The scope of the antimicrobial resistance challenge.

Each year, an estimated 7·7 million deaths are attributed to bacterial infections, of which 4.95 million are associated with drug-resistant pathogens, and 1·27 million are caused by bacterial pathogens resistant to the antibiotics available. Access to effective antibiotics when indicated prolongs life, reduces disability, reduces health-care expenses, and enables access to other life-saving medical innovations. Antimicrobial resistance undoes these benefits and is a major barrier to attainment of the Sustainable Development Goals, including targets for newborn survival, progress on healthy ageing, and alleviation of poverty. Adverse consequences from antimicrobial resistance are seen across the human life course in both health-care-associated and community-associated infections, as well as in animals and the food chain. The small set of effective antibiotics has narrowed, especially in resource-poor settings, and people who are very young, very old, and severely ill are particularly susceptible to resistant infections. This paper, the first in a Series on the challenge of antimicrobial resistance, considers the global scope of the problem and how it should be measured. Robust and actionable data are needed to drive changes and inform effective interventions to contain resistance. Surveillance must cover all geographical regions, minimise biases towards hospital-derived data, and include non-human niches.

The European livestock resistome.

Metagenomic sequencing has proven to be a powerful tool in the monitoring of antimicrobial resistance (AMR). Here, we provide a comparative analysis of the resistome from pigs, poultry, veal calves, turkey, and rainbow trout, for a total of 538 herds across nine European countries. We calculated the effects of per-farm management practices and antimicrobial usage (AMU) on the resistome in pigs, broilers, and veal calves. We also provide an in-depth study of the associations between bacterial diversity, resistome diversity, and AMR abundances as well as co-occurrence analysis of bacterial taxa and antimicrobial resistance genes (ARGs) and the universality of the latter. The resistomes of veal calves and pigs clustered together, as did those of avian origin, while the rainbow trout resistome was different. Moreover, we identified clear core resistomes for each specific food-producing animal species. We identified positive associations between bacterial alpha diversity and both resistome alpha diversity and abundance. Network analyses revealed very few taxa-ARG associations in pigs but a large number for the avian species. Using updated reference databases and optimized bioinformatics, previously reported significant associations between AMU, biosecurity, and AMR in pig and poultry farms were validated. AMU is an important driver for AMR; however, our integrated analyses suggest that factors contributing to increased bacterial diversity might also be associated with higher AMR load. We also found that dispersal limitations of ARGs are shaping livestock resistomes, and future efforts to fight AMR should continue to emphasize biosecurity measures.IMPORTANCEUnderstanding the occurrence, diversity, and drivers for antimicrobial resistance (AMR) is important to focus future control efforts. So far, almost all attempts to limit AMR in livestock have addressed antimicrobial consumption. We here performed an integrated analysis of the resistomes of five important farmed animal populations across Europe finding that the resistome and AMR levels are also shaped by factors related to bacterial diversity, as well as dispersal limitations. Thus, future studies and interventions aimed at reducing AMR should not only address antimicrobial usage but also consider other epidemiological and ecological factors.

Evaluation of Membrane Integrity Monitoring Methods for Hollow Fiber Nanofiltration Membranes: Applicability in Gray Water Reclamation Systems.

Source-separated gray water reclamation using nanofiltration as an advanced post-treatment option has received substantial interest in meeting the growing water demand. During reclamation, membrane integrity is crucial to ensure the water's safety. This study evaluated several chemical and novel microbial indicators as indirect membrane integrity-monitoring methods for hollow fiber nanofiltration membranes in reclamation schemes. Under normal conditions, high retention of divalent ions and organic matter and near-complete removal of Escherichia coli (E. coli) were observed. Limited removal of the antibiotic gene (ARG) tetO was observed due to low feed concentrations and a higher detection limit (LOD). While 16S rRNA and ARG sul1 were not limited by their LODs, lower removals were observed, most likely due to free-floating DNA passing through the membranes. A broken fiber in a pilot-scale module reduced organic matter and microorganism removal substantially, while flux and ion rejection remained similar. Predictions made using the observed results and a previously proposed model allowed for the evaluation of the selected methods in upscaled reclamation systems. Based on these results, it was concluded that microorganisms could be employed as indicators in indirect membrane integrity-monitoring methods in large-scale reclamation schemes, while UV254nm absorbance (used in organic matter determination) could be a viable solution in pilot-scale systems.

The resistome and microbiome of wastewater treatment plant workers - The AWARE study.

Urban wastewater treatment plants harbor a large collection of antibiotic resistant enteric bacteria. It is therefore reasonable to hypothesize that workers at such plants would possess a more diverse set of resistant enteric bacteria, compared to the general population. To address this hypothesis, we have compared the fecal microbiome and resistome of 87 workers at wastewater treatment plants (WWTPs) from Romania and the Netherlands to those of 87 control individuals, using shotgun metagenomics. Controlling for potential confounders, neither the total antibiotic resistance gene (ARG) abundance, nor the overall bacterial composition were significantly different between the two groups. If anything, the ARG richness was slightly lower in WWTP workers, and in a stratified analysis the total ARG abundance was significantly lower in Dutch workers compared to Dutch control participants. We identified country of residence, together with recent antibiotic intake in the Dutch population, as the largest contributing factors to the total abundance of ARGs. A striking side-finding was that sex was associated with carriage of disinfectant resistance genes, with women in both Romania and the Netherlands having significantly higher abundance compared to men. A follow up investigation including an additional 313 publicly available samples from healthy individuals from three additional countries showed that the difference was significant for three genes conferring resistance to chemicals commonly used in cosmetics and cleaning products. We therefore hypothesize that the use of cosmetics and, possibly, cleaning products leads to higher abundance of disinfectant resistance genes in the microbiome of the users. Altogether, this study shows that working at a WWTP does not lead to a higher abundance or diversity of ARGs and no large shifts in the overall gut microbial composition in comparison to participants not working at a WWTP. Instead, other factors such as country of residence, recent antibiotic intake and sex seem to play a larger role.

A comparative study between low- and high-tech methods for the detection and mitigation of illicit connections in stormwater systems.

Illicit connections of wastewater to stormwater systems are the main drawback of separate sewer systems, as they lead to a direct discharge of untreated wastewater to the aquatic environment. Consequently, several inspection methods have been developed for detecting illicit connections. This study simultaneously applied several low- and high-tech methods for the detection of illicit connections in the same catchment (De Heuvel, the Netherlands). The methods included mesh wire screens for capturing coarse contamination, measurements of electroconductivity and temperature, sampling and quantification of Escherichia coli and extended-spectrum ß-lactamase-producing E. coli (ESBL-EC), DNA analysis via quantitative polymerase chain reaction for human-, dog-, and bird-specific fecal indicators, and distributed temperature sensing. Significant illicit connections could be identified using all methods. Nonetheless, hydraulic conditions and, predominantly, the sewage volume determine whether a misconnection can be detected by especially the low-tech methods. Using these results, the identified misconnections were repaired and biological and DNA analyses were repeated. Our results demonstrate that there were no changes in E. coli or ESBL-EC before and after mitigation, suggesting that these common markers of fecal contamination are not specific enough to evaluate the performance of mitigation efforts. However, a marked decrease in human wastewater markers (HF183) was observed.

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.

Wastewater treatment plants, an "escape gate" for ESCAPE pathogens.

Antibiotics are an essential tool of modern medicine, contributing to significantly decreasing mortality and morbidity rates from infectious diseases. However, persistent misuse of these drugs has accelerated the evolution of antibiotic resistance, negatively impacting clinical practice. The environment contributes to both the evolution and transmission of resistance. From all anthropically polluted aquatic environments, wastewater treatment plants (WWTPs) are probably the main reservoirs of resistant pathogens. They should be regarded as critical control points for preventing or reducing the release of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic-resistance genes (ARGs) into the natural environment. This review focuses on the fate of the pathogens Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae spp. (ESCAPE) in WWTPs. All ESCAPE pathogen species, including high-risk clones and resistance determinants to last-resort antibiotics such as carbapenems, colistin, and multi-drug resistance platforms, were detected in wastewater. The whole genome sequencing studies demonstrate the clonal relationships and dissemination of Gram-negative ESCAPE species into the wastewater via hospital effluents and the enrichment of virulence and resistance determinants of S. aureus and enterococci in WWTPs. Therefore, the efficiency of different wastewater treatment processes regarding the removal of clinically relevant ARB species and ARGs, as well as the influence of water quality factors on their performance, should be explored and monitored, along with the development of more effective treatments and appropriate indicators (ESCAPE bacteria and/or ARGs). This knowledge will allow the development of quality standards for point sources and effluents to consolidate the WWTP barrier role against the environmental and public health AR threats.

Geo-referenced simulations of E. coli in a sub-catchment of the Vecht River using a probabilistic approach.

The proportion of wild swimmers at non-official bathing sites has increased during the Covid-19 pandemic. Bathing water quality at designated sites is monitored through analysis of the concentration of fecal indicator bacteria such as E. coli. However, non-official sites are generally not monitored. In a previous work, steady state modelling of E. coli was achieved at catchment scale, enabling a comparison of expected concentrations along an entire catchment for longtime average. However, E. coli concentrations can vary over several orders of magnitude at the same monitoring site throughout the year. To capture the temporal variability of E. coli concentrations on the catchment scale, we extended the existing deterministic E. coli sub-module of the GREAT-ER (Geo-referenced Exposure Assessment tool for European Rivers) model for probabilistic Monte-Carlo simulations. Here, selected model parameters are represented by probability distributions instead of fixed values. Wastewater treatment plant (WWTP) emissions and diffuse emissions were parameterized using selected data from a previous monitoring campaign (calibration data set) and in-stream processes were modeled using literature data. Comparison of simulation results with monitoring data (evaluation data set) indicates that predicted E. coli concentrations well-represent median measured concentrations, although the range of predicted concentrations is slightly larger than the observed concentration variability. The parameters with the largest influence on the range of predicted concentrations are flow rate and E. coli removal efficiency in WWTPs. A comparison of predicted 90th percentiles with the threshold for sufficient bathing water quality (according to the EU Bathing Water Directive) indicates that year-round swimming at sites influenced by WWTP effluents is advisable almost nowhere in the study area. A refinement of the model can be achieved if quantitative relationships between the WWTP removal efficiency and both, the treatment technologies as well as the operating parameters are further established.

In Silico Localization of Perilymph Proteins Enriched in Menier̀e Disease Using Mammalian Cochlear Single-cell Transcriptomics.

Hypothesis: Proteins enriched in the perilymph proteome of Menier̀e disease (MD) patients may identify affected cell types. Utilizing single-cell transcriptome datasets from the mammalian cochlea, we hypothesize that these enriched perilymph proteins can be localized to specific cochlear cell types. Background: The limited understanding of human inner ear pathologies and their associated biomolecular variations hinder efforts to develop disease-specific diagnostics and therapeutics. Perilymph sampling and analysis is now enabling further characterization of the cochlear microenvironment. Recently, enriched inner ear protein expression has been demonstrated in patients with MD compared to patients with other inner ear diseases. Localizing expression of these proteins to cochlear cell types can further our knowledge of potential disease pathways and subsequent development of targeted therapeutics. Methods: We compiled previously published data regarding differential perilymph proteome profiles amongst patients with MD, otosclerosis, enlarged vestibular aqueduct, sudden hearing loss, and hearing loss of undefined etiology (controls). Enriched proteins in MD were cross-referenced against published single-cell/single-nucleus RNA-sequencing datasets to localize gene expression to specific cochlear cell types. Results: In silico analysis of single-cell transcriptomic datasets demonstrates enrichment of a unique group of perilymph proteins associated with MD in a variety of intracochlear cells, and some exogeneous hematologic and immune effector cells. This suggests that these cell types may play an important role in the pathology associated with late MD, suggesting potential future areas of investigation for MD pathophysiology and treatment. Conclusions: Perilymph proteins enriched in MD are expressed by specific cochlear cell types based on in silico localization, potentially facilitating development of disease-specific diagnostic markers and therapeutics.

A potential serological biomarker for inner ear pathologies: OTOLIN-1.

Objective: The goal of the study was to determine whether the level of OTOLIN-1, a protein whose expression is highly restricted to the inner ear,is increased in the body fluids of patients with inner ear disorders in comparison to healthy subjects. Methods: In the preliminary part of the study, OTOLIN-1 levels were measured in the serum, urine, and saliva of patients with an acute onset of Ménière´s disease and in healthy individuals. Subsequently, only serum OTOLIN-1 levels were taken into account and were compared between patients with acute onset of Ménière´s disease, sudden hearing loss, vestibular neuritis and healthy subjects. Results: The most reliable diagnostic parameter was OTOLIN-1 levels in serum. Serum samples of patients with Ménière's disease and sudden hearing loss showed significantly higher OTOLIN-1 levels than those from healthy individuals. In addition, there was no significant difference between the serum concentration of OTOLIN-1 in patients with vestibular neuritis and the control group. Conclusions: Serum levels of OTOLIN-1 can potentially be used as a biomarker for acute onset of inner ear disorders due to its significant increase in patients with acute Meniere´s disease and sudden hearing loss in comparison to healthy individuals.

Insights into the impact of manure on the environmental antibiotic residues and resistance pool.

The intensive use of antibiotics in the veterinary sector, linked to the application of manure-derived amendments in agriculture, translates into increased environmental levels of chemical residues, AR bacteria (ARB) and antibiotic resistance genes (ARG). The aim of this review was to evaluate the current evidence regarding the impact of animal farming and manure application on the antibiotic resistance pool in the environment. Several studies reported correlations between the prevalence of clinically relevant ARB and the amount and classes of antibiotics used in animal farming (high resistance rates being reported for medically important antibiotics such as penicillins, tetracyclines, sulfonamides and fluoroquinolones). However, the results are difficult to compare, due to the diversity of the used antimicrobials quantification techniques and to the different amounts and types of antibiotics, exhibiting various degradation times, given in animal feed in different countries. The soils fertilized with manure-derived products harbor a higher and chronic abundance of ARB, multiple ARG and an enriched associated mobilome, which is also sometimes seen in the crops grown on the amended soils. Different manure processing techniques have various efficiencies in the removal of antibiotic residues, ARB and ARGs, but there is only a small amount of data from commercial farms. The efficiency of sludge anaerobic digestion appears to be dependent on the microbial communities composition, the ARB/ARG and operating temperature (mesophilic vs. thermophilic conditions). Composting seems to reduce or eliminate most of antibiotics residues, enteric bacteria, ARB and different representative ARG in manure more rapidly and effectively than lagoon storage. Our review highlights that despite the body of research accumulated in the last years, there are still important knowledge gaps regarding the contribution of manure to the AMR emergence, accumulation, spread and risk of human exposure in countries with high clinical resistance rates. Land microbiome before and after manure application, efficiency of different manure treatment techniques in decreasing the AMR levels in the natural environments and along the food chain must be investigated in depth, covering different geographical regions and countries and using harmonized methodologies. The support of stakeholders is required for the development of specific best practices for prudent - cautious use of antibiotics on farm animals. The use of human reserve antibiotics in veterinary medicine and of unprescribed animal antimicrobials should be stopped and the use of antibiotics on farms must be limited. This integrated approach is needed to determine the optimal conditions for the removal of antibiotic residues, ARB and ARG, to formulate specific recommendations for livestock manure treatment, storage and handling procedures and to translate them into practical on-farm management decisions, to ultimately prevent exposure of human population.

Horizontal Gene Transfer of an IncP1 Plasmid to Soil Bacterial Community Introduced by Escherichia coli through Manure Amendment in Soil Microcosms.

The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00-2.49 log CFU/g of manured soil, ranging between 10-5 and 10-4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

(Antibiotic-Resistant) E. coli in the Dutch-German Vecht Catchment─Monitoring and Modeling.

Fecally contaminated waters can be a source for human infections. We investigated the occurrence of fecal indicator bacteria (E. coli) and antibiotic-resistant E. coli, namely, extended spectrum beta-lactamase (ESBL)-producing E. coli (ESBL-EC) and carbapenemase-producing E. coli (CP-EC) in the Dutch-German transboundary catchment of the Vecht River. Over the course of one year, bacterial concentrations were monitored in wastewater treatment plant (WWTP) influents and effluents and in surface waters with and without WWTP influence. Subsequently, the GREAT-ER model was adopted for the prediction of (antibiotic-resistant) E. coli concentrations. The model was parametrized and evaluated for two distinct scenarios (average flow scenario, dry summer scenario). Statistical analysis of WWTP monitoring data revealed a significantly higher (factor 2) proportion of ESBL-EC among E. coli in German compared to Dutch WWTPs. CP-EC were present in 43% of influent samples. The modeling approach yielded spatially accurate descriptions of microbial concentrations for the average flow scenario. Predicted E. coli concentrations exceed the threshold value of the Bathing Water Directive for a good bathing water quality at less than 10% of potential swimming sites in both scenarios. During a single swimming event up to 61 CFU of ESBL-EC and less than 1 CFU of CP-EC could be taken up by ingestion.

Bioinformatic Analysis of the Perilymph Proteome to Generate a Human Protein Atlas.

The high complexity of the cellular architecture of the human inner ear and the inaccessibility for tissue biopsy hampers cellular and molecular analysis of inner ear disease. Sampling and analysis of perilymph may present an opportunity for improved diagnostics and understanding of human inner ear pathology. Analysis of the perilymph proteome from patients undergoing cochlear implantation was carried out revealing a multitude of proteins and patterns of protein composition that may enable characterisation of patients into subgroups. Based on existing data and databases, single proteins that are not present in the blood circulation were related to cells within the cochlea to allow prediction of which cells contribute to the individual perilymph proteome of the patients. Based on the results, we propose a human atlas of the cochlea. Finally, druggable targets within the perilymph proteome were identified. Understanding and modulating the human perilymph proteome will enable novel avenues to improve diagnosis and treatment of inner ear diseases.

International Travel as a Risk Factor for Carriage of Extended-Spectrum ß-Lactamase-Producing Escherichia coli in a Large Sample of European Individuals-The AWARE Study.

Antibiotic resistance (AR) is currently a major threat to global health, calling for a One Health approach to be properly understood, monitored, tackled, and managed. Potential risk factors for AR are often studied in specific high-risk populations, but are still poorly understood in the general population. Our aim was to explore, describe, and characterize potential risk factors for carriage of Extended-Spectrum Beta-Lactamase-resistant Escherichia coli (ESBL-EC) in a large sample of European individuals aged between 16 and 67 years recruited from the general population in Southern Germany, the Netherlands, and Romania. Questionnaire and stool sample collection for this cross-sectional study took place from September 2018 to March 2020. Selected cultures of participants' stool samples were analyzed for detection of ESBL-EC. A total of 1183 participants were included in the analyses: 333 from Germany, 689 from the Netherlands, and 161 from Romania. Travels to Northern Africa (adjusted Odds Ratio, aOR 4.03, 95% Confidence Interval, CI 1.67-9.68), Sub-Saharan Africa (aOR 4.60, 95% CI 1.60-13.26), and Asia (aOR 4.08, 95% CI 1.97-8.43) were identified as independent risk factors for carriage of ESBL-EC. Therefore, travel to these regions should continue to be routinely asked about by clinical practitioners as possible risk factors when considering antibiotic therapy.

Antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) abundance in animal faeces, meat, production environments and human faeces in Europe.

BACKGROUND: Real-time quantitative PCR (qPCR) is an affordable method to quantify antimicrobial resistance gene (ARG) targets, allowing comparisons of ARG abundance along animal production chains. OBJECTIVES: We present a comparison of ARG abundance across various animal species, production environments and humans in Europe. AMR variation sources were quantified. The correlation of ARG abundance between qPCR data and previously published metagenomic data was assessed. METHODS: A cross-sectional study was conducted in nine European countries, comprising 9572 samples. qPCR was used to quantify abundance of ARGs [aph(3')-III, erm(B), sul2, tet(W)] and 16S rRNA. Variance component analysis was conducted to explore AMR variation sources. Spearman's rank correlation of ARG abundance values was evaluated between pooled qPCR data and earlier published pooled metagenomic data. RESULTS: ARG abundance varied strongly among animal species, environments and humans. This variation was dominated by between-farm variation (pigs) or within-farm variation (broilers, veal calves and turkeys). A decrease in ARG abundance along pig and broiler production chains ('farm to fork') was observed. ARG abundance was higher in farmers than in slaughterhouse workers, and lowest in control subjects. ARG abundance showed a high correlation (Spearman's ρ > 0.7) between qPCR data and metagenomic data of pooled samples. CONCLUSIONS: qPCR analysis is a valuable tool to assess ARG abundance in a large collection of livestock-associated samples. The between-country and between-farm variation of ARG abundance could partially be explained by antimicrobial use and farm biosecurity levels. ARG abundance in human faeces was related to livestock antimicrobial resistance exposure.

Proteome profile of patients with excellent and poor speech intelligibility after cochlear implantation: Can perilymph proteins predict performance?

Modern proteomic analysis and reliable surgical access to gain liquid inner ear biopsies have enabled in depth molecular characterization of the cochlea microenvironment. In order to clarify whether the protein composition of the perilymph can provide new insights into individual hearing performance after cochlear implantation (CI), computational analysis in correlation to clinical performance after CI were performed based on the proteome profile derived from perilymph samples (liquid biopsies). Perilymph samples from cochlear implant recipients have been analyzed by mass spectrometry (MS). The proteins were identified using the shot-gun proteomics method and quantified and analyzed using Max Quant, Perseus and IPA software. A total of 75 perilymph samples from 68 (adults and children) patients were included in the analysis. Speech perception data one year after implantation were available for 45 patients and these were used for subsequent analysis. According to their hearing performance, patients with excellent (n = 22) and poor (n = 14) performance one year after CI were identified and used for further analysis. The protein composition and statistically significant differences in the two groups were detected by relative quantification of the perilymph proteins. With this procedure, a selection of 287 proteins were identified in at least eight samples in both groups. In the perilymph of the patients with excellent and poor performance, five and six significantly elevated proteins were identified respectively. These proteins seem to be involved in different immunological processes in excellent and poor performer. Further analysis on the role of specific proteins as predictors for poor or excellent performance among CI recipients are mandatory. Combinatory analysis of molecular inner ear profiles and clinical performance data using bioinformatics analysis may open up new possibilities for patient stratification. The impact of such prediction algorithms on diagnosis and treatment needs to be established in further studies.

Risk factors for the abundance of antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) in pig and broiler faeces in nine European countries.

OBJECTIVES: The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. METHODS: We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3')-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. RESULTS: The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. CONCLUSIONS: qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

Determinants for antimicrobial resistance genes in farm dust on 333 poultry and pig farms in nine European countries.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

Ecological Risk Assessment of Pharmaceuticals in the Transboundary Vecht River (Germany and The Netherlands).

Millions of people rely on active pharmaceutical ingredients (APIs) to prevent and cure a wide variety of illnesses in humans and animals, which has led to a steadily increasing consumption of APIs across the globe and concurrent releases of APIs into the environment. In the environment, APIs can have a detrimental impact on wildlife, particularly aquatic wildlife. Therefore, it is essential to assess their potential adverse effects to aquatic ecosystems. The European Water Framework Directive sets out that risk assessment should be performed at the catchment level, crossing borders where needed. The present study defines ecological risk profiles for surface water concentrations of 8 APIs (carbamazepine, ciprofloxacin, cyclophosphamide, diclofenac, erythromycin, 17α-ethinylestradiol, metformin, and metoprolol) in the Vecht River, a transboundary river that crosses several German and Dutch regions. Ultimately, 3 main goals were achieved: 1) the geo-referenced estimation of API concentrations in surface water using the geography-referenced regional exposure assessment tool for European rivers; 2) the derivation of new predicted-no-effect concentrations for 7 of the studied APIs, of which 3 were lower than previously derived values; and 3) the creation of detailed spatially explicit ecological risk profiles of APIs under 2 distinct water flow scenarios. Under average flow conditions, carbamazepine, diclofenac, and 17α-ethinylestradiol were systematically estimated to surpass safe ecological concentration thresholds in at least 68% of the catchment's water volume. This increases to 98% under dry summer conditions. Environ Toxicol Chem 2022;41:648-662. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.