A Metagenomic Approach for Characterizing Antibiotic Resistance Genes in Specific Bacterial Populations: Demonstration with Escherichia coli in Cattle Manure

Bacterial antibiotic resistance genes in the environment are ubiquitous and can pose different levels of human health risks due to their bacterial host association and subsequent mobility. This study introduced a population metagenomic approach to study ARGs and their mobility in specific bacterial populations through a combination of selective cultivation followed by next generation sequencing and bioinformatic analysis of the combined metagenome of isolates. The high diversity of bacterial antibiotic resistance genes (ARGs) and the different health risks due to their association with different bacterial hosts require environmental ARG risk assessment to have capabilities of both high throughput and host differentiation. Current whole genome sequencing of cultivated isolates is low in throughput, while direct metagenomic next generation sequencing (mNGS) of environmental samples is nonselective with respect to bacterial hosts. This study introduced a population metagenomic approach that combines isolate library construction and mNGS of the population metagenomic DNA, which enables studying ARGs and their association with mobile genetic elements (MGEs) in a specific bacterial population. The population metagenomic approach was demonstrated with the E. coli population in cattle manure, which detected the co-location of multiple ARGs on the same MGEs and their correspondence to the prevalence of resistance phenotypes of the E. coli isolates. When compared with direct mNGS of the cattle manure samples, the E. coli population metagenomes exhibited a significantly different resistome and an overall higher relative abundance of ARGs and horizontal gene transfer risks. IMPORTANCE Bacterial antibiotic resistance genes in the environment are ubiquitous and can pose different levels of human health risks due to their bacterial host association and subsequent mobility. This study introduced a population metagenomic approach to study ARGs and their mobility in specific bacterial populations through a combination of selective cultivation followed by next generation sequencing and bioinformatic analysis of the combined metagenome of isolates. The utility of this approach was demonstrated with the E. coli population in cattle manure samples, which showed that ARGs detected in the E. coli population corresponded to the observed resistance phenotypes, co-location of multiple ARGs on the same mobile genetic elements.