Metagenomic characterization of antibiotic resistance genes in full-scale reclaimed water distribution systems and corresponding potable systems

Water reclamation provides a valuable resource for meeting non-potable water demands. However, little is known about the potential for wastewater reuse to disseminate antibiotic resistance genes (ARGs). Here, samples were collected seasonally in 2014-2015 from four U.S. utilities' reclaimed and potable water distribution systems before treatment, after treatment, and at five points of use (POU). Shotgun metagenomic sequencing was used to profile the resistome (i.e., full contingent of ARGs) of a subset (n=38) of samples. Four ARGs (qnrA, bla_TEM, vanA, sul1) were quantified by quantitative polymerase chain reaction. Bacterial community composition (via 16S rRNA gene amplicon sequencing), horizontal gene transfer (via quantification of intI1 integrase and plasmid genes), and selection pressure (via detection of metals and antibiotics) were investigated as potential factors governing the presence of ARGs. Certain ARGs were elevated in all (sul1; p≤0.0011) or some (bla_TEM, qnrA; p≤0.0145) reclaimed POU samples compared to corresponding potable samples. Bacterial community composition was weakly correlated with ARGs (Adonis, R²=0.1424-0.1734) and associations were noted between 193 ARGs and plasmid-associated genes. This study establishes that reclaimed water could convey greater abundances of certain ARGs than potable waters and provides observations regarding factors that likely control ARG occurrence in reclaimed water systems.