What is the Difference between Conventional Drinking Water, Potable Reuse Water, and Nonpotable Reuse Water? A Microbiome Perspective.

As water reuse applications expand, there is a need for more comprehensive means to assess water quality. Microbiome analysis could provide the ability to supplement fecal indicators and pathogen profiling toward defining a "healthy" drinking water microbiota while also providing insight into the impact of treatment and distribution. Here, we utilized 16S rRNA gene amplicon sequencing to identify signature features in the composition of microbiota across a wide spectrum of water types (potable conventional, potable reuse, and nonpotable reuse). A clear distinction was found in the composition of microbiota as a function of intended water use (e.g., potable vs nonpotable) across a very broad range of U.S. water systems at both the point of compliance (Betadisper p > 0.01; ANOSIM p < 0.01, r-stat = 0.71) and point of use (Betadisper p > 0.01; ANOSIM p < 0.01, r-stat = 0.41). Core and discriminatory analysis further served in identifying distinct differences between potable and nonpotable water microbiomes. Taxa were identified at both the phylum (Desulfobacterota, Patescibacteria, and Myxococcota) and genus (Aeromonas and NS11.12_marine_group) levels that effectively discriminated between potable and nonpotable waters, with the most discriminatory taxa being core/abundant in nonpotable waters (with few exceptions, such as Ralstonia being abundant in potable conventional waters). The approach and findings open the door to the possibility of microbial community signature profiling as a water quality monitoring approach for assessing efficacy of treatments and suitability of water for intended use/reuse application.

Metagenomic Profiling of Internationally Sourced Sewage Influents and Effluents Yields Insight into Selecting Targets for Antibiotic Resistance Monitoring.

It has been debated whether wastewater treatment plants (WWTPs) primarily act to attenuate or amplify antibiotic resistance genes (ARGs). However, ARGs are highly diverse with respect to their resistance mechanisms, mobilities, and taxonomic hosts and therefore their behavior in WWTPs should not be expected to be universally conserved. We applied metagenomic sequencing to wastewater influent and effluent samples from 12 international WWTPs to classify the behavior of specific ARGs entering and exiting WWTPs. In total, 1079 different ARGs originating from a variety of bacteria were detected. This included ARGs that could be mapped to assembled scaffolds corresponding to nine human pathogens. While the relative abundance (per 16S rRNA gene) of ARGs decreased during treatment at 11 of the 12 WWTPs sampled and absolute abundance (per mL) decreased at all 12 WWTPs, increases in relative abundance were observed for 40% of the ARGs detected at the 12th WWTP. Also, the relative abundance of mobile genetic elements (MGE) increased during treatment, but the fraction of ARGs known to be transmissible between species decreased, thus demonstrating that increased MGE prevalence may not be generally indicative of an increase in ARGs. A distinct conserved resistome was documented in both influent and effluent across samples, suggesting that well-functioning WWTPs generally attenuate influent antibiotic resistance loads. This work helps inform strategies for wastewater surveillance of antibiotic resistance, highlighting the utility of tracking ARGs as indicators of treatment performance and relative risk reduction.

Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes.

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.

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.

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.

A Pilot Study of Chicago Waterways as Reservoirs of Multidrug-Resistant Enterobacteriaceae (MDR-Ent) in a High-Risk Region for Community-Acquired MDR-Ent Infection in Children.

Community-acquired multidrug resistant Enterobacteriaceae (MDR-Ent) infections continue to increase in the United States. In prior studies, we identified neighboring regions in Chicago, Illinois, where children have 5 to 6 times greater odds of MDR-Ent infections. To prevent community spread of MDR-Ent, we need to identify the MDR-Ent reservoirs. A pilot study of 4 Chicago waterways for MDR-Ent and associated antibiotic resistance genes (ARGs) was conducted. Three waterways (A1 to A3) are labeled safe for "incidental contact recreation" (e.g., kayaking), and A4 is a nonrecreational waterway that carries nondisinfected water. Surface water samples were collected and processed for standard bacterial culture and shotgun metagenomic sequencing. Generally, A3 and A4 (neighboring waterways which are not hydraulically connected) were strikingly similar in bacterial taxa, ARG profiles, and abundances of corresponding clades and genera within the Enterobacteriaceae Additionally, total ARG abundances recovered from the full microbial community were strongly correlated between A3 and A4 (R2 = 0.97). Escherichia coli numbers (per 100 ml water) were highest in A4 (783 most probable number [MPN]) and A3 (200 MPN) relative to A2 (84 MPN) and A1 (32 MPN). We found concerning ARGs in Enterobacteriaceae such as MCR-1 (colistin), Qnr and OqxA/B (quinolones), CTX-M, OXA and ACT/MIR (beta-lactams), and AAC (aminoglycosides). We found significant correlations in microbial community composition between nearby waterways that are not hydraulically connected, suggesting cross-seeding and the potential for mobility of ARGs. Enterobacteriaceae and ARG profiles support the hypothesized concerns that recreational waterways are a potential source of community-acquired MDR-Ent.

Differential Drivers of Antimicrobial Resistance across the World.

Antimicrobial resistance (AMR) is one of the greatest threats faced by humankind. The development of resistance in clinical and hospital settings has been well documented ever since the initial discovery of penicillin and the subsequent introduction of sulfonamides as clinical antibiotics. In contrast, the environmental (i.e., community-acquired) dimensions of resistance dissemination have been only more recently delineated. The global spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) between air, water, soil, and food is now well documented, while the factors that affect ARB and ARG dissemination (e.g., water and air quality, antibiotic fluxes, urbanization, sanitation practices) in these and other environmental matrices are just now beginning to be more fully appreciated. In this Account, we discuss how the global perpetuation of resistance is dictated by highly interconnected socioeconomic risk factors and illustrate that development status should be more fully considered when developing global strategies to address AMR. We first differentiate low to middle income countries (LMICs) and high-income countries (HICs), then we summarize the modes of action of commercially available antibiotics, and then discuss the four primary mechanisms by which bacteria develop resistance to those antibiotics. Resistance is disseminated via both vertical gene transfer (VGT; parent to offspring) as well as by horizontal gene transfer (HGT; cell to cell transference of genetic material). A key challenge hindering attempts to control resistance dissemination is the presence of native, environmental bacteria that can harbor ARGs. Such environmental "resistomes" have potential to transfer resistance to pathogens via HGT. Of particular concern is the development of resistance to antibiotics of last-resort such as the cephalosporins, carbapenems, and polymyxins. We then illustrate how antibiotic use differs in LMICs relative to HICs in terms of the volumes of antibiotics used and their fate within local environments. Antibiotic use in HICs has remained flat over the past 15 years, while in LMICs use over the same period has increased substantially as a result of economic improvements and changes in diet. These use and fate differences impact local citizens and thus the local dissemination of AMR. Various physical, social, and economic circumstances within LMICs potentially favor AMR dissemination. We focus on three physical factors: changing population density, sanitation infrastructure, and solid-waste disposal. We show that high population densities in cities within LMICs that suffer from poor sanitation and solid-waste disposal can potentially impact the dissemination of resistance. In the final section, we discuss potential monitoring approaches to quantify the spread of resistance both within LMICs as well as in HICs. We posit that culture-based approaches, molecular approaches, and cutting-edge nanotechnology-based methods for monitoring ARB and ARGs should be considered both within HICs and, as appropriate, within LMICs.

Demonstrating an Integrated Antibiotic Resistance Gene Surveillance Approach in Puerto Rican Watersheds Post-Hurricane Maria.

Comprehensive surveillance approaches are needed to assess sources, clinical relevance, and mobility of antibiotic resistance genes (ARGs) in watersheds. Here, we examined metrics derived from shotgun metagenomic sequencing and relationship to human fecal markers (HFMs; crAssphage, enterococci) and anthropogenic antibiotic resistance markers (AARMs; intI1, sul1) in three distinct Puerto Rican watersheds as a function of adjacent land use and wastewater treatment plant (WWTP) input 6 months after Hurricane Maria, a category V storm. Relative abundance and diversity of total ARGs increased markedly downstream of WWTP inputs, with ARGs unique to WWTP and WWTP-impacted river samples predominantly belonging to the aminoglycoside and ß-lactam resistance classes. WWTP and other anthropogenic inputs were similarly associated with elevated resistome risk scores and mobility incidence (M%). Contig analysis indicated a wide variety of mobile ß-lactam ARGs associated with pathogens downstream of WWTP discharge that were consistent with regional clinical concern, e.g., Klebsiella pneumoniae contigs containing KPC-2 within an ISKpn6-like transposase. HFMs and AARMs correlated strongly with the absolute abundance of total ARGs, but AARMs better predicted the majority of ARGs in general (85.4 versus <2%) and ß-lactam ARGs in particular. This study reveals sensitive, quantitative, mobile, clinically relevant, and comprehensive targets for antibiotic resistance surveillance in watersheds.

Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016.

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.

Microbial Ecology and Water Chemistry Impact Regrowth of Opportunistic Pathogens in Full-Scale Reclaimed Water Distribution Systems.

Need for global water security has spurred growing interest in wastewater reuse to offset demand for municipal water. While reclaimed (i.e., nonpotable) microbial water quality regulations target fecal indicator bacteria, opportunistic pathogens (OPs), which are subject to regrowth in distribution systems and spread via aerosol inhalation and other noningestion routes, may be more relevant. This study compares the occurrences of five OP gene markers ( Acanthamoeba spp., Legionella spp., Mycobacterium spp., Naegleria fowleri, Pseudomonas aeruginosa) in reclaimed versus potable water distribution systems and characterizes factors potentially contributing to their regrowth. Samples were collected over four sampling events at the point of compliance for water exiting treatment plants and at five points of use at four U.S. utilities bearing both reclaimed and potable water distribution systems. Reclaimed water systems harbored unique water chemistry (e.g., elevated nutrients), microbial community composition, and OP occurrence patterns compared to potable systems examined here and reported in the literature. Legionella spp. genes, Mycobacterium spp. genes, and total bacteria, represented by 16S rRNA genes, were more abundant in reclaimed than potable water distribution system samples ( p ≤ 0.0001). This work suggests that further consideration should be given to managing reclaimed water distribution systems with respect to nonpotable exposures to OPs.

Metagenomic Characterization of Antibiotic Resistance Genes in Full-Scale Reclaimed Water Distribution Systems and Corresponding Potable Systems.

Water reclamation provides a valuable resource for meeting nonpotable water demands. However, little is known about the potential for wastewater reuse to disseminate antibiotic resistance genes (ARGs). Here, samples were collected seasonally in 2014-2015 from four U.S. utilities' reclaimed and potable water distribution systems before treatment, after treatment, and at five points of use (POU). Shotgun metagenomic sequencing was used to profile the resistome (i.e., full contingent of ARGs) of a subset ( n = 38) of samples. Four ARGs ( qnrA, blaTEM, vanA, sul1) were quantified by quantitative polymerase chain reaction. Bacterial community composition (via 16S rRNA gene amplicon sequencing), horizontal gene transfer (via quantification of intI1 integrase and plasmid genes), and selection pressure (via detection of metals and antibiotics) were investigated as potential factors governing the presence of ARGs. Certain ARGs were elevated in all ( sul1; p ≤ 0.0011) or some ( blaTEM, qnrA; p ≤ 0.0145) reclaimed POU samples compared to corresponding potable samples. Bacterial community composition was weakly correlated with ARGs (Adonis, R2 = 0.1424-0.1734) and associations were noted between 193 ARGs and plasmid-associated genes. This study establishes that reclaimed water could convey greater abundances of certain ARGs than potable waters and provides observations regarding factors that likely control ARG occurrence in reclaimed water systems.

Distribution System Operational Deficiencies Coincide with Reported Legionnaires' Disease Clusters in Flint, Michigan.

We hypothesize that the increase in reported Legionnaires' disease from June 2014 to November 2015 in Genesee County, MI (where Flint is located) was directly linked to the switch to corrosive Flint River water from noncorrosive Detroit water from April 2014 to October 2015. To address the lack of epidemiological data linking the drinking water supplies to disease incidence, we gathered physiochemical and biological water quality data from 2010 to 2016 to evaluate characteristics of the Flint River water that were potentially conducive to Legionella growth. The treated Flint River water was 8.6 times more corrosive than Detroit water in short-term testing, releasing more iron, which is a key Legionella nutrient, while also directly causing disinfectant to decay more rapidly. The Flint River water source was also 0.8-6.7 °C warmer in summer months than Detroit water and exceeded the minimum Legionella growth temperature of 20 °C more frequently (average number of days per year for Detroit was 63 versus that for the Flint River, which was 157). The corrosive water also led to 1.3-2.2 times more water main breaks in 2014-2015 compared to 2010-2013; such disruptions have been associated with outbreaks in other locales. Importantly, Legionella spp. and Legionella pneumophila decreased after switching back to Detroit water, in terms of both gene markers and culturability, when August and October 2015 were compared to November 2016.

To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems.

Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems.

A proposed framework for the identification of indicator genes for monitoring antibiotic resistance in wastewater: Insights from metagenomic sequencing.

Antibiotic resistance is one of the greatest threats to global human and animal health of our time. Municipal wastewater has been identified as a hotspot of antibiotic resistance contamination to water bodies. However, there are numerous potential antibiotic resistant pathogens and their associated antibiotic resistance genes (ARGs), making it difficult to implement routine monitoring that addresses the breadth of the problem. The objective of this study was to identify candidate indicator ARGs for monitoring antibiotic resistance in wastewater and receiving water bodies. We developed a framework to identify indicator ARGs that incorporated clinical relevance, abundance in wastewater, geographic ubiquity, environmental relevance, ARG mobility, associations with mobile genetic elements, and the availability of quantitative analytical methods. To identify indicator ARGs, published metagenomic sequencing data from 191 wastewater samples originating from 64 countries across the world were obtained from online public repositories. Through ARG annotation and network analysis, this framework revealed 56 candidate indicator ARGs distributed across four modules of strongly correlated ARGs, with one ARG from each module (oqxA, ermB, sul1, and mexE) proposed as a minimally redundant monitoring target. The results of this study provide the basis for antibiotic resistance surveillance and monitoring framework in wastewater and contaminated waterways.

Microbial source tracking to elucidate the impact of land-use and physiochemical water quality on fecal contamination in a mixed land-use watershed.

Escherichia coli has been widely used as a fecal indicator bacterium (FIB) for monitoring water quality in drinking water sources and recreational water. However, fecal contamination sources remain difficult to identify and mitigate, as millions of cases of infectious diseases are reported yearly due to swimming and bathing in recreational water. The objective of this study was to apply molecular techniques for microbial source tracking (MST) to identify sources of fecal contamination in a representative mixed land-use watershed located in the Appalachian Mountains of the United States of America (USA). Monthly samples were collected over one year at 11 sites, including the confluence of key first-order streams in the study watershed representing distinct land-use types and anticipated fecal sources. Results indicated that coupled monitoring of host-specific MST markers with the FIB E. coli effectively identified sources and quantified fecal contamination in the study watershed. Human-associated MST markers were abundant primarily at developed sites, suggesting septic or sewer failure is a key source of fecal input to the watershed. Across the dataset, samples positive for E. coli and human MST markers were associated with a higher pH than those samples from which each target was not detected, thereby suggesting that acid mine drainage in the watershed likely contributed to inactivation or loss of culturability in E. coli. In addition, this research provides the first evidence that the BacCan-UCD marker is present in fox feces and can influence MST results in areas where substantial wildlife activity is present. Identifying the sources of fecal contamination and better understanding the impact of in-stream physiochemistry throughout this study will help to develop sustainable and effective watershed management plans to control fecal contamination to protect drinking water sources and recreational water.

Tenets of a holistic approach to drinking water-associated pathogen research, management, and communication.

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.

Functional Interrelationships of Microorganisms in Iron-Based Anaerobic Wastewater Treatment.

This study explicated the functional activities of microorganisms and their interrelationships under four previously reported iron reducing conditions to identify critical factors that governed the performance of these novel iron-dosed anaerobic biological wastewater treatment processes. Various iron-reducing bacteria (FeRB) and sulfate reducing bacteria (SRB) were identified as the predominant species that concurrently facilitated organics oxidation and the main contributors to removal of organics. The high organic contents of wastewater provided sufficient electron donors for active growth of both FeRB and SRB. In addition to the organic content, Fe (III) and sulfate concentrations (expressed by Fe/S ratio) were found to play a significant role in regulating the microbial abundance and functional activities. Various fermentative bacteria contributed to this FeRB-SRB synergy by fermenting larger organic compounds to smaller compounds, which were subsequently used by FeRB and SRB. Feammox (ferric reduction coupled to ammonium oxidation) bacterium was identified in the bioreactor fed with wastewater containing ammonium. Organic substrate level was a critical factor that regulated the competitive relationship between heterotrophic FeRB and Feammox bacteria. There were evidences that suggested a synergistic relationship between FeRB and nitrogen-fixing bacteria (NFB), where ferric iron and organics concentrations both promoted microbial activities of FeRB and NFB. A concept model was developed to illustrate the identified functional interrelationships and their governing factors for further development of the iron-based wastewater treatment systems.

Combined effects of composting and antibiotic administration on cattle manure-borne antibiotic resistance genes.

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. Video Abstract.

Evaluation of Metagenomic-Enabled Antibiotic Resistance Surveillance at a Conventional Wastewater Treatment Plant.

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.