Comparison of concentration and extraction workflows for qPCR quantification of intI1 and vanA in untreated wastewater

Quantitative polymerase chain reaction (qPCR) measurement of antibiotic resistance genes (ARGs) in untreated municipal wastewater may prove useful in combating the antimicrobial resistance crisis. However, harmonizing and optimizing qPCR-based workflows is essential to facilitate comparisons across studies, and includes achieving highly-effective ARG capture through efficient concentration and extraction procedures. In the current study, combinations of sample volume, membrane types and DNA extraction kits within filtration and centrifugation-based workflows were used to quantify 16S ribosomal RNA (16S rRNA), class 1 integron-integrase gene (intI1) and an ARG encoding resistance to vancomycin (vanA) in untreated wastewater sampled from three wastewater treatment plants (WWTPs). Highly abundant 16S rRNA and intI1 were detected in 100 % of samples from all three WWTPs using both 2 and 20 mL sample volumes, while lower prevalence vanA was only detected when using the 20 mL volume. When filtering 2 mL of wastewater, workflows with 0.20-/0.40-μm polycarbonate (PC) membranes generally yielded greater concentrations of the three targets than workflows with 0.22-/0.45-μm mixed cellulose ester (MCE) membranes. The improved performance was diminished when the sample volume was increased to 20 mL. Consistently greater concentrations of 16S rRNA, intI1 and vanA were yielded by filtration-based workflows using PC membranes combined with a DNeasy PowerWater (DPW) Kit, regardless of the sample volume used, and centrifugation-based workflows with DNeasy Blood & Tissue Kit for 2-mL wastewater extractions. Within the filtration-based workflows, the DPW kit yielded more detection and quantifiable results for less abundant vanA than the DNeasy PowerSoil Pro Kit and FastDNA™ SPIN Kit for Soil. These findings indicate that the performance of qPCR-based workflows for surveillance of ARGs in wastewater varies across targets, sample volumes, concentration methods and extraction kits. Workflows must be carefully considered and validated considering the target ARGs to be monitored

Front Water

Antimicrobial resistance (AMR) is a world-wide public health threat that is projected to lead to 10 million annual deaths globally by 2050. The AMR public health issue has led to the development of action plans to combat AMR, including improved antimicrobial stewardship, development of new antimicrobials, and advanced monitoring. The National Antimicrobial Resistance Monitoring System (NARMS) led by the United States (U.S) Food and Drug Administration along with the U.S. Centers for Disease Control and U.S. Department of Agriculture has monitored antimicrobial resistant bacteria in retail meats, humans, and food animals since the mid 1990's. NARMS is currently exploring an integrated One Health monitoring model recognizing that human, animal, plant, and environmental systems are linked to public health. Since 2020, the U.S. Environmental Protection Agency has led an interagency NARMS environmental working group (EWG) to implement a surface water AMR monitoring program (SWAM) at watershed and national scales. The NARMS EWG divided the development of the environmental monitoring effort into five areas: (i) defining objectives and questions, (ii) designing study/sampling design, (iii) selecting AMR indicators, (iv) establishing analytical methods, and (v) developing data management/analytics/metadata plans. For each of these areas, the consensus among the scientific community and literature was reviewed and carefully considered prior to the development of this environmental monitoring program. The data produced from the SWAM effort will help develop robust surface water monitoring programs with the goal of assessing public health risks associated with AMR pathogens in surface water (e.g., recreational water exposures), provide a comprehensive picture of how resistant strains are related spatially and temporally within a watershed, and help assess how anthropogenic drivers and intervention strategies impact the transmission of AMR within human, animal, and environmental systems.CC999999/ImCDC/Intramural CDC HHSUnited States

Towards monitoring the invisible threat: a global approach for tackling AMR in water resources and environment

The global threat of antimicrobial resistance (AMR) is now increasingly recognized for the danger posed by its environmental spread. Aquatic environments and wastewater represent a significant diffusion and selection pathway for antibiotic resistance genes and antibiotic resistant bacteria (ARGs and ARBs). During a collaborative hackathon event, the “Innovation Workshop on Water Quality Monitoring & Assessment,” held in September 2023, experts addressed four challenges related to water quality, including the challenge of globalization AMR surveillance in water. This paper, derived from the workshop findings, proposes a globally adaptable model for antimicrobial resistance surveillance intended as an advance to improve future monitoring systems. The new framework aims to address significant challenges, such as the lack of standardized methodological approaches or lack of funding, coordination, and awareness across a short-, medium- and long-term plan, integrating sustainability concepts, extending participation and monitoring capacity of countries, and offering efficient solutions. This vision is first articulated by creating a technical committee that promotes awareness of antimicrobial resistance and develops a single data management and communication platform. Subsequently, by developing local, national, and international policies, centralized laboratories will be established at the regional level, and built based on existing realities. These laboratories will include facilities to make the management of analyses more efficient, from sampling to reporting the final result. In the long term, activities that allow the maintenance of the created framework and continuous technological development and advancement will be promoted. All this will be achieved in collaboration with national and supranational bodies that are already addressing the issue at a global level

Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environment

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3′’)-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.info:eu-repo/semantics/publishedVersio

Towards the definition of an antibiotic resistome signature in wastewater and downstream environments

Domestic wastewater is a significant reservoir of antibiotic resistance genes, which pose environmental and public health risks. We aimed to define an antibiotic resistome signature, represented by core genes, i.e., shared by ≥ 90% of the metagenomes of each of three conceptual environmental compartments – wastewater (influent, sludge, effluent), freshwater, and agricultural soil. The definition of resistome signatures would support the proposal of a framework for monitoring treatment efficacy and assessing the impact of treated wastewater discharge into the environment, such as freshwater and agricultural soil. Metagenomic data from 163 samples originating from wastewater (n = 81), freshwater (n = 58), and agricultural soils (n = 24) across different regions (29 countries, 5 continents), were analysed regarding antibiotic resistance diversity, based on annotation against a database that merged CARD and ResFinder databases. The relative abundance of the total antibiotic resistance genes (corresponding to the ratio between the antibiotic resistance genes and total reads number) was not statistically different between raw and treated wastewater, being significantly higher than in freshwater or agricultural soils. The latter had the significantly lowest relative abundance of antibiotic resistance genes. Genes conferring resistance to aminoglycosides, beta-lactams, and tetracyclines were among the most abundant in wastewater environments, while multidrug resistance was equally distributed across all environments. The wastewater resistome signature included 27 antibiotic resistance genes that were detected in at least 90% of the wastewater resistomes, and that were not frequent in freshwater or agricultural soil resistomes. Among these were genes responsible for resistance to tetracyclines (n = 8), macrolide-lincosamide-streptogramin B (n = 7), aminoglycosides (n = 4), beta-lactams (n = 3), multidrug (n = 2), sulphonamides (n = 2), and polypeptides (n = 1). This comprehensive assessment provides valuable insights into the dynamics of antibiotic resistance in urban wastewater systems and their potential ecological implications in diverse environmental settings. Furthermore, provides guidance for the implementation of One Health monitoring approaches.info:eu-repo/semantics/publishedVersio

Antimicrobial risk assessment –aggregating aquatic chemical and resistome emissions

This is the author accepted manuscriptUrban water systems receive and emit antimicrobial chemicals, resistant bacterial strains, and resistance genes (ARGs), thus representing “antimicrobial hotspots”. Currently, regional environmental risk assessment (ERA) is carried out using drug consumption data and threshold concentrations derived based on chemical-specific minimum inhibitory concentration values. A legislative proposal by the European Commission released in 2022 addresses the need to include selected ARGs besides the chemical concentration-based ERAs. The questions arise as to (A) how to improve chemical concentration-based risk assessment and (B) how to integrate resistome-related information with chemical-based risk – main focal areas of this study. A tiered chemical risk prediction method is proposed by considering effluents of sewer networks and water resource recovery facilities (WRRFs). To improve predicted environmental concentrations (PEC in recipient water bodies), the impact of antimicrobial bio- and re-transformation in WRRFs is assessed using reliable global data. To combine chemical and genetic risks, a new parameter, i.e., the gene response efficiency is proposed. A regression analysis showed higher gene response efficiency (ARG release) for antimicrobials with relatively low consumption rate levels.European Union's Horizon 202

Surveillance of antimicrobial resistance in the environment - Scientific Opinion of the Panel on Microbial Ecology, Norwegian Scientific Committee for Food and Environment

publishedVersio

Surveillance of antimicrobial resistance in the environment - Scientific Opinion of the Panel on Microbial Ecology, Norwegian Scientific Committee for Food and Environment

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Surveillance of antimicrobial resistance in the environment - Scientific Opinion of the Panel on Microbial Ecology, Norwegian Scientific Committee for Food and Environment

publishedVersionpublishedVersio

Surveillance of antimicrobial resistance in the environment - Scientific Opinion of the Panel on Microbial Ecology, Norwegian Scientific Committee for Food and Environment

publishedVersionpublishedVersio