Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Application of three different methods to determine the prevalence, the abundance and the environmental drivers of culturable Vibrio cholerae in fresh and brackish bathing waters.

AIMS: Three cultivation methods were used to study the prevalence and abundance of Vibrio cholerae in Eastern Austrian bathing waters and to elucidate the main factors controlling their distribution. METHODS AND RESULTS: Vibrio cholerae abundance was monitored at 36 inland bathing sites with membrane filtration (MF), a standard most probable number (MPN) approach and direct plating (DP). Membrane filtration yielded the most reliable and sensitive results and allowed V. cholerae detection at 22 sites with concentrations up to 39 000 CFU per 100 ml, all belonging to serogroups other than O1 and O139 and not coding for cholera toxin and toxin coregulated pilus. Direct plating turned out as an easy method for environments with high V. cholerae abundances, conductivity was the only significant predictor of V. cholerae abundance in the bathing waters at warm water temperatures. CONCLUSIONS: Vibrio cholerae nonO1/nonO139 are widely prevalent in Eastern Austrian bathing waters. Instead of the standard MPN approach, MF and DP are recommended for V. cholerae monitoring. Conductivity can be used as a first easy-to-measure parameter to identify potential bathing waters at risk. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio cholerae nonO1/nonO139 infections associated with bathing activities are an increasing public health issue in many countries of the northern hemisphere. However, there are only limited data available on the prevalence and abundance of V. cholerae in coastal and inland bathing waters. For monitoring V. cholerae prevalence and abundance, reliable and simple quantification methods are needed. Moreover, prediction of V. cholerae abundance from environmental parameters would be a helpful tool for risk assessment. This study identified the best culture-based quantification methods and a first quick surrogate parameter to attain these aims.

Multiparametric monitoring of microbial faecal pollution reveals the dominance of human contamination along the whole Danube River.

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.

Automated Sampling Procedures Supported by High Persistence of Bacterial Fecal Indicators and Bacteroidetes Genetic Microbial Source Tracking Markers in Municipal Wastewater during Short-Term Storage at 5°C.

Because of high diurnal water quality fluctuations in raw municipal wastewater, the use of proportional autosampling over a period of 24 h at municipal wastewater treatment plants (WWTPs) to evaluate carbon, nitrogen, and phosphorus removal has become a standard in many countries. Microbial removal or load estimation at municipal WWTPs, however, is still based on manually recovered grab samples. The goal of this study was to establish basic knowledge regarding the persistence of standard bacterial fecal indicators and Bacteroidetes genetic microbial source tracking markers in municipal wastewater in order to evaluate their suitability for automated sampling, as the potential lack of persistence is the main argument against such procedures. Raw and secondary treated wastewater of municipal origin from representative and well-characterized biological WWTPs without disinfection (organic carbon and nutrient removal) was investigated in microcosm experiments at 5 and 21°C with a total storage time of 32 h (including a 24-h autosampling component and an 8-h postsampling phase). Vegetative Escherichia coli and enterococci, as well as Clostridium perfringens spores, were selected as indicators for cultivation-based standard enumeration. Molecular analysis focused on total (AllBac) and human-associated genetic Bacteroidetes (BacHum-UCD, HF183 TaqMan) markers by using quantitative PCR, as well as 16S rRNA gene-based next-generation sequencing. The microbial parameters showed high persistence in both raw and treated wastewater at 5°C under the storage conditions used. Surprisingly, and in contrast to results obtained with treated wastewater, persistence of the microbial markers in raw wastewater was also high at 21°C. On the basis of our results, 24-h autosampling procedures with 5°C storage conditions can be recommended for the investigation of fecal indicators or Bacteroidetes genetic markers at municipal WWTPs. Such autosampling procedures will contribute to better understanding and monitoring of municipal WWTPs as sources of fecal pollution in water resources.

Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium.

Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log10 reduction of biotin-coated CPMs, and a 2.6-log10 reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log10 reduction, while the uncoated CPMs displayed a 3.7-log10 reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water.

A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources.

The realisation of a novel concept for automated on-line monitoring of enzymatic activities in water was successfully demonstrated by long-term field testing at two remote Austrian ground water resources. The ß-D-glucuronidase (GLUC) activity was selected as a representative enzymatic model parameter for the on-line determination. But the device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring, as demonstrated for the ß-D-galactosidase activity. Automated filtration of volumes up to 5 litres supports sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enabled robust enzymatic on-line monitoring during a 2-year period. A proportion of 5,313 out of 5,506 GLUC activity measurements (96.5%) could be positively verified. Hydrological (discharge, gauge, turbidity, temperature, pH, electric conductivity, spectral absorbance coefficient at 254 nm) as well as microbiological parameters (Escherichia coli, coliforms) were concurrently determined to characterise the investigated ground water resources. The enzymatic on-line measurements closely reflected the different hydrological conditions and contamination patterns of the test sites. Contrary to expectations, GLUC did not qualify as a proxy-parameter for the occurrence of cultivation-based E. coli contamination and warrants further detailed investigations on its indication capacity as a rapid means for microbial faecal pollution detection in such aquatic habitats. Microbial on-line monitoring is likely to become more important in the future, complementing existing surveillance strategies for water safety management. Further perspectives on the application of such analytical on-line technologies, such as their connection with event-triggered sampling and standardised diagnostics, are discussed.

Clostridium perfringens is not suitable for the indication of fecal pollution from ruminant wildlife but is associated with excreta from nonherbivorous animals and human sewage.

During a 3-year study, Clostridium perfringens was investigated in defined fecal sources from a temperate alluvial backwater area of a large river system. The results reveal that using C. perfringens as a conservative water quality indicator for total fecal pollution monitoring is no longer justified but suggest that it can be used as a tracer for excreta from nonherbivorous wildlife and human sewage.

Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers.

AIMS: Open cooling towers are frequent sources of infections with Legionella pneumophila. The gold standard for the detection of Leg. pneumophila is based on cultivation lasting up to 10 days and detecting only culturable cells. Alternative fluorescence in situ hybridization (FISH) protocols have been proposed, but they result in faint fluorescence signals and lack specificity because of cross-hybridization with other Legionella species. Our aim was thus to develop a new FISH protocol for rapid and specific detection of Leg. pneumophila in water samples. METHODS AND RESULTS: A novel catalysed reporter deposition FISH (CARD-FISH) protocol for the detection of Leg. pneumophila was developed, which significantly enhanced signal intensity as well as specificity of the probe through the use of a novel competitor probe. The developed protocol was compared with the culture method for monitoring the seasonal development of culturable and nonculturable Leg. pneumophila in two hospital cooling tower systems. Seasonal fluctuations of Leg. pneumophila concentrations detected via CARD-FISH were related to the development of the total bacterial community in both cooling towers, with temperature and biocide as the main factors controlling this development. CONCLUSIONS: Our results clearly showed that the majority of the Leg. pneumophila cells were in a nonculturable state. Thus, detection of Leg. pneumophila with culture methods may underestimate the total numbers of Leg. pneumophila present. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid, sensitive and specific detection and quantification of Leg. pneumophila in water systems is prerequisite for reliable risk estimation. The new protocol significantly improves current methodology and can be used to monitor and screen for Leg. pneumophila concentrations in cooling towers or other water systems.

Inactivation of Pseudomonas aeruginosa in electrochemical advanced oxidation process with diamond electrodes.

The electrochemical advanced oxidation process (EAOP) with diamond electrodes may serve as an additional technology to the currently approved methods for water disinfection. Only few data exist on the microbicidal effect of the EAOP. The aim of our study was to investigate the microbicidal effect of a flow-through oxidation cell with diamond electrodes, using Pseudomonas aeruginosa as the test organism. Without electrical current the EAOP had no measurable effect on investigated microbiological and chemical parameters. For direct electrical current a stronger impact was observed at low flow rate than at higher flow rate. Depending on the contact time of the oxidants and the type of quenching reagent added, inactivation of P. aeruginosa was in the range log 1.6-3.6 at the higher flow rate and log 2.4-4.4 at the lower rate. Direct electrical current showed a stronger microbicidal effect than alternating current (maximum reduction log 4.0 and log 2.9, respectively). The microbiological results of experiments with this EAOP prototype revealed higher standard deviations than expected, based on our experience with standard water disinfection methods. Safe use of an EAOP system requires operating parameters to be defined and used accurately, and thus specific monitoring tests must be developed.

Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water.

Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n=127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method.

Escherichia coli and enterococci are sensitive and reliable indicators for human, livestock and wildlife faecal pollution in alpine mountainous water resources.

AIMS: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. METHODS AND RESULTS: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7-7·0CFU g(-1) ) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0-8·4CFU g(-1) ). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3-7·7 CFUg(-1) . To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi-parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment (Reischer et al. 2008; Environ Microbiol 10:2598-2608). CONCLUSION: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.

Growth response of soda lake bacterial communities to simulated rainfall.

Moderately saline soda lakes harbor extremely abundant and fast growing bacterial communities. An interesting phenomenon of an explosive bacterial growth in shallow soda lakes in Eastern Austria after dilution with rainwater, concomitantly with a significant decrease in temperature was observed in a former study. In the present study, we tried to identify the factors being responsible for this enhanced bacterial growth in laboratory batch cultures. Three experiments were performed with water taken from two different lakes at different seasons. Natural soda lake water was diluted with distilled water, artificial lake water, sterile filtered soda lake water, and grazer-free water to test (1) for the influence of compatible solutes released to the environment and reduced salt stress after osmotic down-shock, (2) for the influence of nutrients, which may be washed in from the dry areas of the lake bottom after rainfall and (3) for the decrease of grazing pressure due to dilution. The potential influence of (4) viruses was indirectly deduced. The response of the bacterial community to the manipulations was measured by changes in bacterial numbers, the incorporation of (3)H-leucine and the concomitant determination of the amount of (3)H-leucine uptaking bacteria by microautoradiography. The influence of the environmental factors enhancing bacterial growth after a simulated rainfall event showed variations between the lakes and over the seasons. The addition of nutrients was, in all experiments, the main factor triggering bacterial growth. The decrease in grazing pressure and viral lysis after dilution was of significant importance in two of three experiments. In the experiment with the highest salinity, we could show that either compatible solutes released after osmotic down-shock and used as a source of nutrients for the soda lake bacterial populations or reduced salt stress were most probably responsible for the observed marked enhancement of bacterial growth.

Benthic bacterial production and protozoan predation in a silty freshwater environment.

The interrelation of heterotrophic bacteria with bacterivorous protists has been widely studied in pelagic environments, but data on benthic habitats, especially in freshwater systems, are still scarce. We present a seasonal study focusing on bacterivory by heterotrophic nanoflagellates (HNF) and ciliates in the silty sediment of a temperate macrophyte-dominated oxbow lake. From January 2001 to February 2002 we monitored the standing stock of bacteria and protozoa, bacterial secondary production (BSP, (3)H-thymidine, and (14)C-leucine incorporation), and grazing rates of HNF and ciliates on bacteria (FLB uptake) in the oxic sediment of the investigated system. BSP ranged from 470 to 4050 micro g C L(-1) wet sediment h(-1). The bacterial compartment turned out to be highly dynamic, indicated by population doubling times (0.6-10.0 d), which were comparable to those in the water column of the investigated system. Yet, the control mechanisms acting upon the bacterial population led to a relative constancy of bacterial standing stock during a year. Ingestion rates of protozoan grazers were 0-20.0 bacteria HNF(-1) h(-1) and 0-97.6 bacteria ciliate(-1) h(-1). HNF and ciliates together cropped 0-14 (mean 4)% of BSP, indicating that they did not significantly contribute to benthic bacterial mortality during any period of the year. The low impact of protozoan grazing was due to the low numbers of HNF and ciliates in relation to bacteria (1.8-3.5 x 10(4) bacteria HNF(-1), 0.9-3.1 x 10(6) bacteria ciliate(-1)). Thus, grazing by HNF and ciliates could be ruled out as a parameter regulating bacterial standing stock or production in the sediment of the investigated system, but the factors responsible for the limitation of benthic protistan densities and the fate of benthic BSP remained unclear.

Factors controlling extremely productive heterotrophic bacterial communities in shallow soda pools.

Dilute soda lakes are among the world's most productive environments and are usually dominated by dense blooms of cyanobacteria. Up to now, there has been little information available on heterotrophic bacterial abundance, production, and their controlling factors in these ecosystems. In the present study the main environmental factors responsible for the control of the heterotrophic bacterial community in five shallow soda pools in Eastern Austria were investigated during an annual cycle. Extremely high cyanobacterial numbers and heterotrophic bacterial numbers up to 307 x 10(9) L(-1) and 268 x 10(9) L(-1) were found, respectively. Bacterial secondary production rates up to 738 micro g C L(-1) h(-1) and specific growth rates up to 1.65 h(-1) were recorded in summer and represent the highest reported values for natural aquatic ecosystems. The combination of dense phytoplankton blooms, high temperature, high turbidity, and nutrient concentration due to evaporation is supposed to enable the development of such extremely productive microbial populations. By principal component analysis containing the data set of all five investigated pools, two factors were extracted which explained 62.5% of the total variation of the systems. The first factor could be interpreted as a turbidity factor; the second was assigned to as concentration factor. From this it was deduced that bacterial and cyanobacterial abundance were mainly controlled by wind-induced sediment resuspension and turbidity stabilized by the high pH and salinity and less by evaporative concentration of salinity and dissolved organic carbon. Bacterial production was clustered with temperature in factor 3, showing that bacterial growth was mainly controlled by temperature. The concept of describing the turbid water columns of the shallow soda pools as "fluid sediment" is discussed.

Extremely productive microbial communities in shallow saline pools respond immediately to changing meteorological conditions.

Diel changes in bacterial and cyanobacterial numbers, as well as heterotrophic bacterial production, were examined in two shallow alkaline pools, harbouring dense populations of cyanobacteria (up to 1100 x 109 cells l-1) and bacteria (up to 500 x 109 cells l-1). Together with the recorded bacterial production rates (925 micro gC l-1x h-1), these values are the highest reported for natural aquatic ecosystems. The investigations were performed during a fair-weather situation, and during a rapid change after a long-term fair-weather situation to thunderstorms and heavy rainfall. During fair weather, bacterial growth was significantly correlated to the diurnal light and temperature cycle. Prokaryotic abundances were fairly constant, and loss by grazing and viral lysis must have been of significant importance. During the invasion of rainy weather, the prokaryotic community showed a strong and immediate response. A significant enhancement of bacterial growth followed after rainfall, suggesting that the high salt concentrations had inhibited bacterial activity. Changes in bacterial and cyanobacterial numbers were consistent with this pattern. From comparison with the available literature, we conclude that diel changes of bacterioplankton are regulated by a complex combination of environmental factors specific for each investigated ecosystem. In the soda pools investigated, external abiotic factors were dominant on a diel scale. In larger ecosystems, such factors are much more buffered and internal biotic interactions may prevail.

Weak Coupling between Heterotrophic Nanoflagellates and Bacteria in a Eutrophic Freshwater Environment.

In a study on the dynamics and trophic role of the heterotrophic nanoflagellate (HNAN) assemblage in the microbial food web of a eutrophic oxbow lake abundances, biomass, and production rates of HNAN and their potential prey organisms, namely heterotrophic bacteria and autotrophic picoplankton, were monitored for a period of 2 years. No coupling between HNAN abundance and biomass and the abundance and biomass of their picoplanktonic prey was observed for the investigation period. The ratio of heterotrophic bacterial to HNAN abundance ranged from 2.2 x 103 to 8.6 x 103 (mean: 4.2 x 103 +/- 1.8 x 103). HNAN carbon consumption could account for only 10% to 40% of bacterial secondary production. The lack of coupling between HNAN and their potential prey and the low HNAN abundance relative to bacterial abundance suggested (a) that HNAN grazing was an insignificant factor in the regulation of bacterial abundance and (b) that HNAN abundance was regulated by predation rather than by prey abundance. This hypothesis was supported by the fact that HNAN growth rates were high (in the range of 0.45 d-1 to 1.00 d-1 during spring and summer, yearly mean: 0.52 d-1), and only weakly correlated with prey abundance and biomass. The results indicated strong top-down control of HNAN and consequently a weak coupling of HNAN and picoplankton in the investigated eutrophic freshwater environment.