PROBING ANTIBIOTIC RESIDUES AND RESISTANCE TRANSFER IN AQUATIC ENVIRONMENTS

Aquatic ecosystems are subjected to intense pressure by human activities in and around the aquatic environment. A plethora of pernicious substances can persist in the environment for long periods and some of these, such as residues of antibiotics, are known to contribute to increased resistance to antibiotics in bacterial populations. Antibiotic resistance is increasing in pathogenic, commensal, and environmental bacteria. The problem has escalated by recent overuse of antibiotics, causing selective pressure on bacterial populations, both in antibiotic-treated human and (farmed) animal hosts, and by contamination of residues in the environment. Today we face a real risk that the emergence of multi-resistance in bacterial pathogens may render some infections untreatable. This project aims to improve our knowledge on presence of bacteria and plasmids with antibiotic resistance genes (ARG) and concentrations of antibiotic residues in various environments, and to better understand how they are selected for and spread in the environment. Sampling will include groundwater, surface water, wastewater, marine water environments in the North Sea and the Atlantic including ports, and aquaculture facilities. Samples will be collected from sites with assumed high and low load of antibiotic residues (influenced by human activity). Culture – and sequencing methods will be used to identify resistance properties. Transfer of ARG plasmids will be analyzed using the indicator bacteria Escherichia coli, Vibrio, and Shewanella as recipients. UHPLC-MS/MS will quantify the levels of different antibiotics in water and sediment samples. We hypothesize that local water microbiota, antibiotic residues, and recipients will affect the type of plasmids transferred. The consequences of antibiotic treatment with respect to changes in the community structure of water microbiota, will be tested experimentally by use of RAS microcosms. Effects on bacteria by acquired plasmids will be analyzed to evaluate the effect on fitness and virulence in an animal host model. The project will determine common ARG plasmids in Europe and their inherent properties as a fundament to prevent their dissemination. In the initial project period, procedures for water sampling, culture and sequence-based methods are harmonized between the partners and sampling is initiated. The consortium has successfully collected samples from several lake and river water locations in Sweden, Norway, and Belgium, and from marine locations at Gran Canary and in Norway. Additionally, wastewater has been sampled in Sweden, Norway, and Gran Canary. Collectively, the project has generated a substantial strain collection of the chosen indicator bacteria, and the isolates are now analysed with respect to antibiotic resistance properties. Preliminary results points towards low levels of indicator bacteria and phenotypic antibiotic resistance in water samples from environments with assumed low anthropogenic impact. In contrast, a majority of the E. coli isolates from wastewater samples analysed so far were ESBL (extended spectrum beta-lactamase) positive and multiresistant, and residues of several clinically relevant antibiotics are detected in wastewater. The project is now entering the next phase focusing on sequencing of selected resistant bacterial isolates and studies of horizontal gene transfer of antibiotic resistance genes and the impact of retrieved mobile genetic elements on recipient bacteria.

This project aims to improve our knowledge on presence of bacteria and plasmids with antibiotic resistance genes (ARG) and concentrations of antibiotic residues in various environments, and to better understand how they are selected for and spread in the environment. Sampling will include groundwater, surface water, wastewater, marine water environments in the North Sea and the Atlantic including ports, and aquaculture facilities. Samples will be collected from sites with high and low load of antibiotic residues. Culture- and sequencing methods will be used to identify ARG. Transfer of ARG plasmids will be analyzed using the indicator bacteria E. coli, Vibrio, and Shewanella as recipients. LC-MC will quantify the levels of different antibiotics. We hypothesize that local water microbiota, antibiotic residues, and recipients will affect the type of plasmids transferred. The consequences of antibiotic treatment with respect to changes in the community structure of water microbiota, will be tested experimentally by use of RAS microcosms. Effects on bacteria by acquired plasmids will be analyzed in experimental lab systems, to evaluate the effect on fitness and virulence of an animal host model with a shrimp (Artemia). The project will determine common ARG plasmids in Europe and their inherent properties as a fundament to prevent their dissemination.