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

Lucia Maestre-Carballa; Vicente Navarro-López; Manuel Martinez-Garcia

doi:10.1186/s40793-024-00557-6

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

Environ Microbiome. 2024 Mar 15;19(1):16. doi: 10.1186/s40793-024-00557-6.

Authors

Lucia Maestre-Carballa^{1

2}, Vicente Navarro-López³, Manuel Martinez-Garcia^{4

5}

Affiliations

¹ Department of Physiology, Genetics, and Microbiology, University of Alicante, Carretera San Vicente del Raspeig, San Vicente del Raspeig, Alicante, 03690, Spain.
² Instituto Multidisciplinar para el Estudio del Medio Ramon Margalef, University of Alicante, San Vicente del Raspeig, Alicante, 03690, Spain.
³ Clinical Microbiology and Infectious Disease Unit, Hospital Universitario Vinalopó, Elche, Spain.
⁴ Department of Physiology, Genetics, and Microbiology, University of Alicante, Carretera San Vicente del Raspeig, San Vicente del Raspeig, Alicante, 03690, Spain. m.martinez@ua.es.
⁵ Instituto Multidisciplinar para el Estudio del Medio Ramon Margalef, University of Alicante, San Vicente del Raspeig, Alicante, 03690, Spain. m.martinez@ua.es.

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 urban 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 dPCR and metagenomics.

Results: The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated 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 and 18,600 copies per ng of sewage DNA (≈10^5-7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point.

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.

Keywords: sul2; tetW; ARG; Antibiotic resistance gene; Bacteria; DPCR; Digital PCR; Metagenomics; Resistome; Virus; Wastewater.