Antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) abundance in animal faeces, meat, production environments and human faeces in Europe

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.

Figure 1.

Relative abundance of four targets [aph(3′)-III, erm(B), sul2, tet(W)] in all samples. Relative abundance of gene target was calculated by log₁₀ (gene copies/16S copies). Asterisk shows the mean by sample type. Pooled faeces, slaughterhouse pig and broiler faeces, and slaughterhouse carcass samples were not included in this figure.

Figure 2.

Relative abundance of four targets [aph(3′)-III, erm(B), sul2, tet(W)] in samples related to pig production in the Netherlands. Relative abundance of gene target was calculated by log₁₀ (gene copies/16S copies). Only pig farms and slaughterhouses in the Netherlands were involved. Human faeces of control subjects were collected from the ‘Lifelines’ cohort in the Netherlands. Asterisk shows the mean by sample type in the Netherlands.

Figure 3.

Relative abundance of four targets [aph(3′)-III, erm(B), sul2, tet(W)] in samples related to broiler production in Germany. Relative abundance of gene target was calculated by log₁₀ (gene copies/16S copies). Only broiler farms and slaughterhouses in Germany were involved. Asterisk shows the mean by sample type in Germany.

Figure 4.

PCA biplot of relative ARG abundance in animal faecal samples. ARG targets: aph(3′)-III, erm(B), sul2, tet(W). Relative abundance of gene target was calculated by log₁₀ (gene copies/16S copies). Symmetric scaling was used. Circles indicate 95% confidence ellipses that were computed with the assumption of multivariate normal distribution of the data.

Figure 5.

Variance component percentages of relative ARG abundance in faecal samples from pigs and broilers. Relative abundance of gene target was calculated by log₁₀ (gene copies/16S copies). Variance percentages of three components (between-country variance, between-farm variance and within-farm variance) were calculated using VCA.

Figure 6.

Spearman’s rank correlation of relative ARG abundance and ARG FPKM abundance in pooled faecal samples from pig and broiler farms. ARG targets: aph(3′)-III, erm(B), sul2, tet(W). Relative abundance of gene target was calculated by log₁₀ (gene copies/16S copies). FPKM was log₁₀ transformed after adding a pseudocount of 1. BE, Belgium; BG, Bulgaria; DE, Germany; DK, Denmark; ES, Spain; FR, France; IT, Italy; NL, the Netherlands; PL, Poland.