{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T05:18:42Z","timestamp":1788585522304,"version":"build-2803163510"},"reference-count":38,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T00:00:00Z","timestamp":1788134400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Ministero della Salute","doi-asserted-by":"publisher","award":["SG-2021-12375470"],"award-info":[{"award-number":["SG-2021-12375470"]}],"id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]},{"award":["SG-2021-12375470"],"award-info":[{"award-number":["SG-2021-12375470"]}],"id":[{"id":"https:\/\/ror.org\/00789fa95","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background\/Objectives: Wastewater-based epidemiology (WBE) has emerged as a valuable One Health approach for monitoring antimicrobial resistance (AMR) at the population level. Although quantitative PCR (qPCR) and shotgun (SG) metagenomics are widely used for wastewater surveillance, studies integrating these complementary approaches remain limited. This study aimed to investigate the occurrence, seasonal dynamics, and diversity of antimicrobial resistance genes (ARGs) in municipal wastewater from Central Italy by combining targeted qPCR and SG metagenomic sequencing. Methods: Influent wastewater samples were collected monthly from eight municipal wastewater treatment plants in Central Italy between April 2025 and March 2026. Clinically relevant antimicrobial resistance genes were quantified by quantitative real-time PCR, while SG metagenomic sequencing was used to characterize resistome composition, resistance gene families, and ARG sequence diversity using bioinformatic pipelines. Results: All investigated ARGs were detected in every sample. Significant seasonal variation was observed for all investigated markers, including qnrS, blaKPC, blaCTX-M and intI1. Metagenomic analysis revealed broadly similar resistome profiles across sampling sites and time points, dominated by resistance genes to macrolide\u2013lincosamide\u2013streptogramin, aminoglycosides, \u03b2-lactams, and fluoroquinolones. High sequence diversity was observed within the dominant ARG families, highlighting the complementary value of SG metagenomics for comprehensive resistome characterization. Conclusions: The integration of targeted qPCR and SG metagenomics provided a comprehensive characterization of antimicrobial resistance in municipal wastewater. While qPCR enabled sensitive quantification of clinically relevant ARGs and revealed seasonal trends, metagenomics expanded resistome characterization by identifying dominant resistance classes, gene families, and sequence variants. These findings support the implementation of integrated molecular approaches for routine wastewater-based AMR surveillance within a One Health framework.<\/jats:p>","DOI":"10.3390\/antibiotics15090850","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T10:32:18Z","timestamp":1788258738000},"page":"850","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated qPCR and Shotgun Metagenomics for Surveillance of Antimicrobial Resistance in Municipal Wastewater from Central Italy"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-1725-3956","authenticated-orcid":false,"given":"Federica Di","family":"Timoteo","sequence":"first","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emanuela","family":"Di Giulio","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4795-8323","authenticated-orcid":false,"given":"Marco","family":"Di Domenico","sequence":"additional","affiliation":[{"name":"National Reference Centre (NRC) for Whole Genome Sequencing of Microbial Pathogens: Database and Bioinformatics Analysis (GENPAT), Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise (IZSAM), 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1750-6695","authenticated-orcid":false,"given":"Barbara","family":"Secondini","sequence":"additional","affiliation":[{"name":"National Reference Centre (NRC) for Whole Genome Sequencing of Microbial Pathogens: Database and Bioinformatics Analysis (GENPAT), Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise (IZSAM), 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2044-7024","authenticated-orcid":false,"given":"Francesca","family":"Marotta","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-3834-073X","authenticated-orcid":false,"given":"Giusy","family":"Matteucci","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katiuscia","family":"Zilli","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teresa","family":"Romualdi","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dalia","family":"Palmieri","sequence":"additional","affiliation":[{"name":"Prevention, Food Safety and Veterinary Service, Abruzzo Region Health Department, 65124 Pescara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuliano","family":"Garofolo","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7263-3814","authenticated-orcid":false,"given":"Anna","family":"Janowicz","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Campylobacter, Istituto Zooprofilattico Sperimentale dell\u2019Abruzzo e del Molise \u201cG. Caporale\u201d, 64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,31]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2022). WHO Strategic Priorities on Antimicrobial Resistance: Preserving Antimicrobials for Today and Tomorrow, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/nrmicro3439","article-title":"Tackling antibiotic resistance: The environmental framework","volume":"13","author":"Berendonk","year":"2015","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","unstructured":"European Parliamentary Research Service (2023). Stepping Up EU Action to Combat Antimicrobial Resistance: The \u201cOne Health\u201d Approach, European Parliamentary Research Service."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"146964","DOI":"10.1016\/j.scitotenv.2021.146964","article-title":"Monitoring antibiotic resistance genes in wastewater treatment: Current strategies and future challenges","volume":"783","author":"Nguyen","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1016\/j.envint.2018.03.044","article-title":"Antibiotic resistance in wastewater treatment plants: Tackling the black box","volume":"115","author":"Manaia","year":"2018","journal-title":"Environ. Int."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kalli, M., Noutsopoulos, C., and Mamais, D. (2023). The fate and occurrence of antibiotic-resistant bacteria and antibiotic resistance genes during advanced wastewater treatment and disinfection: A review. Water, 15.","DOI":"10.3390\/w15112084"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1016\/j.scitotenv.2013.01.032","article-title":"Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: A review","volume":"447","author":"Rizzo","year":"2013","journal-title":"Sci. Total Environ."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1052","DOI":"10.1016\/j.scitotenv.2018.08.130","article-title":"Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread: A review","volume":"648","author":"Krzeminski","year":"2019","journal-title":"Sci. Total Environ."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1038\/s41467-019-08853-3","article-title":"Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage","volume":"10","author":"Hendriksen","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"161419","DOI":"10.1016\/j.scitotenv.2023.161419","article-title":"Understanding the distribution of antibiotic resistance genes in an urban community using a wastewater-based epidemiological approach","volume":"868","author":"Javvadi","year":"2023","journal-title":"Sci. Total Environ."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Smith, A.M., Ramudzulu, M., Munk, P., Avot, B.J., Esterhuyse, K.C., Van Blerk, N., Kwenda, S., and Sekwadi, P. (2024). Metagenomics analysis of sewage for surveillance of antimicrobial resistance in South Africa. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0309409"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Elbait, G.D., Daou, M., Abuoudah, M., Elmekawy, A., Hasan, S.W., Everett, D.B., Alsafar, H., Henschel, A., and Yousef, A.F. (2024). Comparison of qPCR and metagenomic sequencing methods for quantifying antibiotic resistance genes in wastewater. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0298325"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"121989","DOI":"10.1016\/j.watres.2024.121989","article-title":"National-scale antimicrobial resistance surveillance in wastewater: A comparative analysis of HT qPCR and metagenomic approaches","volume":"262","author":"Knight","year":"2024","journal-title":"Water Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"fiw060","DOI":"10.1093\/femsec\/fiw060","article-title":"Seasonality of antibiotic prescriptions for outpatients and resistance genes in sewers and wastewater treatment plant outflow","volume":"92","author":"Caucci","year":"2016","journal-title":"FEMS Microbiol. Ecol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"122598","DOI":"10.1016\/j.watres.2024.122598","article-title":"Seasonal variation of antibiotic resistance genes in activated sludge of a full-scale municipal wastewater treatment plant: Contribution of activated sludge functional taxa and clinically relevant taxa","volume":"268","author":"Guruge","year":"2025","journal-title":"Water Res."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Malcom, H.B., and Bowes, D.A. (2025). Use of wastewater to monitor antimicrobial resistance trends in communities and implications for wastewater-based epidemiology: A review of the recent literature. Microorganisms, 13.","DOI":"10.3390\/microorganisms13092073"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"173217","DOI":"10.1016\/j.scitotenv.2024.173217","article-title":"Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR","volume":"933","author":"Bonomo","year":"2024","journal-title":"Sci. Total Environ."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.watres.2019.04.031","article-title":"Effluents of wastewater treatment plants promote the rapid stabilization of the antibiotic resistome in receiving freshwater bodies","volume":"158","author":"Corno","year":"2019","journal-title":"Water Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"35294","DOI":"10.1007\/s11356-022-24650-w","article-title":"Occurrence of antibiotic-resistant bacteria and resistance genes in the urban water cycle","volume":"30","author":"Bonetta","year":"2023","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.watres.2016.02.049","article-title":"Co-occurrence of integrase 1, antibiotic and heavy metal resistance genes in municipal wastewater treatment plants","volume":"94","author":"Eckert","year":"2016","journal-title":"Water Res."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Magnano San Lio, R., Maugeri, A., Barchitta, M., Favara, G., La Rosa, M.C., La Mastra, C., and Agodi, A. (2025). Monitoring antibiotic resistance in wastewater: Findings from three treatment plants in Sicily, Italy. Int. J. Environ. Res. Public Health, 22.","DOI":"10.3390\/ijerph22030351"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e70319","DOI":"10.1002\/mbo3.70319","article-title":"Wastewater-based assessment of antimicrobial resistance and bacterial communities in urban and rural areas in the Province of Trento (Italy)","volume":"15","author":"Petricciuolo","year":"2026","journal-title":"MicrobiologyOpen"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"125051","DOI":"10.1016\/j.envpol.2024.125051","article-title":"Antibiotic resistance and pathogen spreading in a wastewater treatment plant designed for wastewater reuse","volume":"363","author":"Bonetta","year":"2024","journal-title":"Environ. Pollut."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"126096","DOI":"10.1016\/j.envpol.2025.126096","article-title":"High-throughput qPCR profiling of antimicrobial resistance genes and bacterial loads in wastewater and receiving environments","volume":"373","author":"Silvester","year":"2025","journal-title":"Environ. Pollut."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"127943","DOI":"10.1016\/j.envpol.2026.127943","article-title":"Longitudinal wastewater metagenomics reveals distinct environmental and anthropogenic associations with resistance, virulence, and viral communities","volume":"397","author":"Freitas","year":"2026","journal-title":"Environ. Pollut."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1269","DOI":"10.1038\/ismej.2014.226","article-title":"Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution","volume":"9","author":"Gillings","year":"2015","journal-title":"ISME J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1016\/j.watres.2017.07.002","article-title":"Metagenomic analysis reveals wastewater treatment plants as hotspots of antibiotic resistance genes and mobile genetic elements","volume":"123","author":"Guo","year":"2017","journal-title":"Water Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Magnano San Lio, R., Maugeri, A., Barchitta, M., Favara, G., La Rosa, M.C., La Mastra, C., Ferrante, M., and Agodi, A. (2026). The wastewater resistome: A shotgun metagenomics analysis of urban treatment plants in Sicily. Antibiotics, 15.","DOI":"10.3390\/antibiotics15020148"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4763","DOI":"10.2147\/IDR.S370669","article-title":"Metagenomic analysis of urban wastewater treatment plant effluents in Tokyo","volume":"15","author":"Sekizuka","year":"2022","journal-title":"Infect. Drug Resist."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"D744","DOI":"10.1093\/nar\/gkac1047","article-title":"MEGARes and AMR++, v3.0: An updated comprehensive database of antimicrobial resistance determinants and an improved software pipeline for classification using high-throughput sequencing","volume":"51","author":"Bonin","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1186\/s13073-025-01480-2","article-title":"Simultaneous detection of pathogens and antimicrobial resistance genes with the open-source, cloud-based CZ ID platform","volume":"17","author":"Lu","year":"2025","journal-title":"Genome Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e01672-25","DOI":"10.1128\/mbio.01672-25","article-title":"Metagenomic analysis after selective culture enrichment of hospital and community wastewater enhances antimicrobial resistance gene detection","volume":"16","author":"Acosta","year":"2025","journal-title":"mBio"},{"key":"ref_33","first-page":"356","article-title":"Sequencing-based methods and resources to study antimicrobial resistance","volume":"20","author":"Boolchandani","year":"2019","journal-title":"Nat. Rev. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1186\/s40168-021-01216-5","article-title":"Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge","volume":"10","author":"Dai","year":"2022","journal-title":"Microbiome"},{"key":"ref_35","unstructured":"Federici, E., Petricciuolo, M., La Rosa, G., Bonanno Ferraro, G., and GdL Geni di Antibiotico Resistenza (2025). Ricerca e Quantificazione di Geni di Antibiotico Resistenza nei Reflui Urbani non Trattati, Ministero della Salute."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments","volume":"55","author":"Bustin","year":"2009","journal-title":"Clin. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"D574","DOI":"10.1093\/nar\/gkw1009","article-title":"MEGARes: An antimicrobial resistance database for high-throughput sequencing","volume":"45","author":"Lakin","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"giaa111","DOI":"10.1093\/gigascience\/giaa111","article-title":"IDseq\u2014An open-source, cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring","volume":"9","author":"Kalantar","year":"2020","journal-title":"GigaScience"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/9\/850\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T05:01:08Z","timestamp":1788584468000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/9\/850"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,31]]},"references-count":38,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["antibiotics15090850"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics15090850","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,31]]}}}