An Extraction Method to Quantify the Fraction of Extracellular and Intracellular Antibiotic Resistance Genes in Aquatic Environments

Abstract

As the abundance and diversity of antibiotic resistance genes increases in the environment, there is a concurrent increase in the threat to public and ecosystem health. Extracellular antibiotic resistance genes (eARGs) are cell-free DNA that can promote the development of antibiotic resistance via transformation by competent bacterial cells. Despite this role, eARGs have not been well characterized in different environmental waters. Their small size and low concentrations in some aquatic environments render them difficult to extract. The aim of this research was to modify an eARG extraction method to determine the abundance of both eARGs and intracellular ARGs (iARGs) in the same water sample. The modified method, consisting of sequential filtration to separate iARGs from eARGs, adsorption-elution with aluminum hydroxide–coated silica gel, and precipitation, extracted eARGs and iARGs with a recovery rate between 79.5% and 99.0%. The novel method was then utilized for the extraction of the extracellular and intracellular fractions of four ARGs, one mobile genetic element, and the 16S rRNA in tap water, river surface water, lake surface water, stormwater, and wastewater effluent. This is the first instance in which the extracellular and intracellular fractions of the 16S rRNA, intI1, blaTEM, ermF, sul1, and tetC genes in stormwater and lake surface water are reported. In addition, this modified method enabled the quantification of the extracellular concentration of the erythromycin resistance gene ermF in environmental waters for the first time; the gene’s abundance ranged from 1.26×105 to 8.82×106 gene copies/L across the aquatic waters sampled. The extracellular abundance of the mobile genetic element intI1, moreover, was quantified in tap water (7.00×104 gene copies/L) for the first time. The validation and application of this method to diverse environmental matrices should allow for further research to be conducted to better understand the role of eARGs in the spread of antibiotic resistance.