Uncovering the prevalence and drivers of antibiotic resistance genes in soils across different land-use types.

The emergence and spread of antibiotic resistance genes (ARGs) in soil due to animal excreta and organic waste is a major threat to human health and ecosystems, and global efforts are required to tackle the issue. However, there is limited knowledge of the variation in ARG prevalence and diversity resulting from different land-use patterns and underlying driving factors in soils. This study aimed to comprehensively characterize the profile of ARGs and mobile genetic elements and their drivers in soil samples collected from 11 provinces across China, representing three different land-use types, using high-throughput quantitative polymerase chain reaction and 16S rRNA amplicon sequencing. Our results showed that agricultural soil had the highest abundance and diversity of ARGs, followed by tea plantation and forest land. A total of 124 unique ARGs were detected in all samples, with shared subtypes among different land-use patterns indicating a common origin or high transmission frequency. Moreover, significant differences in ARG distribution were observed among different geographical regions, with the greatest enrichment of ARGs found in southern China. Biotic and abiotic factors, including soil properties, climatic factors, and bacterial diversity, were identified as the primary drivers associated with ARG abundance, explaining 71.8% of total ARG variation. The findings of our study demonstrate that different land-use patterns are associated with variations in ARG abundance in soil, with agricultural practices posing the greatest risk to human health and ecosystems regarding ARGs. Our identification of biotic and abiotic drivers of ARG abundance provides valuable insights into strategies for mitigating the spread of these genes. This study emphasizes the need for coordinated and integrated approaches to address the global antimicrobial resistance crisis.

Interspecific competition can drive plasmid loss from a focal species in a microbial community.

Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids' ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

Assessing the impact of recreational water use on carriage of antimicrobial resistant organisms.

Understanding the role of exposure to natural recreational waters in the acquisition and transmission of antimicrobial resistance (AMR) is an area of increasing interest. A point prevalence study was carried out in the island of Ireland to determine the prevalence of colonisation with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE) in recreational water users (WU) and matched controls. A total of 411 adult participants (199 WU, 212 controls) submitted at least one faecal sample between September 2020 - October 2021. In total, 80 Enterobacterales were isolated from 73 participants. ESBL-PE were detected in 29 (7.1 %) participants (7 WU, 22 controls), and CRE were detected in nine (2.2 %) participants (4 WU, 5 controls). No carbapenemase-producing Enterobacterales (CPE) were detected. WU were significantly less likely to harbour ESBL-PE than controls (risk ratio = 0.34, 95 % CI 0.148 to 0.776, χ2 7.37, p = 0.007). This study demonstrates the occurrence of ESBL-PE and CRE in healthy participants in Ireland. Recreational exposure to bathing water in Ireland was associated with a decreased prevalence of colonisation with ESBL-PE and CRE.

Functional metagenomic libraries generated from anthropogenically impacted environments reveal importance of metabolic genes in biocide and antibiotic resistance.

Anthropogenic activities result in the release of antimicrobial resistant bacteria and a cocktail of antimicrobial compounds into the environment that may directly select or indirectly co-select for antimicrobial resistance (AMR). Many studies use metagenome sequencing or qPCR-based approaches to study the environmental resistome but these methods are limited by a priori knowledge. In this study, a functional metagenomic approach was used to explore biocide resistance mechanisms in two contaminated environments and a pristine site, and to identify whether potentially novel genes conferring biocide resistance also conferred resistance or reduced susceptibility to antibiotics. Resistance was predominately mediated through novel mechanisms exclusive of the well-known qac efflux genes. UDP-galactose 4-epimerase (galE) -like genes were identified in both contaminated environments and were shown to confer cross-resistance to biocides and clinically important antibiotics for the first time (to our knowledge), compared to knockout mutants. GalE -like genes were also co-located with transposons, suggesting mobilisation potential. These results show that housekeeping genes may play a significant yet underappreciated role in AMR in environmental microbiomes.

The Complex Interplay Between Antibiotic Resistance and Pharmaceutical and Personal Care Products in the Environment.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are important environmental contaminants. Nonetheless, what drives the evolution, spread, and transmission of antibiotic resistance dissemination is still poorly understood. The abundance of ARB and ARGs is often elevated in human-impacted areas, especially in environments receiving fecal wastes, or in the presence of complex mixtures of chemical contaminants, such as pharmaceuticals and personal care products. Self-replication, mutation, horizontal gene transfer, and adaptation to different environmental conditions contribute to the persistence and proliferation of ARB in habitats under strong anthropogenic influence. Our review discusses the interplay between chemical contaminants and ARB and their respective genes, specifically in reference to co-occurrence, potential biostimulation, and selective pressure effects, and gives an overview of mitigation by existing man-made and natural barriers. Evidence and strategies to improve the assessment of human health risks due to environmental antibiotic resistance are also discussed. Environ Toxicol Chem 2023;00:1-16. © 2022 SETAC.

Non-antibiotic pharmaceuticals promote conjugative plasmid transfer at a community-wide level.

BACKGROUND: Horizontal gene transfer (HGT) plays a critical role in the spread of antibiotic resistance and the evolutionary shaping of bacterial communities. Conjugation is the most well characterized pathway for the spread of antibiotic resistance, compared to transformation and transduction. While antibiotics have been found to induce HGT, it remains unknown whether non-antibiotic pharmaceuticals can facilitate conjugation at a microbial community-wide level. RESULTS: In this study, we demonstrate that several commonly consumed non-antibiotic pharmaceuticals (including carbamazepine, ibuprofen, naproxen and propranolol), at environmentally relevant concentrations (0.5 mg/L), can promote the conjugative transfer of IncP1-α plasmid-borne antibiotic resistance across entire microbial communities. The over-generation of reactive oxygen species in response to these non-antibiotic pharmaceuticals may contribute to the enhanced conjugation ratios. Cell sorting and 16S rRNA gene amplicon sequencing analyses indicated that non-antibiotic pharmaceuticals modulate transconjugant microbial communities at both phylum and genus levels. Moreover, microbial uptake ability of the IncP1-α plasmid was also upregulated under non-antibiotic pharmaceutical exposure. Several opportunistic pathogens, such as Acinetobacter and Legionella, were more likely to acquire the plasmid conferring multidrug resistance. CONCLUSIONS: Considering the high possibility of co-occurrence of pathogenic bacteria, conjugative IncP1-α plasmids and non-antibiotic pharmaceuticals in various environments (e.g., activated sludge systems), our findings illustrate the potential risk associated with increased dissemination of antibiotic resistance promoted by non-antibiotic pharmaceuticals in complex environmental settings. Video abstract.

Stereoselective metabolism of chloramphenicol by bacteria isolated from wastewater, and the importance of stereochemistry in environmental risk assessments for antibiotics.

Wastewater treatment plants have been highlighted as a potential hotspot for the development and spread of antibiotic resistance. Although antibiotic resistant bacteria in wastewater present a public health threat, it is also possible that these bacteria play an important role in the bioremediation through the metabolism of antibiotics before they reach the wider environment. Here we address this possibility with a particular emphasis on stereochemistry using a combination of microbiology and analytical chemistry tools including the use of supercritical-fluid chromatography coupled with mass spectrometry for chiral analysis and high-resolution mass spectrometry to investigate metabolites. Due to the complexities around chiral analysis the antibiotic chloramphenicol was used as a proof of concept to demonstrate stereoselective metabolism due to its relatively simple chemical structure and availability over the counter in the U.K. The results presented here demonstrate the chloramphenicol can be stereoselectively transformed by the chloramphenicol acetyltransferase enzyme with the orientation around the first stereocentre being key for this process, meaning that accumulation of two isomers may occur within the environment with potential impacts on ecotoxicity and emergence of bacterial antibiotic resistance within the environment.

Natural recreational waters and the risk that exposure to antibiotic resistant bacteria poses to human health.

Antimicrobial resistance (AMR) is widely recognised as a considerable threat to human health, wellbeing and prosperity. Many clinically important antibiotic resistance genes are understood to have originated in the natural environment. However, the complex interactions between humans, animals and the environment makes the health implications of environmental AMR difficult to quantify. This narrative review focuses on the current state of knowledge regarding antibiotic resistant bacteria (ARB) in natural bathing waters and implications for human health. It considers the latest research focusing on the transmission of ARB from bathing waters to humans. The limitations of existing evidence are discussed, as well as research priorities. The authors are of the opinion that future studies should include faecally contaminated bathing waters and people exposed to these environments to accurately parameterise environment-to-human transmission.

Antifungal Exposure and Resistance Development: Defining Minimal Selective Antifungal Concentrations and Testing Methodologies.

This scoping review aims to summarise the current understanding of selection for antifungal resistance (AFR) and to compare and contrast this with selection for antibacterial resistance, which has received more research attention. AFR is an emerging global threat to human health, associated with high mortality rates, absence of effective surveillance systems and with few alternative treatment options available. Clinical AFR is well documented, with additional settings increasingly being recognised to play a role in the evolution and spread of AFR. The environment, for example, harbours diverse fungal communities that are regularly exposed to antifungal micropollutants, potentially increasing AFR selection risk. The direct application of effect concentrations of azole fungicides to agricultural crops and the incomplete removal of pharmaceutical antifungals in wastewater treatment systems are of particular concern. Currently, environmental risk assessment (ERA) guidelines do not require assessment of antifungal agents in terms of their ability to drive AFR development, and there are no established experimental tools to determine antifungal selective concentrations. Without data to interpret the selective risk of antifungals, our ability to effectively inform safe environmental thresholds is severely limited. In this review, potential methods to generate antifungal selective concentration data are proposed, informed by approaches used to determine antibacterial minimal selective concentrations. Such data can be considered in the development of regulatory guidelines that aim to reduce selection for AFR.

Spatiotemporal profiling of antibiotics and resistance genes in a river catchment: Human population as the main driver of antibiotic and antibiotic resistance gene presence in the environment.

Studies to understand the role wastewater treatment plants (WWTPs) play in the dissemination of antibiotics (ABs), and in the emergence of antibiotic resistance (ABR), play an important role in tackling this global crisis. Here we describe the abundance and distribution of 16 ABs, and 4 corresponding antibiotic resistance genes (ARGs), sampled from the influent to five WWTPs within a single river catchment. We consider four classes of antibiotics: fluroquinolones, macrolides, sulfamethoxazole and chloramphenicol, as well the corresponding antibiotic resistance genes qnrS, ermB, sul1 and catA. All antibiotics, apart from four fluroquinolones (besifloxacin, lomefloxacin, ulifloxacin, prulifloxacin), were detected within all influent wastewater from the 5 cities (1 city = 1 WWTP), as were the corresponding antibiotic resistance genes (ARGs). Strong correlations were observed between the daily loads of ABs and ARGs versus the size of the population served by each WWTP, as well as between AB and ARG loads at a single site. The efficiency of ABs and ARGs removal by the WWTPs varied according to site (and treatment process utilized) and target, although strong correlations were maintained between the population size served by WWTPs and daily loads of discharged ABs and ARGs into the environment. We therefore conclude that population size is the main determinant of the magnitude of AB and ARG burden in the environment.

Monitoring SARS-CoV-2 in municipal wastewater to evaluate the success of lockdown measures for controlling COVID-19 in the UK.

SARS-CoV-2 and the resulting COVID-19 pandemic represents one of the greatest recent threats to human health, wellbeing and economic growth. Wastewater-based epidemiology (WBE) of human viruses can be a useful tool for population-scale monitoring of SARS-CoV-2 prevalence and epidemiology to help prevent further spread of the disease, particularly within urban centres. Here, we present a longitudinal analysis (March-July 2020) of SARS-CoV-2 RNA prevalence in sewage across six major urban centres in the UK (total population equivalent 3 million) by q(RT-)PCR and viral genome sequencing. Our results demonstrate that levels of SARS-CoV-2 RNA generally correlated with the abundance of clinical cases recorded within the community in large urban centres, with a marked decline in SARS-CoV-2 RNA abundance following the implementation of lockdown measures. The strength of this association was weaker in areas with lower confirmed COVID-19 case numbers. Further, sequence analysis of SARS-CoV-2 from wastewater suggested that multiple genetically distinct clusters were co-circulating in the local populations covered by our sample sites, and that the genetic variants observed in wastewater reflected similar SNPs observed in contemporaneous samples from cases tested in clinical diagnostic laboratories. We demonstrate how WBE can be used for both community-level detection and tracking of SARS-CoV-2 and other virus' prevalence, and can inform public health policy decisions. Although, greater understanding of the factors that affect SARS-CoV-2 RNA concentration in wastewater are needed for the full integration of WBE data into outbreak surveillance. In conclusion, our results lend support to the use of routine WBE for monitoring of SARS-CoV-2 and other human pathogenic viruses circulating in the population and assessment of the effectiveness of disease control measures.

Stereoselective Bacterial Metabolism of Antibiotics in Environmental Bacteria - A Novel Biochemical Workflow.

Although molecular genetic approaches have greatly increased our understanding of the evolution and spread of antibiotic resistance genes, there are fewer studies on the dynamics of antibiotic - bacterial (A-B) interactions, especially with respect to stereochemistry. Addressing this knowledge gap requires an interdisciplinary synthesis, and the development of sensitive and selective analytical tools. Here we describe SAM (stereoselective antimicrobial metabolism) workflow, a novel interdisciplinary approach for assessing bacterial resistance mechanisms in the context of A-B interactions that utilise a combination of whole genome sequencing and mass spectrometry. Chloramphenicol was used to provide proof-of-concept to demonstrate the importance of stereoselective metabolism by resistant environmental bacteria. Our data shows that chloramphenicol can be stereoselectively transformed via microbial metabolism with R,R-(-)-CAP being subject to extensive metabolic transformation by an environmental bacterial strain. In contrast S,S-(+)-CAP is not metabolised by this bacterial strain, possibly due to the lack of previous exposure to this isomer in the absence of historical selective pressure to evolve metabolic capacity.

Dawning of a new ERA: Environmental Risk Assessment of antibiotics and their potential to select for antimicrobial resistance.

Antibiotics and antimicrobials are used, misused and overused in human and veterinary medicine, animal husbandry and aquaculture. These compounds can persist in both human and animal waste and then enter the environment through a variety of mechanisms. Though generally measured environmental concentrations (MECs) of antibiotics in aquatic systems are significantly lower than point of therapeutic use concentrations, there is increasing evidence that suggests these concentrations may still enrich antimicrobial resistant bacteria. In light of this evidence, a rigorous and standardised novel methodology needs to be developed which can perform environmental risk assessment (ERA) of antimicrobials in terms of their selective potential as well as their environmental impact, to ensure that diffuse and point source discharges are safe. This review summarises and critically appraises the current methodological approaches that study selection at below point of therapeutic use, or sub-inhibitory, concentrations of antibiotics. We collate and compare selective concentration data generated to date. We recommend how these data can be interpreted in line with current ERA guidelines; outlining and describing novel concepts unique to risk assessment of AMR (such as direct selection of AMR or increased persistence of AMR). We consolidate terminology used thus far into a single framework that could be adopted moving forward, by proposing predicted no effect concentrations for resistance (PNECRs) and predicted no effect concentrations for persistence (PNECPs) be determined in AMR risk assessment. Such a framework will contribute to antibiotic stewardship and by extension, protection of human health, food security and the global economy.

Human Health and Ocean Pollution.

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas.

The 'SELection End points in Communities of bacTeria' (SELECT) Method: A Novel Experimental Assay to Facilitate Risk Assessment of Selection for Antimicrobial Resistance in the Environment.

BACKGROUND: Antimicrobial resistance (AMR) is one of the most significant health threats to society. A growing body of research demonstrates selection for AMR likely occurs at environmental concentrations of antibiotics. However, no standardized experimental approaches for determining selective concentrations of antimicrobials currently exist, preventing appropriate environmental and human health risk assessment of AMR. OBJECTIVES: We aimed to design a rapid, simple, and cost-effective novel experimental assay to determine selective effect concentrations of antibiotics and to generate the largest experimental data set of selective effect concentrations of antibiotics to date. METHODS: Previously published methods and data were used to validate the assay, which determines the effect concentration based on reduction of bacterial community (wastewater) growth. Risk quotients for test antibiotics were generated to quantify risk. RESULTS: The assay (SELection End points in Communities of bacTeria, or the SELECT method) was used to rapidly determine selective effect concentrations of antibiotics. These were in good agreement with quantitative polymerase chain reaction effect concentrations determined within the same experimental system. The SELECT method predicted no effect concentrations were minimally affected by changes in the assay temperature, growth media, or microbial community used as the inoculum. The predicted no effect concentrations for antibiotics tested ranged from 0.05µg/L for ciprofloxacin to 1,250µg/L for erythromycin. DISCUSSION: The lack of evidence demonstrating environmental selection for AMR, and of associated human health risks, is a primary reason for the lack of action in the mitigation of release of antibiotics into the aquatic environment. We present a novel method that can reliably and rapidly fill this data gap to enable regulation and subsequent mitigation (where required) to lower the risk of selection for, and human exposure to, AMR in aquatic environments. In particular, ciprofloxacin and, to a lesser extent, azithromycin, cefotaxime, and trimethoprim all pose a significant risk for selection of AMR in the environment. https://doi.org/10.1289/EHP6635.

Evolution of antibiotic resistance at low antibiotic concentrations including selection below the minimal selective concentration.

Determining the selective potential of antibiotics at environmental concentrations is critical for designing effective strategies to limit selection for antibiotic resistance. This study determined the minimal selective concentrations (MSCs) for macrolide and fluoroquinolone antibiotics included on the European Commission's Water Framework Directive's priority hazardous substances Watch List. The macrolides demonstrated positive selection for ermF at concentrations 1-2 orders of magnitude greater (>500 and <750 µg/L) than measured environmental concentrations (MECs). Ciprofloxacin illustrated positive selection for intI1 at concentrations similar to current MECs (>7.8 and <15.6 µg/L). This highlights the need for compound specific assessment of selective potential. In addition, a sub-MSC selective window defined by the minimal increased persistence concentration (MIPC) is described. Differential rates of negative selection (or persistence) were associated with elevated prevalence relative to the no antibiotic control below the MSC. This increased persistence leads to opportunities for further selection over time and risk of human exposure and environmental transmission.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.