Expanding one health environmental surveillance of amr: from quantitative detection to comprehensive resistome profiling across oceanic waters

Highlights: • ARGs detected even in remote oceanic regions worldwide • Combined dPCR and targeted metagenomics for AMR surveillance • Over 900 unique ARGs identified across marine environments Within Node 4 (Epidemiology, Monitoring, Mathematical Models) of the INF-ACT project, Work Package 1 focuses on developing innovative One Health genomic surveillance models. In this context, Task 4 – Wastewater and Surface Water: Environmental Surveillance aims to explore the potential of environmental and wastewater monitoring for the detection of emerging viral pathogens and antimicrobial resistance (AMR). As part of this objective, and within the framework of the SEA CARE project, (https://sdgs.un.org/partnerships/sea-care-model-partnership-investigating-global-ocean-strengthen-public-health), our group investigated the presence, abundance, and diversity of antimicrobial resistance genes (ARGs) in marine environments, extending the concept of wastewater surveillance to coastal and open-ocean waters. SEA CARE focuses on assessing the health of marine ecosystems through the integration of microbiological, chemical, and ecological indicators, providing a One Health perspective that links ocean health and public health. The activity was articulated in two complementary phases. The first phase, provided a quantitative baseline of ARGs occurrence across oceanic regions. A total of 43 seawater samples were collected worldwide, including both coastal and open-ocean sites. Using a multiplex digital PCR (dPCR), five ARGs were targeted — sul1, blaTEM, blaOXA48, blaCTX-M-1 and tetA — representing key antibiotic classes (sulfonamides, β-lactams and tetracyclines). The study revealed that ARGs are ubiquitous even in remote marine areas, with higher concentrations detected near coastal zones and maritime routes. The sul1 gene was both ubiquitous and more abundant than other ARGs emerging as an effective proxy for assessing anthropogenic impact on oceans. This work established a global quantitative overview of ARGs in oceanic waters, demonstrating that the marine environment acts both as a reservoir for resistance genes introduced from sources of anthropogenic impact and as a medium for their long-range dispersion through oceanic circulation. Building on these findings, the second phase expanded the analytical scope from a limited set of targets to a comprehensive resistome assessment through targeted metagenomics. We applied the QIAseq xHYB AMR panel (QIAGEN), a hybrid-capture sequencing platform enabling the enrichment and detection of over 2,786 ARGs. This approach bridges dPCR quantification and untargeted metagenomics, enhancing sensitivity for low-abundance genes. A total of 22 marine samples (16 with preliminary results) were analyzed from the Atlantic Ocean, Mediterranean Sea, and Indian Ocean. Between 262 and 957 unique ARGs sequences were identified per sample, indicating remarkable gene diversity. The most frequently detected determinants conferred resistance to aminoglycosides, β-lactams, and tetracyclines. Functional classification of the identified genes revealed that the predominant mechanisms of resistance included antibiotic inactivation (e.g., β-lactamases, aminoglycoside-modifying enzymes), efflux pumps, and target protection or modification. Samples from the Atlantic Ocean exhibited the greatest ARGs and mechanism diversity, suggesting complex environmental and anthropogenic drivers. By integrating quantitative dPCR and targeted metagenomics, this work demonstrates the feasibility and added value of applying environmental genomic surveillance to aquatic ecosystems beyond wastewater.