Upscaling Transport of Bacillus subtilis Endospores and Coliphage phiX174 in Heterogeneous Porous Media from the Column to the Field Scale.

Groundwater contamination and transport of viruses and bacteria in aquifers are a major concern worldwide. To ascertain the ability of these aquifers to remove pathogens, tracer tests with microbial surrogates are carried out. These tests are laborious and may require special permits, and therefore, column tests are often done instead. Unfortunately, results from column tests tend to grossly overestimate removal rates when compared to the field scale, which can lead to an underestimation of groundwater contamination risks. Scale is an important consideration when examining pathogen transport through porous media, as pathogen removal is rarely a linear process. In this study, field tests were carried out with endospores of Bacillus subtilis and coliphage phiX174 over a distance of 25 m in an alluvial gravel aquifer near Vienna, Austria. The sandy gravel material from the field site was also used in column tests with the same tracers. Both attachment-detachment and colloid filtration theory were used to model these tests, as well as log-removal rates per meter. The results show that the spatial removal rate (log/m) is approximately 2 orders of magnitude higher on the column scale, when compared to the field. A comparison with the literature showed a correlation between the heterogeneity of the porous media and the difference in removal rates between the column and field scale.

Microbial faecal pollution of river water in a watershed of tropical Ethiopian highlands is driven by diffuse pollution sources.

Tropical communities in the developing world depend heavily on riverine systems for their socioeconomic development. However, these resources are poorly protected from diffuse pollution, and there is a lack of quantitative information regarding the microbial pollution characteristics of riverine water, despite frequently reported gastrointestinal diseases. The aim of our study was to apply faecal taxation (i.e., faecal pellet counting in representative test areas to estimate the potential availability of diffuse pollution sources) in combination with a detailed microbiological faecal pollution analysis in a riverine environment to elucidate the importance of diffuse pollution. To realize this approach, ambient faecal pellets, a multiparametric data set for standard faecal indicator bacteria (SFIB), including Escherichia coli, Clostridium perfringens spores and enterococci from catchment soil and river water, and a number of riverine water physicochemical variables were analysed during a one-year cycle. We demonstrated that the abundance of ambient faecal pellets, which were consistently counted at reference sites in the catchment, was associated with faecal pollution in the river water. Water SFIB, dissolved oxygen, nutrients, conductivity and total suspended solids were strongly linked with the abundance of ambient faecal pellets in the river catchment, as demonstrated by principal component analysis (PCA). Elevated concentrations of SFIB in the riverine water in the absence of rainfall also suggested the direct input of faecal bacteria into the riverine water by livestock (e.g., during watering) and humans (e.g., during bathing). Statistical analyses further revealed that the microbiological water quality of the investigated riverine water was not influenced by SFIB potentially occurring in the soil. This study demonstrates the importance of diffuse faecal pollution sources as major drivers of the microbiological quality of riverine water in the Ethiopian highlands. In addition, the new successfully applied integrated approach could be very useful for developing predictive models, which would aid in forecasting riverine microbiological quality in tropical developing countries.

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis.

Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract ᅟ.

Poikilothermic Animals as a Previously Unrecognized Source of Fecal Indicator Bacteria in a Backwater Ecosystem of a Large River.

Quantitative information regarding the presence of Escherichia coli, intestinal enterococci, and Clostridium perfringens in poikilotherms is notably scarce. Therefore, this study was designed to allow a systematic comparison of the occurrence of these standard fecal indicator bacteria (SFIB) in the excreta of wild homeothermic (ruminants, boars, carnivores, and birds) and poikilothermic (earthworms, gastropods, frogs, and fish) animals inhabiting an alluvial backwater area in eastern Austria. With the exception of earthworms, the average concentrations of E. coli and enterococci in the excreta of poikilotherms were equal to or only slightly lower than those observed in homeothermic excreta and were 1 to 4 orders of magnitude higher than the levels observed in the ambient soils and sediments. Enterococci reached extraordinarily high concentrations in gastropods. Additional estimates of the daily excreted SFIB (E. coli and enterococcus) loads (DESL) further supported the importance of poikilotherms as potential pollution sources. The newly established DESL metric also allowed comparison to the standing stock of SFIB in the sediment and soil of the investigated area. In agreement with its biological characteristics, the highest concentrations of C. perfringens were observed in carnivores. In conclusion, the long-standing hypothesis that only humans and homeothermic animals are primary sources of SFIB is challenged by the results of this study. It may be necessary to extend the fecal indicator concept by additionally considering poikilotherms as potential important primary habitats of SFIB. Further studies in other geographical areas are needed to evaluate the general significance of our results. We hypothesize that the importance of poikilotherms as sources of SFIB is strongly correlated with the ambient temperature and would therefore be of increased significance in subtropical and tropical habitats and water resources.IMPORTANCE The current fecal indicator concept is based on the assumption that the standard fecal indicator bacteria (SFIB) Escherichia coli, intestinal enterococci, and Clostridium perfringens multiply significantly only in the guts of humans and other homeothermic animals and can therefore indicate fecal pollution and the potential presence of pathogens from those groups. The findings of the present study showed that SFIB can also occur in high concentrations in poikilothermic animals (i.e., animals with body temperatures that vary with the ambient environmental temperature, such as fish, frogs, and snails) in an alluvial backwater area in a temperate region, indicating that a reconsideration of this long-standing indicator paradigm is needed. This study suggests that poikilotherms must be considered to be potential primary sources of SFIB in future studies.

Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents.

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long-distance transfer of strains.

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non-O1/non-O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot-spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non-O1/non-O139 isolates from Austria and other European countries and added sequences of seven genome-sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.

A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters.

We report a novel molecular assay, based on helicase-dependent amplification (HDA), for the detection of enterococci as markers for fecal pollution in water. This isothermal assay targets the same Enterococcus 23S rRNA gene region as the existing quantitative polymerase chain reaction (qPCR) assays of U.S. Environmental Protection Agency Methods 1611 and 1609 but can be entirely performed on a simple heating block. The developed Enterococcus HDA assay successfully discriminated 15 enterococcal from 15 non-enterococcal reference strains and reliably detected 48 environmental isolates of enterococci. The limit of detection was 25 target copies per reaction, only 3 times higher than that of qPCR. The applicability of the assay was tested on 30 environmental water sample DNA extracts, simulating a gradient of fecal pollution. Despite the isothermal nature of the reaction, the HDA results were consistent with those of the qPCR reference. Given this performance, we conclude that the developed Enterococcus HDA assay has great potential as a qualitative molecular screening method for resource-limited settings when combined with compatible up- and downstream processes. This amplification strategy can pave the way for developing a new generation of rapid, low-cost, and field-deployable molecular diagnostic tools for water quality monitoring.

QMRAcatch: Human-Associated Fecal Pollution and Infection Risk Modeling for a River/Floodplain Environment.

Protection of drinking water resources requires addressing all relevant fecal pollution sources in the considered catchment. A freely available simulation tool, QMRAcatch, was recently developed to simulate concentrations of fecal indicators, a genetic microbial source tracking (MST) marker, and intestinal pathogens in water resources and to conduct a quantitative microbial risk assessment (QMRA). At the same time, QMRAcatch was successfully applied to a region of the Danube River in Austria, focusing on municipal wastewater emissions. Herein, we describe extension of its application to a Danube River floodplain, keeping the focus on fecal sources of human origin. QMRAcatch was calibrated to match measured human-associated MST marker concentrations for a dry year and a wet year. Appropriate performance characteristics of the human-associated MST assay were proven by simulating correct and false-positive marker concentrations, as determined in human and animal feces. With the calibrated tool, simulated and measured enterovirus concentrations in the rivers were compared. Finally, the calibrated tool allowed demonstrating that 4.5 log enterovirus and 6.6 log norovirus reductions must be achieved to convert current surface water to safe drinking water that complies with a health-based target of 10 infections person yr. Simulations of the low- and high-pollution scenarios showed that the required viral reductions ranged from 0 to 8 log. This study has implications for water managers with interests in assessing robust catchment protection measures and water treatment criteria by considering the fate of fecal pollution from its sources to the point of abstraction.

Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry.

In order to elucidate the main predictors of Vibrio cholerae dynamics and to estimate the risk of Vibrio 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. Vibrio 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 × 10(6) V. cholerae per L in Neusiedler See and 7.59 × 10(7) 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.

Bacterial diversity along a 2600 km river continuum.

The bacterioplankton diversity in large rivers has thus far been under-sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA-gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free-living and particle-associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the Polynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater-affiliated bacteria. Based on views of the meta-community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.

A novel triplex quantitative PCR strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments.

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6×10(2) to 2.3×10(4) cell equivalents liter(-1), whereas GR-corrected abundances ranged from 4.7×10(3) to 1.6×10(6) cell equivalents liter(-1). GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.

QMRAcatch: Microbial Quality Simulation of Water Resources including Infection Risk Assessment.

Given the complex hydrologic dynamics of water catchments and conflicts between nature protection and public water supply, models may help to understand catchment dynamics and evaluate contamination scenarios and may support best environmental practices and water safety management. A catchment model can be an educative tool for investigating water quality and for communication between parties with different interests in the catchment. This article introduces an interactive computational tool, QMRAcatch, that was developed to simulate concentrations in water resources of , a human-associated microbial source tracking (MST) marker, enterovirus, norovirus, , and as target microorganisms and viruses (TMVs). The model domain encompasses a main river with wastewater discharges and a floodplain with a floodplain river. Diffuse agricultural sources of TMVs that discharge into the main river are not included in this stage of development. The floodplain river is fed by the main river and may flood the plain. Discharged TMVs in the river are subject to dilution and temperature-dependent degradation. River travel times are calculated using the Manning-Gauckler-Strickler formula. Fecal deposits from wildlife, birds, and visitors in the floodplain are resuspended in flood water, runoff to the floodplain river, or infiltrate groundwater. Fecal indicator and MST marker data facilitate calibration. Infection risks from exposure to the pathogenic TMVs by swimming or drinking water consumption are calculated, and the required pathogen removal by treatment to meet a health-based quality target can be determined. Applicability of QMRAcatch is demonstrated by calibrating the tool for a study site at the River Danube near Vienna, Austria, using field TMV data, including a sensitivity analysis and evaluation of the model outcomes.

Attachment and Detachment Behavior of Human Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material.

The transport of human adenovirus, nanoparticles, and PRD1 and MS2 bacteriophages was tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, South Australia. Comparison of transport and removal of virus surrogates with the pathogenic virus is necessary to understand the differences between the virus and surrogate. Because experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow-through soil columns were used. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material but not under high ionic strength or high pH conditions. It was also found that straining due to size and the charge of the colloid were not dominant removal mechanisms in this system. Implications of this study indicate that a certain surrogate may not represent a specific pathogen solely based on similar size, morphology, and/or surface charge. Moreover, if a particular surrogate is representative of a pathogen in one aquifer system, it may not be the most appropriate surrogate in another porous media system. This was apparent in the inferior performance of MS2 as a surrogate, which is commonly used in virus transport studies.

Detection of the food allergen celery via loop-mediated isothermal amplification technique.

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Introducing bacterial community turnover times to elucidate temporal and spatial hotspots of biological instability in a large Austrian drinking water distribution network.

Ensuring biological stability in drinking water distribution systems (DWDSs) is important to reduce the risk of aesthetic, operational and hygienic impairments of the distributed water. Drinking water after treatment often changes in quality during transport due to interactions with pipe-associated biofilms, temperature increases and disinfectant residual decay leading to potential biological instability. To comprehensively assess the potential for biological instability in a large chlorinated DWDS, a tool-box of bacterial biomass and activity parameters was applied, introducing bacterial community turnover times (BaCTT) as a direct, sensitive and easy-to-interpret quantitative parameter based on the combination of 3H-leucine incorporation with bacterial biomass. Using BaCTT, hotspots and periods of bacterial growth and potential biological instability could be identified in the DWDS that is fed by water with high bacterial growth potential. A de-coupling of biomass from activity parameters was observed, suggesting that bacterial biomass parameters depict seasonally fluctuating raw water quality rather than processes related to biological stability of the finished water in the DWDS. BaCTT, on the other hand, were significantly correlated to water age, disinfectant residual, temperature and a seasonal factor, indicating a higher potential of biological instability at more distant sampling sites and later in the year. As demonstrated, BaCTT is suggested as a novel, sensitive and very useful parameter for assessing the biological instability potential. However, additional studies in other DWDSs are needed to investigate the general applicability of BaCTT depending on water source, applied treatment processes, biofilm growth potential on different pipe materials, or size, age and complexity of the DWDS.

The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

A comparative study on antibiotic resistant Escherichia coli isolates from Austrian patients and wastewater-influenced Danube River water and biofilms.

Antimicrobial resistance (AMR) poses a major threat to human health worldwide. AMR can be introduced into natural aquatic ecosystems, for example, from clinical facilities via wastewater emissions. Understanding AMR patterns in environmental populations of bacterial pathogens is important to elucidate propagation routes and develop mitigation strategies. In this study, AMR patterns of Escherichia coli isolates from urinary tract infections and colonised urinary catheters of inpatients and outpatients were compared to isolates from the Danube River within the same catchment in Austria to potentially link environmental with clinical resistance patterns. Susceptibility to 20 antibiotics was tested for 697 patient, 489 water and 440 biofilm isolates. The resistance ratios in patient isolates were significantly higher than in the environmental isolates and higher resistance ratios were found in biofilm in comparison to water isolates. The role of the biofilm as potential sink of resistances was reflected by two extended-spectrum beta-lactamase (ESBL) producing isolates in the biofilm while none were found in water, and by higher amoxicillin/clavulanic acid resistance ratios in biofilm compared to patient isolates. Although, resistances to last-line antibiotics such as carbapenems and tigecycline were found in the patient and in the environmental isolates, they still occurred at low frequency.

Domestic hot-water boilers harbour active thermophilic bacterial communities distinctly different from those in the cold-water supply.

Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.

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.