City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics

Abstract

Background. Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics. Methods. ARG hot-spots were sampled from the water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by digital PCR and metagenomics. To complement, the potential ARG dispersion through airborne aerosols was addressed. Results. The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP input (up to »6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. ARG abundance in DNA and RNA viruses was up to three order of magnitude lower compared to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (»125-225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000-18,600 copies per ng of sewage DNA (»105-7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. dPCR data suggest that despite a large amount of gene copies are discharged to nature, marine autochthonous bacteria do not seem to significantly acquire these ARGs. Airborne particle metagenomics showed a broad range of ARGs potentially disseminated with higher abundances at the WWTP (outdoor; 32 ARG hits per Gb and m3) followed by the indoor hospital sample. Conclusions. dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2<0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments. Finally, non-ventilated and populated environments could represent a human thread in ARG spreading that should be considered within the One-Health perspective.