Robustness in quantifying the abundance of antimicrobial resistance genes in pooled faeces samples from batches of slaughter pigs using metagenomics analysis

Abstract

ObjectivesWith the continued spread of antimicrobial resistance (AMR) in animals, it is important to assess its occurrence throughout a microbiome quantitatively in order to evaluate significantly affecting factors, e.g. antimicrobial usage. Metagenomics methods make it possible to measure the abundance of AMR genes in complex samples such as pooled faeces samples from batches of slaughter pigs. This study was performed to determine the random error in pooled samples from batches of pigs at slaughter and the measurement error from the metagenomics processes. MethodsIn four farms, two pooled samples were obtained from a batch of slaughter pigs by two individual samplers, and each pooled sample was thereafter processed twice. Hierarchically clustered heatmaps were applied to evaluate dissimilarities between samples. The coefficient of variation was used to calculate the percentage difference between samples from the same farm. ResultsResults of the analysis revealed that it was not possible to quantitatively separate the variation arising from sampling and metagenomics processes. They both contributed to the overall measurement error in batches of slaughter pigs. ConclusionSampling of single pigs in 30 randomly selected pig pens within the farms provides a composition representative for frequently occurring AMR genes present within the farms, while rare genes were not dispersed in a similar manner. Aggregating the resistance abundance at gene family or antimicrobial class level will reduce the apparent variation originating from errors in sampling and metagenomics processing.