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.

Risk Factors for Antimicrobial Resistance in Turkey Farms: A Cross-Sectional Study in Three European Countries.

Food-producing animals are an important reservoir and potential source of transmission of antimicrobial resistance (AMR) to humans. However, research on AMR in turkey farms is limited. This study aimed to identify risk factors for AMR in turkey farms in three European countries (Germany, France, and Spain). Between 2014 and 2016, faecal samples, antimicrobial usage (AMU), and biosecurity information were collected from 60 farms. The level of AMR in faecal samples was quantified in three ways: By measuring the abundance of AMR genes through (i) shotgun metagenomics sequencing (n = 60), (ii) quantitative real-time polymerase chain reaction (qPCR) targeting ermB, tetW, sul2, and aph3'-III; (n = 304), and (iii) by identifying the phenotypic prevalence of AMR in Escherichia coli isolates by minimum inhibitory concentrations (MIC) (n = 600). The association between AMU or biosecurity and AMR was explored. Significant positive associations were detected between AMU and both genotypic and phenotypic AMR for specific antimicrobial classes. Beta-lactam and colistin resistance (metagenomics sequencing); ampicillin and ciprofloxacin resistance (MIC) were associated with AMU. However, no robust AMU-AMR association was detected by analyzing qPCR targets. In addition, no evidence was found that lower biosecurity increases AMR abundance. Using multiple complementary AMR detection methods added insights into AMU-AMR associations at turkey farms.

Assigning Defined Daily/Course Doses for Antimicrobials in Turkeys to Enable a Cross-Country Quantification and Comparison of Antimicrobial Use.

Antimicrobial resistance (AMR) threatens our public health and is mainly driven by antimicrobial usage (AMU). For this reason the World Health Organization calls for detailed monitoring of AMU over all animal sectors involved. Therefore, we aimed to quantify AMU on turkey farms. First, turkey-specific Defined Daily Dose (DDDturkey) was determined. These were compared to the broiler alternative from the European Surveillance of Veterinary Antimicrobial Consumption (DDDvet), that mention DDDvet as a proxy for other poultry species. DDDturkey ranged from being 81.5% smaller to 48.5% larger compared to its DDDvet alternative for broilers. Second, antimicrobial treatments were registered on 60 turkey farms divided over France, Germany and Spain between 2014 and 2016 (20 flocks per country). Afterwards, AMU was quantified using treatment incidence (TI) per 100 days. TI expresses the percentage of the rearing period that the turkeys were treated with a standard dose of antimicrobials. Minimum, median and maximum TI at flock level and based on DDDturkey = 0.0, 10.0 and 65.7, respectively. Yet, a huge variation in amounts of antimicrobials used at flock level was observed, both within and between countries. Seven farms (12%) did not use any antimicrobials. Aminopenicillins, polymyxins, and fluoroquinolones were responsible for 72.2% of total AMU. The proportion of treating farms peaked on week five of the production cycle (41.7%), and 79.4% of the total AMU was administered in the first half of production. To conclude, not all DDDvet values for broilers can be applied to turkeys. Additionally, the results of AMU show potential for reducing and improving AMU on turkey farms, especially concerning the usage of critically important antimicrobials.

Genomic evolution of antimicrobial resistance in Escherichia coli.

The emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

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.

Evaluation of a simplified Herd Health and Welfare Index for benchmarking in pig herds.

OBJECTIVE: Animal welfare is a topic of increasing public interest, especially interventions for improving the welfare of food-producing animals are demanded. The Herd Health and Welfare Index (HHWI) was developed for the EU-research project "EFFORT" (Ecology from Farm to Fork Of microbial drug Resistance and Transmission) to measure health and welfare without interfering with the daily work of farmers. As improving welfare by increasing animal health can contribute to the reduction of antimicrobial usage, it is of great importance to measure the quality of welfare and health of pig herds as precondition for implementing improvement measures. MATERIAL AND METHODS: The HHWI was tested under field conditions by assessing 20 German pig herds. The results were used for benchmarking to encourage continuous improvement. To test a future field of application, the HHWI results (HHWI points: minimum 10 points [very good] to maximum 30 points [very bad]) were compared to the amount of antimicrobial substances in the study herds measured by the Antimicrobial Treatment Index (ATI). RESULTS: The HHWI points of the study herds ranged from 11 to 25. When comparing the HHWI with data of antimicrobial treatments (measured by the ATI) no strong association could be shown. CONCLUSIONS: The preliminary data indicate that the HHWI cannot be statistically related to antimicrobial usage in the field. The number of risk factors for a higher antimicrobial use that are not directly linked to health and welfare parameters is so high that the theoretical influence of the welfare and health status is masked by the influence of many factors (the farmer's attitude, routine usage, group treatments). But preliminary results show a potential to use the index to measure the outcome of interventions within one herd. CLINICAL RELEVANCE: The HHWI is an easy-to-use measuring tool to evaluate animal health and welfare. It can help to increase the continuity of monitoring results of health and welfare at herd level. In the long term, the HHWI could be used for regional or national assessments that could help to identify pig herds that are in need of improvement of their health and welfare status in order to increase the overall life quality of pigs in these herds.

Association of intestinal colonization of ESBL-producing Enterobacteriaceae in poultry slaughterhouse workers with occupational exposure-A German pilot study.

BACKGROUND: Bacteria that have acquired antimicrobial resistance, in particular ESBL-producing Enterobacteriaceae, are an important healthcare concern. Therefore, transmission routes and risk factors are of interest, especially for the carriage of ESBL-producing E. coli. Since there is an enhanced risk for pig slaughterhouse employees to carry ESBL-producing Enterobacteriaceae, associated with animal contact as potential risk factor, the present study investigated the occurrence of ESBL-producing Enterobacteriaceae in poultry slaughterhouse employees. Due to the higher level of resistant Enterobacteriaceae in primary poultry production than in pig production, a higher risk of intestinal colonization of poultry slaughterhouse employees was expected. RESULTS: ESBL-producing Enterobacteriaceae were detected in 5.1% (5 of 99) of the fecal samples of slaughterhouse workers. The species of these isolates was confirmed as E. coli. PCR assays revealed the presence of the genes blaCTX-M-15 (n = 2) and blaSHV-12 (n = 3) in these isolates, partly in combination with the ß-lactamase gene blaTEM-135. Participants were divided into two groups according to their occupational exposure and results indicated an increased probability of colonization with ESBL-producing Enterobacteriaceae for the group of 'higher exposure' (OR 3.7, exact 95% CI 0.6-23.5; p = 0.4). For intestinal colonization with ESBL-producing Enterobacteriaceae, a prevalence of 10% (3/30) was observed in the group of 'higher exposure' versus 2.9% (2/69) in the group of 'lower exposure'. Employees in working steps such as 'hanging' poultry in the process of slaughter and 'evisceration' seemed to have a higher risk for intestinal colonization with ESBL-producing Enterobacteriaceae compared to the group of 'lower exposure'. CONCLUSION: This study is the first of its kind to collect data on the occupational exposure of slaughterhouse workers to ESBL-producing Enterobacteriaceae in Europe. The results suggested that colonization with ESBL-producing Enterobacteriaceae is associated with occupational exposure in poultry slaughterhouses. However, the presence of ESBL-producing E. coli isolates in only 5.1% (5/99) of the tested employees in poultry slaughterhouses suggests a lower transmission risk than in pig slaughterhouses.

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.