The antimicrobial resistome in relation to antimicrobial use and biosecurity in pig farming, a metagenome-wide association study in nine European countries

Objectives Previous studies in food-producing animals have shown associations between antimicrobial use (AMU) and resistance (AMR) in specifically isolated bacterial species. Multi-country data are scarce and only describe between-country differences. Here we investigate associations between the pig faecal mobile resistome and characteristics at the farm-level across Europe. Methods A cross-sectional study was conducted among 176 conventional pig farms from nine European countries. Twenty-five faecal samples from fattening pigs were pooled per farm and acquired resistomes were determined using shotgun metagenomics and the Resfinder reference database, i.e. the full collection of horizontally acquired AMR genes (ARGs). Normalized fragments resistance genes per kilobase reference per million bacterial fragments (FPKM) were calculated. Specific farm-level data (AMU, biosecurity) were collected. Random-effects meta-analyses were performed by country, relating farm-level data to relative ARG abundances (FPKM). Results Total AMU during fattening was positively associated with total ARG (total FPKM). Positive associations were particularly observed between widely used macrolides and tetracyclines, and ARGs corresponding to the respective antimicrobial classes. Significant AMU-ARG associations were not found for -lactams and only few colistin ARGs were found, despite high use of these antimicrobial classes in younger pigs. Increased internal biosecurity was directly related to higher abundances of ARGs mainly encoding macrolide resistance. These effects of biosecurity were independent of AMU in mutually adjusted models. Conclusions Using resistome data in association studies is unprecedented and adds accuracy and new insights to previously observed AMU-AMR associations. Major components of the pig resistome are positively and independently associated with on-farm AMU and biosecurity conditions

Associations between antimicrobial use and the faecal resistome on broiler farms from nine European countries

Objectives: To determine associations between farm-and flock-level antimicrobial usage (AMU), farmbiosecurity status and the abundance of faecal antimicrobial resistance genes (ARGs) on broiler farms. Methods: In the cross-sectional pan-European EFFORT study, conventional broiler farms were visited and faeces, AMU information and biosecurity records were collected. The resistomes of pooled faecal samples were determined by metagenomic analysis for 176 farms. A meta-analysis approach was used to relate total and classspecific ARGs (expressed as fragments per kb reference per million bacterial fragments, FPKM) to AMU (treatment incidence per DDD, TIDDDvet) per country and subsequently across all countries. In a similar way, the association between biosecurity status (Biocheck. UGent) and the resistome was explored. Results: Sixty-six (38%) flocks did not report group treatments but showed a similar resistome composition and roughly similar ARG levels to antimicrobial-treated flocks. Nevertheless, we found significant positive associations between beta-lactam, tetracycline, macrolide and lincosamide, trimethoprim and aminoglycoside antimicrobial flock treatments and ARG clusters conferring resistance to the same class. Similar associations were found with purchased products. In gene-level analysis for beta-lactams and macrolides, lincosamides and streptogramins, a significant positive association was found with the most abundant gene clusters blaTEM and erm(B). Little evidence was found for associations with biosecurity. Conclusions: The faecal microbiome in European broilers contains a high diversity of ARGs, even in the absence of current antimicrobial selection pressure. Despite this, the relative abundance of genes and the composition of the resistome is positively related to AMU in European broiler farms for several antimicrobial classes

Erratum to: Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries (Nature Microbiology, (2018), 3, 8, (898-908), 10.1038/s41564-018-0192-9)

In the version of this Article originally published, the surname of author Oksana Lukjancenko was spelt incorrectly as ‘Lukjacenko’. This has now been corrected

Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries

Antimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. The use of antimicrobials in livestock selects for AMR that can subsequently be transferred to humans. This flow of AMR between reservoirs demands surveillance in livestock and in humans. We quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, sequencing more than 5,000 Gb of DNA using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a second database constructed for this study, consisting of AMR genes identified through screening environmental DNA. The pig and poultry resistomes were very different in abundance and composition. There was a significant country effect on the resistomes, more so in pigs than in poultry. We found higher AMR loads in pigs, whereas poultry resistomes were more diverse. We detected several recently described, critical AMR genes, including mcr-1 and optrA, the abundance of which differed both between host species and between countries. We found that the total acquired AMR level was associated with the overall country-specific antimicrobial usage in livestock and that countries with comparable usage patterns had similar resistomes. However, functionally determined AMR genes were not associated with total drug use

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

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Author Correction: Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries

In the version of this Article originally published, the surname of author Oksana Lukjancenko was spelt incorrectly as ‘Lukjacenko’. This has now been corrected.</p