Metagenomics reveal triclosan-induced changes in the antibiotic resistome of anaerobic digesters

Highlights: • ARGs resistomes of anaerobic digesters were altered by TCS additions. • The changes in the resistome were dependent on TCS concentration. • Previously known ARGs, including fabV, were enriched under the highest TCS concentration. • Shotgun metagenomics revealed novel genes potentially involved in TCS resistance. • Novel genes include metal resistance genes, transporters, and mobile elements. Triclosan (TCS) is a broad-spectrum antimicrobial used in a variety of consumer products. While it was recently banned from hand soaps in the US, it is still a key ingredient in a top-selling toothpaste. TCS is a hydrophobic micropollutant that is recalcitrant under anaerobic digestion thereby resulting in high TCS concentrations in biosolids. The objective of this study was to determine the impact of TCS on the antibiotic resistome and potential cross-protection in lab-scale anaerobic digesters using shotgun metagenomics. It was hypothesized that metagenomics would reveal selection for antibiotic resistance genes (ARGs) not previously found in pure culture studies or mixed-culture studies using targeted qPCR. In this study, four different levels of TCS were continuously fed to triplicate lab-scale anaerobic digesters to assess the effect of TCS levels on the antibiotic resistance gene profiles (resistome). Blasting metagenomic reads against antibiotic/metal resistance gene database (BacMet) revealed that ARG diversity and abundance changed along the TCS concentration gradient. While loss of bacterial diversity and digester function were observed in the digester treated with the highest TCS concentration, FabV, which is a known TCS resistance gene, increased in this extremely high TCS environment. The abundance of several other known ARG or metal resistance genes (MRGs), including corA and arsB, also increased as the concentrations of TCS increased. Analysis of other functional genes using SEED database revealed the increase of potentially key genes for resistance including different types of transporters and transposons. These results indicate that antimicrobials can alter the abundance of multiple resistance genes in anaerobic digesters even when function (i.e. methane production) is maintained. This study also suggests that enriched ARGs could be released into environments with biosolids land application.