The microbial faecal pollution of rivers has wide-ranging impacts on a variety of human activities that rely on appropriate river water quality. Thus, detailed knowledge of the extent and origin of microbial faecal pollution is crucial for watershed management activities to maintain safe water use. In this study, the microbial faecal pollution levels were monitored by standard faecal indicator bacteria (SFIB) along a 2580 km stretch of the Danube, the world's most international river, as well as the Danube's most important tributaries. To track the origin of faecal pollution, host-associated Bacteroidetes genetic faecal marker qPCR assays for different host groups were applied in concert with SFIB. The spatial resolution analysis was followed by a time resolution analysis of faecal pollution patterns over 1 year at three selected sites. In this way, a comprehensive faecal pollution map of the total length of the Danube was created, combining substantiated information on both the extent and origin of microbial faecal pollution. Within the environmental data matrix for the river, microbial faecal pollution constituted an independent component and did not cluster with any other measured environmental parameters. Generally, midstream samples representatively depicted the microbial pollution levels at the respective river sites. However, at a few, somewhat unexpected sites, high pollution levels occurred in the lateral zones of the river while the midstream zone had good water quality. Human faecal pollution was demonstrated as the primary pollution source along the whole river, while animal faecal pollution was of minor importance. This study demonstrates that the application of host-associated genetic microbial source tracking markers in concert with the traditional concept of microbial faecal pollution monitoring based on SFIB significantly enhances the knowledge of the extent and origin of microbial faecal pollution patterns in large rivers. It constitutes a powerful tool to guide target-oriented water quality management in large river basins.

SummaryIn order to elucidate the main predictors of V ibrio cholerae dynamics and to estimate the risk of V ibrio cholera‐related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid‐phase cytometry (CARD‐FISH/SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. V ibrio cholerae attached to crustacean zooplankton was quantified via FISH and epifluorescence microscopy. Concentrations obtained by CARD‐FISH/SPC were significantly higher than those obtained by culture in 2011, but were mostly of similar magnitude in 2012. Maximum cell numbers were 1.26 × 106 V . cholerae per L in Neusiedler See and 7.59 × 107 V . cholerae per L in the shallow alkaline lakes. Only on a few occasions during summer was the crustacean zooplankton the preferred habitat for V . cholerae. In winter, V . cholerae was not culturable but could be quantified at all sites with CARD‐FISH/SPC. Beside temperature, suspended solids, zooplankton and ammonium were the main predictors of V . cholerae abundance in Neusiedler See, while in the shallow alkaline lakes it was organic carbon, conductivity and phosphorus. Based on the obtained concentrations a first estimation of the health risk for visitors of the lake could be performed.

DNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. This approach is demonstrated on Enterococcus faecalis (E. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. Based on NGS-derived data, we identified 16 aptamer candidates. Among these, aptamer EF508 exhibited high binding affinity to E. faecalis cells (KD-value: 37 nM) and successfully discriminated E. faecalis from 20 different Enterococcus and non-Enterococcus spp. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.

The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.