, Michael J. Jr.; Snow, Daniel D.; Watson, John E.; Dungan, Robert S. (2016-03)
The presence of antibiotic drug residues, antibiotic resistant bacteria, and antibiotic resistance genes in agroecosystems has become a significant area of research in recent years and is a growing public health concern. While antibiotics are used in both human medicine and agricultural practices, the majority of their use occurs in animal production where historically they have been used for growth promotion, in addition to the prevention and treatment of disease. The widespread use of antibiotics and the application of animal wastes to agricultural lands play major roles in the introduction of antibiotic-related contamination into the environment. Overt toxicity in organisms directly exposed to antibiotics in agroecosystems is typically not a major concern because environmental concentrations are generally lower than therapeutic doses. However, the impacts of introducing antibiotic contaminants into the environment are unknown, and concerns have been raised about the health of humans, animals, and ecosystems. Despite increased research focused on the occurrence and fate of antibiotics and antibiotic resistance over the past decade, standard methods and practices for analyzing environmental samples are limited and future research needs are becoming evident. To highlight and address these issues in detail, this special collection of papers was developed with a framework of five core review papers that address the (i) overall state of science of antibiotics and antibiotic resistance in agroecosystems using a causal model, (ii) chemical analysis of antibiotics found in the environment, (iii) need for background and baseline data for studies of antibiotic resistance in agroecosystems with a decision-making tool to assist in designing research studies, as well as (iv) culture- and (v) molecular-based methods for analyzing antibiotic resistance in the environment. With a focus on the core review papers, this introduction summarizes the current state of science for analyzing antibiotics and antibiotic resistance in agroecosystems, discusses current knowledge gaps, and develops future research priorities. This introduction also contains a glossary of terms used in the core reivew papers of this special section. The purpose of the glossary is to provide a common terminology that clearly characterizes the concepts shared throughout the narratives of each review paper.
Antimicrobial resistance in soil: long-term effects on microbial communities, interactions with soil properties, and transport of antimicrobial elements
Shawver, Sarah Elizabeth (Virginia Tech, 2022-06-08)
Since penicillin was discovered in 1928, antibiotic usage in human and veterinary medicine and prevalence of antibiotic resistant bacteria (ARB), has been increasing. While antibiotics and antibiotic resistance genes (ARGs) naturally occur in soils, increasing abundances of ARGs correlate with increased antibiotic usage in agricultural settings. When livestock are treated with antibiotics, the antibiotic compounds, ARB, and ARGs can enter soil via manure excreted onto pastures or applied to other fields as fertilizer, thereby spreading antimicrobial resistance (AMR) in the environment. In addition to human health implications, increased AMR has negative impacts on ecosystem services such as carbon and nitrogen cycling. While many studies have researched antibiotic persistence in agricultural systems and their impacts on soil microbial communities, there are still significant knowledge gaps around the long-term effects of antibiotic exposure in soils, how those impacts differ among soils, and how elements of AMR may differentially transport through soil. To address these knowledge gaps, our objectives were to 1) examine the impact of multi-year repeated additions of manure from cattle administered antibiotics on soil microbial communities, 2) determine the interactive effects of soil moisture and type on soil microbial communities exposed to antibiotics and manure, and 3) differentiate between vertical transport of AMR in the form of viable ARB or ARGs in extracellular plasmids. Our results demonstrate that soil bacterial community structures were consistently altered by 3-year additions of manure from cattle administered antibiotics compared to soil amended with antibiotic-free manure. Furthermore, ARG abundances were higher in soils with manure additions compared to soil without manure, although this was true regardless of whether the cattle were administered antibiotics, suggesting that manure and antibiotic impacts on soil microbial communities can persist over multi-year of repeated manure applications. Additionally, in microcosms, effects of manure from cattle administered antibiotics on ARG abundances, microbial community structures, respiration, and nitrogen pools in soil were seen across multiple soil types and moisture contents, suggesting environmental conditions can alter how manure and antibiotics impact microbial community structure and nutrient cycling. Finally, ARB flowed readily through saturated soil, but were also detectable in the top 5 cm of soil columns. However, ARGs on extracellular plasmids did not flow through soil columns and were not detected in soil, indicating that extracellular DNA does not persist or transport through the soil to any meaningful degree. Overall, these results indicate a nuanced approach is required to mitigate the environmental spread of AMR. Soil management strategies for addressing the AMR crisis should consider the broader context of manure management, as high ARG abundances can come from application of manure from antibiotic-free cattle, and soil microbial communities in individual environments may have varied responses to manure antibiotic exposure. Furthermore, the transport of AMR through soil is complex and dynamic, as elements of AMR may transport differently through soil and require separate consideration in modeling and management. Future AMR management practices that consider diverse factors that affect persistence and spread of AMR in the environment can help protect livestock productivity and maintain the efficacy of antibiotics to protect human and animal health.
Antimicrobial Resistance Mitigation [ARM] Concept Paper
Vikesland, Peter J.; Alexander, Kathleen A.; Badgley, Brian D.; Krometis, Leigh-Anne H.; Knowlton, Katharine F.; Gohlke, Julia M.; Hall, Ralph P.; Hawley, Dana M.; Heath, Lenwood S.; Hession, W. Cully; Hull, Robert Bruce IV; Moeltner, Klaus; Ponder, Monica A.; Pruden, Amy; Schoenholtz, Stephen H.; Wu, Xiaowei; Xia, Kang; Zhang, Liqing (Virginia Tech, 2017-05-15)
The development of viable solutions to the global threat of antimicrobial resistance requires a transdisciplinary approach that simultaneously considers the clinical, biological, social, economic, and environmental drivers responsible for this emerging threat. The vision of the Antimicrobial Resistance Mitigation (ARM) group is to build upon and leverage the present strengths of Virginia Tech in ARM research and education using a multifaceted systems approach. Such a framework will empower our group to recognize the interconnectedness and interdependent nature of this threat and enable the delineation, development, and testing of resilient approaches for its mitigation. We seek to develop innovative and sustainable approaches that radically advance detection, characterization, and prevention of antimicrobial resistance emergence and dissemination in human-dominated and natural settings...
Antimicrobial Resistance Monitoring of Water Environments: A Framework for Standardized Methods and Quality Control
Liguori, Krista; Keenum, Ishi M.; Davis, Benjamin C.; Calarco, Jeanette; Milligan, Erin; Harwood, Valerie J.; Pruden, Amy (American Chemical Society, 2022-06)
Antimicrobial resistance (AMR) is a grand societal challenge with important dimensions in the water environment that contribute to its evolution and spread. Environmental monitoring could provide vital information for mitigating the spread of AMR; this includes assessing antibiotic resistance genes (ARGs) circulating among human populations, identifying key hotspots for evolution and dissemination of resistance, informing epidemiological and human health risk assessment models, and quantifying removal efficiencies by domestic wastewater infrastructure. However, standardized methods for monitoring AMR in the water environment will be vital to producing the comparable data sets needed to address such questions. Here we sought to establish scientific consensus on a framework for such standardization, evaluating the state of the science and practice of AMR monitoring of wastewater, recycled water, and surface water, through a literature review, survey, and workshop leveraging the expertise of academic, governmental, consulting, and water utility professionals.
Antimicrobial Resistance: What is it and What's at Stake?
Maurer, Ellen; Vallotton, Amber; Pruden, Amy (Virginia Cooperative Extension, 2017-10-25)
Examines the issue of bacterial resistance to antimicrobial drugs, and the implications for human health.
ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization
Liang, Xiao; Zhang, Jingyi; Kim, Yoonjin; Ho, Josh; Liu, Kevin; Keenum, Ishi M.; Gupta, Suraj; Davis, Benjamin; Hepp, Shannon L.; Zhang, Liqing; Xia, Kang; Knowlton, Katharine F.; Liao, Jingqiu; Vikesland, Peter J.; Pruden, Amy; Heath, Lenwood S. (Frontiers, 2023-09-15)
Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem.
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.
Balancing Bromate Formation, Organics Oxidation, and Pathogen Inactivation: The Impact of Bromate Suppression Techniques on Ozonation System Performance in Reuse Waters
Buehlmann, Peter Hamilton (Virginia Tech, 2019-09-10)
Ozonation is an integral process in ozone-biofiltration treatment systems and is beginning to be widely adopted worldwide for water reuse applications. Ozone is effective for pathogenic inactivation and organics oxidation: both increasing assimilable organic carbon for biofiltration and eliminating trace organic contaminants which may pose a threat to human health. However, ozone can also form disinfection byproducts such as bromate from the oxidation of naturally occurring anion bromide. Bromate is a known human carcinogen and is regulated by the EU, WHO, and USEPA to a maximum limit of 10µg/L. In waters high in bromide, especially above 100µg/L, bromate formation becomes a major concern. In the secondary wastewater effluent studied, bromide concentration may exceed 500µg/L. Several bromate suppression techniques have been devised in previous work, including free ammonia addition, monochloramination, and the chlorine-ammonia process. While free ammonia addition was not found to adequately reduce bromate formation below the required MCL, monochloramine addition and the chlorine-ammonia process were found to be effective. However, the impact of these chemical suppression techniques on organics oxidation and disinfection has not been fully studied. This study explored the impact of these bromate suppression techniques at a wide range of ozone doses on bromate formation, pathogenic inactivation, ozone-refractory organics oxidation through the surrogate 1,4-dioxane, and N-nitrosodimethylamine (NDMA) formation. Additionally, bromate suppression mechanisms of monochloramine were explored further through a variety of different water quality parameters, such as through hydroxyl radical exposure and ultraviolet absorption spectrum measurements, which were correlated and utilized to develop a hydroxyl radical exposure predictive model.
Bioelectrochemical Systems: Microbiology, Catalysts, Processes and Applications
Yuan, Heyang (Virginia Tech, 2017-11-01)
The treatment of water and wastewater is energy intensive, and there is an urgent need to develop new approaches to address the water-energy challenges. Bioelectrochemical systems (BES) are energy-efficient technologies that can treat wastewater and simultaneously achieve multiple functions such as energy generation, hydrogen production and/or desalination. The objectives of this dissertation are to understand the fundamental microbiology of BES, develop cost-effective cathode catalysts, optimize the process engineering and identify the application niches. It has been shown in Chapter 2 that electrochemically active bacteria can take advantage of shuttle-mediated EET and create optimal anode salinities for their dominance. A novel statistical model has been developed based on the taxonomic data to understand and predict functional dynamics and current production. In Chapter 3, 4 and 5, three cathode catalyst (i.e., N- and S- co-doped porous carbon nanosheets, N-doped bamboo-like CNTs and MoS2 coated on CNTs) have been synthesized and showed effective catalysis of oxygen reduction reaction or hydrogen evolution reaction in BES. Chapter 6, 7 and 8 have demonstrated how BES can be combined with forward osmosis to enhance desalination or achieve self-powered hydrogen production. Mathematical models have been developed to predict the performance of the integrated systems. In Chapter 9, BES have been used as a research platform to understand the fate and removal of antibiotic resistant genes under anaerobic conditions. The studies in this dissertation have collectively demonstrated that BES may hold great promise for energy-efficient water and wastewater treatment.
Characterization of opportunistic Pathogens in Drinking Water Supplied by Private Wells
Mapili, Kristine Irene Manzano (Virginia Tech, 2019-07-03)
Private wells are understudied potential sources of opportunistic pathogen (OP) infections. OPs, including Legionella and Mycobacterium, are of particular concern for immunocompromised individuals and are known to proliferate in drinking water systems. Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems, which primarily use surface water sources and are always treated with primary and secondary disinfection in United States. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems will translate to private well systems with rare and infrequent exposure to chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well field surveys were conducted to document the incidence of OPs in private well systems. One survey conducted in North Carolina private wells with no history of recent flooding was focused on molecular and culture-based detection of Legionella spp. and Legionella pneumophila. The other survey was a broader molecular (i.e., DNA-based) characterization of the incidence of Legionella spp., L. pneumophila, Mycobacterium spp., Mycobacterium avium (the most commonly nontuberculous mycobacteria associated with disease), and Naegleria fowleri in private wells with recent history of flooding (i.e., Hurricanes Harvey and Irma in 2017, or the Great Louisiana Floods in 2016, extending to Texas, Florida, and Louisiana). All samples in both studies were analyzed for total bacterial 16S rRNA genes, indicator bacteria (e.g., total coliform and Escherichia coli) and inorganic constituents. Information about well system characteristics were obtained through questionnaires sent to participating residents. Widespread detection of OP DNA markers were noted in the flooded well survey. Legionella spp. (detectable in 50-100% of well waters, depending on the flood event) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly detected among the OPs targets. At the genus level, L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) were less commonly detected, but still highly variable. It is not possible to judge whether these OP levels were elevated as a result of the flooding because the sampling was carried out as an emergency response and background levels were not previously tested. Also of interest was whether well characteristics could predict OP levels, including well depth, well type, or treatment. However, none of these emerged as significant predictors of OP detection frequency or levels. Similarly, these OP DNA markers were not elevated in homes reporting submerged wellheads or system damage, suggesting that detection of these OPs is more dependent on the groundwater that supplies these private wells than influx of contaminated surface water. The incidence of DNA markers pertaining to N. fowleri, the "brain eating amoeba" that causes rare incidences of primary amebic meningoencephalitis (PAM), tended to be lower (5.0-12.7%) than that of other OPs targeted, but was more frequently detected in wells reporting submerged well heads, suggesting its occurrence was related to contamination from flood water. A positive association between total bacteria and occurrence of both Legionella spp. and Mycobacterium spp., was observed in private wells of all surveyed areas, contrary to observations in municipal drinking water systems with secondary disinfectant residuals. On the other hand, Legionella reportedly has an optimal growth range of 20-42 °C in municipal systems and recent surveys of municipal systems reported a strong association between Legionella spp. and temperature that was not observed for private wells in this study. We speculate that the essentially "infinite" water age and lack of disinfectant for well water, may contribute to these differences relative to municipal water supplies. The results presented in this work are likely an overestimation of OPs numbers in private wells, as molecular detection of OPs does not distinguish between live and dead cells. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables impacting the occurrence of OPs in private wells, given that our study shows that these pathogens are relatively common, might someday help limit the risk of infections.

Browsing by Author "Pruden, Amy"

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