Deciphering the extracellular and intracellular antibiotic resistance genes in multiple environments reveals the persistence of extracellular ones

Abstract

The extracellular and intracellular antibiotic resistance genes (eARGs and iARGs) together constitute the entire resistome in environments. However, the systematic analysis of eARGs and iARGs was still inadequate. Three kinds of environments, i.e., livestock manure, sewage sludge, and lake sediment, were analyzed to reveal the comprehensive characteristics of eARGs and iARGs. Based on the metagenomic data, the diversities, relative abundances, and compositions of eARGs and iARGs were similar. The extracellular and intracellular integrons and insertion sequences (ISs) also did not show any significant differences. However, the degree and significance of the correlation between total relative abundances of integrons/ISs and ARGs were lower outside than inside the cells. Gene cassettes carried by class 1 integron were amplified in manure and sludge samples, and sequencing results showed that the identified ARGs extracellularly and intracellularly were distinct. By analyzing the genetic contexts, most ARGs were found located on chromosomes. Nevertheless, the proportion of ARGs carried by plasmids increased extracellularly. qPCR was employed to quantify the absolute abundances of sul1, sul2, tetO, and tetW, and their extracellular proportions were found highest in sludge samples. These findings together raised the requirements of considering eARGs and iARGs separately in terms of risk evaluation and removal management.