DisPersal of AntIbiotic Resistance and antibiotics in Water ecosystems and Influence on liveStock and aquatic wildlife

The PAIRWISE project aims to generate knowledge about antimicrobial resistance (AMR) as a contaminant in aquatic environments, wildlife, and livestock. PAIRWISE focuses on the spread and dynamics of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARG), and antibiotics (ATB) in aquatic environments affected by wastewater treatment plants (WWTPs). The main objectives are to understand: i) the spread of ARB, ARG, and ATB in surface waters downstream of WWTPs; ii) the transmission of ARB and ARG in livestock associated with surface waters affected by WWTPs; iii) the role of aquatic birds in the spread of ARB and ARG. Topics from the JPI call addressed by PAIRWISE include: sources and fate of ARB, ARG, and ATB; identification of ARG and ARB useful as indicators; insight into the fate and transport of ARB, ARG, and ATB in and from aquatic ecosystems; and the role of aquatic birds in such cases. PAIRWISE will investigate the impact of AMR in the iodine use environment and improve understanding of the spread and maintenance of AMR at the interface between humans, wildlife and livestock in the 'One Health' perspective. It will generate knowledge for policymakers and end-users, facilitating informed decisions on intervention strategies. NIVA is leading the work on the spread of ARGs, ARBs and ATBs in water. The main tasks include the development of method protocols and harmonisation, characterisation of study sites; determining background levels, abundances and diversity of selected ARGs, ARBs, ATBs in aquatic environments; investigation of sources and levels; and evaluation of influencing factors and spread of ARGs, ARBs and ATBs with regard to environmental, geographical, climatic and temporal dimensions. To date, water-related sampling campaigns have been carried out in two locations in Norway and the samples are being prepared for the dedicated AMR analyses. Sampling of water as well as feces from grazing cattle and waterfowl was carried out at several sampling points and times during 2022-2023. In total, approximately 450 water samples, 900 cattle feces samples and 2100 bird feces samples were collected. In Norway, samples were mainly taken from different types of aquatic environments at two locations. The collected water samples included freshwater, brackish water, seawater and wastewater, all of which have been subjected to dedicated AMR analyses. Bacterial cultivation was performed to identify growth of Escherichia coli and Klebsiella pneumoniae bacteria that produce extended-spectrum beta-lactamases (ESBL) or carbapenemases (CARBA), indicating resistance to important antibiotics (ATB). Such bacteria were further characterized by antibacterial susceptibility testing (AST) and whole genome sequencing (WGS). Water and faecal samples were also analysed for the presence of a range of ARGs using high-throughput HT-qPCR smartchip assays. Furthermore, the presence of ATB in water samples is analysed using HPLC-MS-MS. In Spain and Sweden, approximately 120 waterbirds (mainly black-backed gulls) were fitted with GPS transmitters and their movements were tracked in relation to the presence of wastewater treatment plants (WWTPs) and similar sites.