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 o...
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | Environmental Microbiome |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s40793-024-00557-6 |
| _version_ | 1827315756548751360 |
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| author | Lucia Maestre-Carballa Vicente Navarro-López Manuel Martinez-Garcia |
| author_facet | Lucia Maestre-Carballa Vicente Navarro-López Manuel Martinez-Garcia |
| author_sort | Lucia Maestre-Carballa |
| collection | DOAJ |
| description | 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 (≈105–7 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. |
| first_indexed | 2024-04-24T23:02:40Z |
| format | Article |
| id | doaj.art-77e350df026b4b0582ea00b5eac866f0 |
| institution | Directory Open Access Journal |
| issn | 2524-6372 |
| language | English |
| last_indexed | 2024-04-24T23:02:40Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Environmental Microbiome |
| spelling | doaj.art-77e350df026b4b0582ea00b5eac866f02024-03-17T12:38:51ZengBMCEnvironmental Microbiome2524-63722024-03-0119111210.1186/s40793-024-00557-6City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomicsLucia Maestre-Carballa0Vicente Navarro-López1Manuel Martinez-Garcia2Department of Physiology, Genetics, and Microbiology, University of AlicanteClinical Microbiology and Infectious Disease Unit, Hospital Universitario VinalopóDepartment of Physiology, Genetics, and Microbiology, University of AlicanteAbstract 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 (≈105–7 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.https://doi.org/10.1186/s40793-024-00557-6Antibiotic resistance geneMetagenomicsDigital PCRDPCRARGBacteria |
| spellingShingle | Lucia Maestre-Carballa Vicente Navarro-López Manuel Martinez-Garcia City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics Environmental Microbiome Antibiotic resistance gene Metagenomics Digital PCR DPCR ARG Bacteria |
| title | City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics |
| title_full | City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics |
| title_fullStr | City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics |
| title_full_unstemmed | City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics |
| title_short | City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics |
| title_sort | city scale monitoring of antibiotic resistance genes by digital pcr and metagenomics |
| topic | Antibiotic resistance gene Metagenomics Digital PCR DPCR ARG Bacteria |
| url | https://doi.org/10.1186/s40793-024-00557-6 |
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