Antimicrobial resistance (AMR) is one of the greatest threats to human health and requires a comprehensive One Health approach for thorough investigation and control. However, surveillance of AMR in bacteria is limited to the clinical setting both in Croatia and worldwide, while little attention is paid to the environment. Despite the important role of groundwater as a drinking water supply, its contribution to the global AMR crisis is still largely unexplored. To fill this gap, the ARES project will adopt a holistic, interdisciplinary approach to investigate the resistome, microbiome and mobilome of four groundwater sources, including three karst sources used for water supply in Croatia. We will apply culture-independent methods such as high-throughput qPCR arrays and standard qPCR in combination with 16S rRNA amplicon sequencing to investigate the resistome and microbiome of groundwater over several seasons. This will allow us to assess temporal trends in the diversity and abundance of the microbiome and resistome in the studied groundwater sources. In addition, we will use culture-dependent approaches to assess the AMR profile of clinically important pathogens (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterococcus spp.) isolated from groundwater and investigate the molecular mechanisms of their resistance and virulence potential using whole-genome sequencing. Finally, the direct capture of antibiotic-resistant plasmids from groundwater bacteria to E. coli and their subsequent complete sequencing will be crucial to draw conclusions on the potential exchange of AMR between environmental reservoirs and the community. We believe that this knowledge will provide important insights for management strategies aimed at mitigating the emergence and spread of AMR and important human pathogens via drinking water reservoirs.

The increasing prevalence of antibiotic resistant bacteria is currently one of the most serious health threats. There is growing evidence that continuous environmental discharge of antibiotics and heavy metals contributes to this issue. The selection pressure imposed by these pollutants has promoted the development and spread of antibiotic resistance among commensal and pathogenic bacteria. Although pharmaceutical waste is recognized as the most important point source of these pollutants in the receiving aquatic environment, its impact on the composition and antibiotic resistance profile of exposed microbial communities is not known. To fill this important research gap, we propose to take an interdisciplinary approach focusing on freshwater sediments impacted by wastewaters of two local pharmaceutical industries. We will assess the prevalence of antibiotics and heavy metals in these sediments and identify potential hot spots for resistance evolution. Antibiotic resistance genes from hot spots and reference sites will be discovered using functional metagenomics. This will lay the groundwork for a quantitative study that will establish spatio-temporal relationships between industrial discharge and the occurrence of antibiotic resistance. This, in combination with direct capturing of resistance plasmids from sediment bacteria to a model pathogens will be critical to draw conclusions about the spread of resistance genes among bacteria. Complete sequencing of transferable plasmids will furthermore assist in identifying novel plasmids that carry clustered antibiotic and heavy metal resistance gene loci. Finally, the impact of discharge on dynamics of sediment community composition will be analyzed by Illumina sequencing of 16S rRNA genes. We believe that the obtained knowledge will have vital implications for the development of effective management strategies to reduce the spread of antibiotics and antibiotic resistance determinants via environmental pathways.

The extensive use of antibiotics for human, veterinary and agricultural purposes results in their continuous release into the environment. There is a widespread concern that this environmental pollution contributes to the increasingly problematic development of antibiotic resistance genes and bacteria, which reduce the therapeutical effectiveness of antibiotics. Many studies have shown that the elimination of antibiotics in conventional wastewater treatment plants (WWTPs) is poor simply because these units have not been designed to remove such compounds. Consequently, WWTPs are considered as one of the main ˝hotspots˝ for the release of antibiotics into the aquatic environment. As the maximum allowable concentrations of antibiotics in environmental waters are not regulated yet, scientists are concerned about negative effects of these compounds on ecosystems and public health. Therefore, in order to reduce the prevalence and dispersion of antibiotics from WWTPs, it is imperative to better understand the mechanisms relevant for their removal within WWTPs.