At present, very little is known about the fate and persistence of multiresistant bacteria (MRB) and their resistance genes in natural aquatic environments. Treated, but partly also untreated sewage of the city of Lausanne, Switzerland is discharged into Vidy Bay (Lake Geneva) resulting in high levels of contamination in this part of the lake. In the present work we have studied the prevalence of MRB and resistance genes in the wastewater stream of Lausanne. Samples from hospital and municipal raw sewage, treated effluent from Lausanne’s wastewater treatment plant (WTP) as well as lake water and sediment samples obtained close to the WTP outlet pipe and a remote site close to a drinking water pump were evaluated for the prevalence of MRB. Selected isolates were identified (16S rRNA gene fragment sequencing) and characterized with regards to further resistances, resistance genes, and plasmids. Mostly, studies investigating this issue have relied on cultivation-based approaches. However, the limitations of these tools are well known, in particular for environmental microbial communities, and cultivation-independent molecular tools should be applied in parallel in order to take non-culturable organisms into account. Here we directly quantified the sulfonamide resistance genes sul1 and sul2 from environmental DNA extracts using TaqMan real-time quantitative PCR. Hospital sewage contained the highest load of MRB and antibiotic resistance genes (ARGs). Wastewater treatment reduced the total bacterial load up to 78% but evidence for selection of extremely multiresistant strains and accumulation of resistance genes was observed. Our data clearly indicated pollution of sediments with ARGs in the vicinity of the WTP outlet. The potential of lakes as reservoirs of MRB and potential risks are discussed.

Antibiotic-resistance genes (ARGs) are currently discussed as emerging environmental contaminants. Hospital and municipal sewage are important sources of ARGs for the receiving freshwater bodies. We investigated the spatial distribution of different ARGs (sul1, sul2, tet(B), tet(M), tet(W) and qnrA) in freshwater lake sediments in the vicinity of a point source of treated wastewater. ARG contamination of Vidy Bay, Lake Geneva, Switzerland was quantified using real-time PCR and compared with total mercury (THg), a frequently particle-bound inorganic contaminant with known natural background levels. Two-dimensional mapping of the investigated contaminants in lake sediments with geostatistical tools revealed total and relative abundance of ARGs in close proximity of the sewage discharge point were up to 200-fold above levels measured at a remote reference site (center of the lake) and decreased exponentially with distance. Similar trends were observed in the spatial distribution of different ARGs, whereas distributions of ARGs and THg were only moderately correlated, indicating differences in the transport and fate of these pollutants or additional sources of ARG contamination. The spatial pattern of ARG contamination and supporting data suggest that deposition of particle-associated wastewater bacteria rather than co-selection by, for example, heavy metals was the main cause of sediment ARG contamination.

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Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

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