(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.

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.

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.

Association of antimicrobial usage with faecal abundance of aph(3')-III, ermB, sul2 and tetW resistance genes in veal calves in three European countries.

BACKGROUND: High antimicrobial use (AMU) and antimicrobial resistance (AMR) in veal calves remain a source of concern. As part of the EFFORT project, the association between AMU and the abundance of faecal antimicrobial resistance genes (ARGs) in veal calves in three European countries was determined. METHODS: In 2015, faecal samples of veal calves close to slaughter were collected from farms located in France, Germany and the Netherlands (20 farms in France, 20 farms in the Netherlands and 21 farms in Germany; 25 calves per farm). Standardized questionnaires were used to record AMU and farm characteristics. In total, 405 faecal samples were selected for DNA extraction and quantitative polymerase chain reaction to quantify the abundance (16S normalized concentration) of four ARGs [aph(3')-III, ermB, sul2 and tetW] encoding for resistance to frequently used antimicrobials in veal calves. Multiple linear mixed models with random effects for country and farm were used to relate ARGs to AMU and farm characteristics. RESULTS: A significant positive association was found between the use of trimethoprim/sulfonamides and the concentration of sul2 in faeces from veal calves. A higher weight of calves on arrival at the farm was negatively associated with aph(3')-III and ermB. Lower concentrations of aph(3')-III were found at farms with non-commercial animals present. Furthermore, farms using only water for the cleaning of stables had a significantly lower abundance of faecal ermB and tetW compared with other farms. CONCLUSION: A positive association was found between the use of trimethoprim/sulfonamides and the abundance of sul2 in faeces in veal calves. Additionally, other relevant risk factors associated with ARGs in veal calves were identified, such as weight on arrival at the farm and cleaning practices.

Occupational Exposure and Carriage of Antimicrobial Resistance Genes (tetW, ermB) in Pig Slaughterhouse Workers.

OBJECTIVES: Slaughterhouse staff is occupationally exposed to antimicrobial resistant bacteria. Studies reported high antimicrobial resistance gene (ARG) abundances in slaughter pigs. This cross-sectional study investigated occupational exposure to tetracycline (tetW) and macrolide (ermB) resistance genes and assessed determinants for faecal tetW and ermB carriage among pig slaughterhouse workers. METHODS: During 2015-2016, 483 faecal samples and personal questionnaires were collected from workers in a Dutch pig abattoir, together with 60 pig faecal samples. Human dermal and respiratory exposure was assessed by examining 198 carcass, 326 gloves, and 33 air samples along the line, next to 198 packed pork chops to indicate potential consumer exposure. Samples were analyzed by qPCR (tetW, ermB). A job exposure matrix was created by calculating the percentage of tetW and ermB positive carcasses or gloves for each job position. Multiple linear regression models were used to link exposure to tetW and ermB carriage. RESULTS: Workers are exposed to tetracycline and macrolide resistance genes along the slaughter line. Tetw and ermB gradients were found for carcasses, gloves, and air filters. One packed pork chop contained tetW, ermB was non-detectable. Human faecal tetW and ermB concentrations were lower than in pig faeces. Associations were found between occupational tetW exposure and human faecal tetW carriage, yet, not after model adjustments. Sampling round, nationality, and smoking were determinants for ARG carriage. CONCLUSION: We demonstrated clear environmental tetracycline and macrolide resistance gene exposure gradients along the slaughter line. No robust link was found between ARG exposure and human faecal ARG carriage.

Confirmed and Potential Sources of Legionella Reviewed.

Legionella bacteria are ubiquitous in natural matrices and man-made systems. However, it is not always clear if these reservoirs can act as source of infection resulting in cases of Legionnaires' disease. This review provides an overview of reservoirs of Legionella reported in the literature, other than drinking water distribution systems. Levels of evidence were developed to discriminate between potential and confirmed sources of Legionella. A total of 17 systems and matrices could be classified as confirmed sources of Legionella. Many other man-made systems or natural matrices were not classified as a confirmed source, since either no patients were linked to these reservoirs or the supporting evidence was weak. However, these systems or matrices could play an important role in the transmission of infectious Legionella bacteria; they might not yet be considered in source investigations, resulting in an underestimation of their importance. To optimize source investigations it is important to have knowledge about all the (potential) sources of Legionella. Further research is needed to unravel what the contribution is of each confirmed source, and possibly also potential sources, to the LD disease burden.

Soil as a source of Legionella pneumophila sequence type 47.

Legionella pneumophila sequence type (ST) 47 was isolated from soil in a garden. We speculate that this strain was transmitted from soil to the whirlpool in the garden where it caused an outbreak of Legionnaires' disease and Pontiac fever. In the Netherlands, ST47 is frequently isolated from patients, but hardly ever from environmental sources. It is possible that human pathogenic Legionella strains, with ST47 as one of the predominant strains, are transmitted to humans from sources such as natural soil that are currently not targeted in outbreak investigations.