•Food chain workers are exposed to antimicrobial resistant (AMR) bacteria in livestock.•We describe faecal resistomes and microbiomes of farmers and slaughterhouse workers.•Faecal AMR gene loads in ...pork exposed workers exceed those in broiler farmers/controls.•Resistome and microbiome differences are shown between and within occupational groups.•On-farm working hours and working/living on a pig farm determinethe faecal resistome.•Contact with livestock may be a determinant for human AMR gene carriage.
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.
Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, β-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.
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.
Residues of malachite green (MG) were extracted from homogenized animal tissues with a mixture of McIlvaine buffer (pH 3.0)–acetonitrile, and purified over an aromatic sulfonic acid solid-phase ...extraction column followed by HPLC or LC–ESI-MS–MS analysis. Ascorbic acid and
N,
N,
N′,
N′-tetramethyl-1,4-phenylenediamine dihydrochloride were added to reduce de-methylation of the dye. Responses were recorded at 620 nm (HPLC) or by multiple-reaction-monitoring (LC–MS–MS) after post-column oxidation using PbO
2. MG and its primary metabolite leuco-malachite green (LMG) were successfully determined at 2.5–2000 μg/kg in catfish, eel, rainbow trout, salmon, tropical prawns and turbot, with a limit of detection at 1 μg/kg (HPLC) and 0.2 μg/kg (LC–MS–MS) for both MG and LMG. Recoveries for LMG were between 86±15% (prawn) and 105±14% (eel). Freeze–thawing cycles, and storage at 4
°C and −20
°C affected the recovery of both MG and LMG. Analyses of eel, trout and (processed) salmon field samples collected at local retailers, fish-market and -shops demonstrated trace levels of MG-residues.
A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of leuco-malachite green (LMG) in various fish tissues is described. LMG, which is the primary metabolite of ...the parasiticide and fungicide malachite green (MG), is the targeted analyte to reveal abuse of this veterinary drug in fish. After extraction using McIlvaine buffer and acetonitrile, the extract was purified on an aromatic sulphonic acid solid-phase extraction column. After conversion of LMG into MG by post-column oxidation with PbO
2, the effluent was analysed by LC-MS/MS in the multiple reaction monitoring (MRM) mode. The MS–MS trace
m/z 329 →
m/z 313 was used for quantification of LMG and, for salmon, gave an averaged decision limits (CC
α;
α 1%) and detection capability (CC
β;
β 5%) of 0.11 and 0.18
μg
kg
−1, respectively, with a measurement each of three consecutive days. The last values were comparable for those for MG. Other traces were used to collect sufficient identification points to establish the identity of this prohibited veterinary drug, which was achieved at CC
β and higher. These values were comparable for other tested species, including pangasius, tilapia, trout and Victoria perch. Recoveries ranged from 66% in trout at 0.4
μg
kg
−1 to 112% in pangasius at 0.1
μg
kg
−1. Three out of nineteen samples including pangasius, salmon, shrimps and trout bought in local shops, revealed detectable amounts of residues, i.e. in excess of CC
α, and were considered non-compliant. The findings demonstrate the suitability of the presented analytical method to detect residues of malachite green in various aquatic species at relatively low residue levels.
Metabolism plays an important role in the toxic effects caused by 3,4-methylenedioxymethamphetamine (MDMA). Most research has focused on the involvement of CYP2D6 enzyme in MDMA bioactivation, and ...less is known about the contribution of other cytochrome P450 (P450) and phase II metabolism. In this study, we researched the differential roles of phase I P450 enzymes CYP1A2, CYP3A4, and CYP2D6 and phase II enzymes glutathione S-transferase (GST) and catechol-O-methyltransferase (COMT) on the toxic potential of MDMA. MDMA acts as inhibitor of its own metabolism with a relative potency of inhibition of CYP2D>CYP3A>> CYP1A in rat liver microsomes and in human liver immortalized human liver epithelial cells (THLE) cells transfected with individual CYP1A2, CYP3A4, or CYP2D6. Cytotoxicity measurements by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in THLE cells showed that the inhibition of phase I enzymes CYP1A2 by alpha-naphthoflavone and CYP3A4 by troleandomycin does not affect MDMA-induced cytotoxicity. MDMA metabolism by CYP2D6 significantly increased cytotoxicity, which was counteracted by CYP2D6 inhibition by quinidine. Inhibition of COMT by 2'-fluoro-3,4-dihydroxy-5-nitrobenzophenone (Ro-41-0960) and GST by buthionine sulfoximine showed that COMT is mainly involved in detoxification of CYP2D6-formed MDMA metabolites, whereas glutathione (GSH) is mainly involved in detoxification of CYP3A4-formed MDMA metabolites. Liquid chromatography/tandem mass spectrometry analyses of MDMA-metabolites in the THLE cell culture media confirmed formation of the specific MDMA metabolites and corroborated the observed cytotoxicity. Our data suggest that CYP2D6 as well as CYP3A4 play an important role in MDMA bioactivation. In addition, further studies are needed to address the differential roles of CYP3A4 and GSH/GST in MDMA bioactivation and detoxification.
The occurrence of the steroid hormones estrone (E1), 17α-estradiol (αE2), 17β́-estradiol (βE2), and estriol (E3) in processed bovine milk with different fat contents and in raw milk from ...(non)gestated cows was investigated. Following liquid extraction, optional enzymatical deconjugation, C18 solid-phase extraction, and derivatization, estrogens were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free and deconjugated E1 (6.2-1266 ng/L) was the major estrogen followed by αE2 (7.2-322 ng/L) and βE2 (5.6-51 ng/L), whereas E3 was detected regularly at the detection limit of 10 ng/L. The lowest and highest concentrations were determined in raw milk from nonpregnant and from cows in the third trimester of gestation, respectively. The estrogen concentration in processed milk coincides with that of raw milk between first and second trimesters, reflecting the contribution of lactating pregnant cows to the final consumable product. The daily intake of total investigated estrogens through milk is 372 ng, which is dramatically more than currently recognized.
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.
•A large-scale epidemiological study that analyzed AMR in farm dust with EDCs and qPCR.•ARGs in animal feces were found to be positively associated to ARGs in farm dust.•Higher ARG levels were observed in pig farms with higher antimicrobial usage.•Other factors, such as the summer season and biosecurity, were related to AMR in dust.•Farm dust is a large reservoir for antimicrobial resistance genes.
Fish bioconcentration factors (BCFs) are commonly used in chemical hazard and risk assessment. For neutral organic chemicals BCFs are positively correlated with the octanol-water partition ratio (
), ...but
is not a reliable parameter for surfactants. Membrane lipid-water distribution ratios (
) can be accurately measured for all kinds of surfactants, using phospholipid-based sorbents. This study first demonstrates that
values for ionic surfactants are more than 100 000 times higher than the partition ratio to fish-oil, representing neutral storage lipid. A non-ionic alcohol ethoxylate surfactant showed almost equal affinity for both lipid types. Accordingly, a baseline screening BCF value for surfactants (BCF
) can be approximated for ionic surfactants by multiplying
by the phospholipid fraction in tissue, and for non-ionic surfactants by multiplying
by the total lipid fraction. We measured
values for surfactant structures, including linear and branched alkylbenzenesulfonates, an alkylsulfoacetate and an alkylethersulfate, bis(2-ethylhexyl)-surfactants (
, docusate), zwitterionic alkylbetaines and alkylamine-oxides, and a polyprotic diamine. Together with sixty previously published
values for surfactants, structure-activity relationships were derived to elucidate the influence of surfactant specific molecular features on
. For 23 surfactant types, we established the alkyl chain length at which BCF
would exceed the EU REACH bioaccumulation (B) threshold of 2000 L kg
, and would therefore require higher tier assessments to further refine the BCF estimate. Finally, the derived BCF
are compared with measured literature
BCF data where available, suggesting that refinements, most notably reliable estimates of biotransformation rates, are needed for most surfactant types.
Working with and analysis of cationic surfactants can be problematic since aqueous concentrations are difficult to control, both when taking environmental aqueous samples as well as performing ...laboratory work with spiked concentrations. For a selection of 32 amine based cationic surfactants (including C8- to C18-alkylamines, C14-dialkyldimethylammonium, C8-tetraalkylammonium, benzalkonium and pyridinium compounds), the extraction from aqueous samples was studied in detail. Aqueous concentrations were determined using solid phase extraction (SPE; 3 mL/60 mg Oasis WCX-SPE cartridges) with recoveries of ≥80% for 30 compounds, and ≥90% for 16 compounds. Sorption to glassware was evaluated in 120 mL flasks, 40 mL vials and 1.5 mL autosampler vials, using 15 mM NaCl, where the glass binding of simple primary amines and quaternary ammonium compounds increased with alkyl chain length. Sorption to the outside of pipette tips (≤20% of total amount in solution) when sampling aqueous solutions may interfere with accurate measurements. Polyacrylate solid phase microextraction (PA-SPME) fibers with two coating thicknesses (7 and 35 μm) were tested as potential extraction devices. The uptake kinetics, pH-dependence and influence of ionic strength on sorption to PA fibers were studied. Changing medium from 100 mM Na+ to 10 mM Ca2+ decreases Kfw with one order of magnitude. Results indicate that for PA-SPME neutral amines are absorbed rather than adsorbed, although the exact sorption mechanism remains to be elucidated. Further research remains necessary to establish a definitive applicability domain for PA-SPME. However, results indicate that alkyl chain lengths ≥14 carbon atoms and multiple alkyl chains become problematic. A calibration curve should always be measured together with the samples. In conclusion, it seems that for amine based surfactants PA-SPME does not provide the reliability and reproducibility necessary for precise sorption experiments, specifically for alkyl chain lengths beyond 12 carbon atoms.
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•An SPE method to measure 32 amine based cationic surfactants is proposed.•Sorption of cationic surfactants to pipette tips and glassware is quantified.•Charged compounds adsorb to and neutral compounds absorb into PA-SPME fibers.•pH-dependency of sorption of cationic surfactants to PA-SPME fibers is explored.•PA-SPME shows pH-dependency even for quaternary ammonium surfactants.
Abstract
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.
Abstract
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.
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