Pilot-scale assessment reveals effects of anode type and orthophosphate in governing antimicrobial capacity of copper for Legionella pneumophila control.

Copper (Cu) is sometimes applied as an antimicrobial for controlling Legionella in hot water plumbing systems, but its efficacy is inconsistent. Here we examined the effects of Cu (0 - 2 mg/L), orthophosphate corrosion inhibitor (0 or 3 mg/L as phosphate), and water heater anodes (aluminum, magnesium, and powered anodes) on both bulk water and biofilm-associated L. pneumophila in pilot-scale water heater systems. Soluble, but not total, Cu was a good predictor of antimicrobial capacity of Cu. Even after months of exposure to very high Cu levels (>1.2 mg/L) and low pH (<7), which increases solubility and enhances bioavailability of Cu, culturable L. pneumophila was only reduced by ∼1-log. Cu antimicrobial capacity was shown to be limited by various factors, including binding of Cu ions by aluminum hydroxide precipitates released from corrosion of aluminum anodes, higher pH due to magnesium anode corrosion, and high Cu tolerance of the outbreak-associated L. pneumophila strain that was inoculated into the systems. L. pneumophila numbers were also higher in several instances when Cu was dosed together with orthophosphate (e.g., with an Al anode), revealing at least one scenario where high levels of total Cu appeared to stimulate Legionella. The controlled, pilot-scale nature of this study provides new understanding of the limitations of Cu as an antimicrobial in real-world plumbing systems.

Towards risk assessment for antibiotic resistant pathogens in recycled water: a systematic review and summary of research needs.

Risk assessment is critical for identifying target concentrations of antibiotic resistant pathogens necessary for mitigating potential harmful exposures associated with water reuse. However, there is currently limited available data characterizing the concentrations of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in recycled water to support robust efforts at risk assessment. The objective of this systematic review was to identify and synthesize the existing literature documenting the presence and abundance of ARB and ARGs in recycled water. In addition, this review identifies best practices and explores monitoring targets for studying ARB and ARGs in recycled water to guide future work and identifies key research needs aimed at better supporting quantitative microbial risk assessment focused on recycled water and antibiotic resistance. Future efforts to collect data about ARB and ARG prevalence in recycled water should report concentration data per unit volume. Sample metadata should also be provided, including a description of treatment approach, a description of planned water uses (e.g., potable, irrigation), methods for conveyance to the point of use, and available physicochemical water quality data. Additional research is needed aimed at identifying recommended ARB and ARG monitoring targets and for developing approaches to incorporate metagenomic data into risk assessment.

Integrated Metagenomic Assessment of Multiple Pre-harvest Control Points on Lettuce Resistomes at Field-Scale.

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.

Source-to-Tap Assessment of Microbiological Water Quality in Small Rural Drinking Water Systems in Puerto Rico Six Months After Hurricane Maria.

Maria made a landfall in Puerto Rico on September 20, 2017 as a category 4 hurricane, causing severe flooding, widespread electricity outages, damage to infrastructure, and interruptions in water and wastewater treatment. Small rural community water systems face unique challenges in providing drinking water, which intensify after natural disasters. The purpose of this study was to evaluate the functionality of six very small rural public water systems and one large regulated system in Puerto Rico six months after Maria and survey a broad sweep of fecal, zoonotic, and opportunistic pathogens from the source to tap. Samples were collected from surface and groundwater sources, after water treatment and after distribution to households. Genes indicative of pathogenic Leptospira spp. were detected by polymerase chain reaction (PCR) in all systems reliant on surface water sources. Salmonella spp. was detected in surface and groundwater sources and some distribution system water both by culture and PCR. Legionella spp. and Mycobacteria spp. gene numbers measured by quantitative PCR were similar to nonoutbreak conditions in the continental U.S. Amplicon sequencing provided a nontarget screen for other potential pathogens of concern. This study aids in improving future preparedness, assessment, and recovery operations for small rural water systems after natural disasters.

Molecular Methods for Assessment of Antibiotic Resistance in Agricultural Ecosystems: Prospects and Challenges.

Agricultural ecosystems are of special interest for monitoring the potential for antibiotic resistance to spread through the environment and contribute to human exposure. Molecular methods, which target DNA, RNA, and other molecular components of bacterial cells, present certain advantages for characterizing and quantifying markers of antibiotic resistance and their horizontal gene transfer. These include rapid, unambiguous detection of targets; consistent results; and avoidance of culture bias. However, molecular methods are also subject to limitations that are not always clearly addressed or taken into consideration in the interpretation of scientific data. In particular, DNA-based methods do not directly assess viability or presence within an intact bacterial host, but such information may be inferred based on appropriate experimental design or in concert with complementary methods. The purpose of this review is to provide an overview of existing molecular methods for tracking antibiotic resistance in agricultural ecosystems, to define their strengths and weaknesses, and to recommend a path forward for future applications of molecular methods and standardized reporting in the literature. This will guide research along the farm-to-fork continuum and support comparability of the growing number of studies in the literature in a manner that informs management decisions and policy development.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.