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.

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.

Description and determinants of the faecal resistome and microbiome of farmers and slaughterhouse workers: A metagenome-wide cross-sectional study.

BACKGROUND: By studying the entire human faecal resistome and associated microbiome, the diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be comprehensively characterized. Prior culture-based studies have shown associations between occupational exposure to livestock and carriage of specific antimicrobial resistant bacteria. Using shotgun metagenomics, the present study investigated 194 faecal resistomes and bacteriomes from humans occupationally exposed to ARGs in livestock (i.e. pig and poultry farmers, employees and family members and pig slaughterhouse workers) and a control population (Lifelines cohort) in the Netherlands. In addition, we sought to identify determinants for the human resistome and bacteriome composition by applying a combination of multivariate (NMDS, PERMANOVA, SIMPER and DESeq2 analysis) and multivariable regression analysis techniques. RESULTS: Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, ß-lactam and macrolide resistance gene clusters dominated the resistome of all studied groups. No significant resistome alpha diversity differences were found among the four populations. However, the resistome beta diversity showed a separation of the mean resistome composition of pig and pork exposed workers from broiler farmers and controls, independent of their antimicrobial use. We demonstrated differences in resistome composition between slaughter line positions, pig versus poultry exposed workers, as well as differences between farmers and employees versus family members. In addition, we found a significant correlation between the bacteriome and resistome, and significant differences in the bacteriome composition between and within the studied subpopulations. Finally, an in-depth analysis of pig and poultry farms - of which also farm livestock resistomes were analysed - showed positive associations between the number of on-farm working hours and human faecal AMR loads. CONCLUSION: We found that the total normalized faecal ARG carriage was larger in persons working in the Dutch pork production chain compared to poultry farmers and controls. Additionally, we showed significant differences in resistome and bacteriome composition of pig and pork exposed workers compared to a control group, as well as within-population (farms, slaughterhouse) compositional differences. The number of on-farm working hours and the farm type (pig or broiler) that persons live or work on are determinants for the human faecal resistome. Overall, our results may suggest direct or indirect livestock contact as a determinant for human ARG carriage. Future studies should further focus on the connection between the human and livestock resistome (i.e. transmission routes) to substantiate the evidence for livestock-associated resistome acquisition.

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.

Functional recovery of biofilm bacterial communities after copper exposure.

Potential of bacterial communities in biofilms to recover after copper exposure was investigated. Biofilms grown outdoor in shallow water on glass dishes were exposed in the laboratory to 0.6, 2.1, 6.8 micromol/l copper amended surface water and a reference and subsequently to un-amended surface water. Transitions of bacterial communities were characterised with denaturing gradient gel electrophoresis (DGGE) and community-level physiological profiles (CLPP). Exposure to 6.8 micromol/l copper provoked distinct changes in DGGE profiles of bacterial consortia, which did not reverse upon copper depuration. Exposure to 2.1 and 6.8 micromol/l copper was found to induce marked changes in CLPP of bacterial communities that proved to be reversible during copper depuration. Furthermore, copper exposure induced the development of copper-tolerance, which was partially lost during depuration. It is concluded that bacterial communities exposed to copper contaminated water for a period of 26 days are capable to restore their metabolic attributes after introduction of unpolluted water in aquaria for 28 days.

Effects of copper and temperature on aquatic bacterial communities.

The present study aimed to characterise effects of copper and temperature on bacterial communities in photosynthetic biofilms using a suit of supplementary methods: pollution-induced community tolerance (PICT), DNA profiles with denaturing gradient gel electrophoresis (DGGE) and physiological profiles with community-level physiological profiling (CLPP). Biofilms of algae and bacteria were grown in a ditch of a Dutch polder and exposed in the laboratory to copper (3 microM and a reference) at three different temperatures (10, 14 and 20 degrees C). Bacterial communities sampled from the field showed heterogeneity in their physiological profiles, however the heterogeneity decreased during laboratory incubation. After 3 days laboratory incubation, the copper treated biofilms were different from the reference biofilms, as revealed by DGGE and CLPP analyses. Effects of temperature were not observed in the CLPPs, or in the DGGE profiles. PICT was observed for the bacterial communities at all temperatures. The copper-tolerance at 10 and 14 degrees C increased about 3 times, whereas copper-tolerance at 20 degrees C increased about 6 times. Temperature had an effect on the community tolerance, but not on the structure or on the physiological profile, suggesting that temperature was not a major factor causing successional changes under these laboratory conditions. In contrast, temperature had an effect on tolerance development indicating that the exposure to copper was enhanced at higher temperature.

Combined effects of lowered oxygen and toxicants (copper and diazinon) on the mayfly Ephoron virgo.

In many large European rivers, the number of typical riverine insect species, such as mayflies, stoneflies, and caddisflies, is greatly reduced compared to historic records. This can no longer be explained by high concentrations of a relatively small number of dominant toxicants since many rivers have changed from heavily contaminated systems with a few selected key toxicants to systems with a complex contamination. This contamination consists of many substances in low concentrations coinciding with other unfavorable conditions, such as low oxygen concentrations. It was hypothesized that the joint adverse effects of such multiple stressors may be a steering factor in the distribution of riverine insect species. The aim of this study was therefore to determine the combined effects of toxicants and oxygen depletion. To this purpose, larvae of the indigenous riverine mayfly Ephoron virgo were exposed to two different model toxicants, copper and diazinon, under normoxia and hypoxia (50% air saturation) conditions. The median effective concentrations for mortality for copper were significantly lower in the hypoxia treatments than in the normoxia treatments. For diazinon, no differences were observed between two treatments differing in dissolved oxygen levels, and therefore we argue that interactions between multiple stressors may be compound specific. It is concluded that the combination of toxicants and lowered oxygen may have a stronger impact than can be expected based on the adverse effects of the individual factors and that standard toxicity tests may be insufficient alone to determine the impact of human activities on the ecological state of riverine communities. Instead, attention needs to be paid to more environmentally realistic nonoptimal conditions in toxicity testing to adequately fulfill the needs of ecological recovery programs.