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.

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.

Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains.

BACKGROUND: Legionella pneumophila, the causative agent of Legionnaire's disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water. RESULTS: With ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb- stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population. CONCLUSIONS: The results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.

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.

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.

Optimized methods for Legionella pneumophila release from its Acanthamoeba hosts.

BACKGROUND: Free-living amoebae (FLA) and particularly acanthamoebae serve as vehicles and hosts for Legionella pneumophila, among other pathogenic microorganisms. Within the amoebae, L. pneumophila activates a complex regulatory pathway that enables the bacteria to resist amoebal digestion and to replicate. Moreover, the amoebae provide the bacteria protection against harsh environmental conditions and disinfectants commonly used in engineered water systems. To study this ecological relationship, co-culture and infection models have been used. However, there is a lack of data regarding the effectiveness of the different methods used to release intracellular bacteria from their amoebal hosts. The aim of this study was to evaluate the impact of the methods used to release intracellular L. pneumophila cells on the culturability of the bacteria. Furthermore, the standard method ISO 11731:1998 for the recovery and enumeration of Legionella from water samples was evaluated for its suitability to quantify intracellular bacteria. RESULTS: The effectiveness of the eight release treatments applied to L. pneumophila and Acanthamoeba strains in a free-living state varied between bacterial strains. Moreover, the current study provides numerical data on the state of co-culture suspensions at different time points. The release treatments enhanced survival of both microorganisms in co-cultures of L. pneumophila and Acanthamoeba. Passage through a needle (21G, 27G) and centrifugation at 10,000 × g showed the highest bacterial counts when releasing the bacteria from the intracellular state. Regarding the ISO 11731:1998 method, one of the tested strains showed no differences between the recovery rates of associated and free-living L. pneumophila. However, a reduced bacterial recovery rate was observed for the second L. pneumophila strain used, and this difference is likely linked to the survival of the amoebae. CONCLUSIONS: Mechanical release treatments were the most effective methods for providing bacterial release without the use of chemicals that could compromise further study of the intracellular bacteria. The current results demonstrated that the recovery of L. pneumophila from water systems may be underestimated if protozoal membranes are not disrupted.

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.

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.

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.

Assessing the impact of inland navigation on the faecal pollution status of large rivers: A novel integrated field approach.

The contribution of ships to the microbial faecal pollution status of water bodies is largely unknown but frequently of human health concern. No methodology for a comprehensive and target-orientated system analysis was available so far. We developed a novel approach for integrated and multistage impact evaluation. The approach includes, i) theoretical faecal pollution source profiling (PSP, i.e., size and pollution capacity estimation from municipal vs. ship sewage disposal) for impact scenario estimation and hypothesis generation, ii) high-resolution field assessment of faecal pollution levels and chemo-physical water quality at the selected river reaches, using standardized faecal indicators (cultivation-based) and genetic microbial source tracking markers (qPCR-based), and iii) integrated statistical analyses of the observed faecal pollution and the number of ships assessed by satellite-based automated ship tracking (i.e., automated identification system, AIS) at local and regional scales. The new approach was realised at a 230 km long Danube River reach in Austria, enabling detailed understanding of the complex pollution characteristics (i.e., longitudinal/cross-sectional river and upstream/downstream docking area analysis). Faecal impact of navigation was demonstrated to be remarkably low at regional and local scale (despite a high local contamination capacity), indicating predominantly correct disposal practices during the investigated period. Nonetheless, faecal emissions were sensitively traceable, attributable to the ship category (discriminated types: cruise, passenger and freight ships) and individual vessels (docking time analysis) at one docking area by the link with AIS data. The new innovative and sensitive approach is transferrable to any water body worldwide with available ship-tracking data, supporting target-orientated monitoring and evidence-based management practices.

Performance characteristics of qPCR assays targeting human- and ruminant-associated bacteroidetes for microbial source tracking across sixteen countries on six continents.

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

PVC-piping promotes growth of Ralstonia pickettii in dialysis water treatment facilities.

Biofilms forming inside dialysis water treatment systems are one of the main sources of microbiological contamination. Among the bacteria found in biofilms, Ralstonia pickettii is frequently encountered in dialysis water treatment systems and has been shown to develop extreme oligotrophic talents. In Austria, R. pickettii was exclusively detected in high numbers in dialysis water treatment facilities equipped with chlorinated polyvinyl chloride (PVC-C) piping. In this laboratory study it was shown that PVC-C effectively promotes growth of R. pickettii biofilms, while residual organic carbon in purified dialysis water is sufficient for promoting substantial growth of planktic R. pickettii. This provides evidence that PVC-C is an unsuitable material for piping in dialysis water treatment systems.

Probabilistic fecal pollution source profiling and microbial source tracking for an urban river catchment.

We developed an innovative approach to estimate the occurrence and extent of fecal pollution sources for urban river catchments. The methodology consists of 1) catchment surveys complemented by literature data where needed for probabilistic estimates of daily produced fecal indicator (FIBs, E. coli, enterococci) and zoonotic reference pathogen numbers (Campylobacter, Cryptosporidium and Giardia) excreted by human and animal sources in a river catchment, 2) generating a hypothesis about the dominant sources of fecal pollution and selecting a source targeted monitoring design, and 3) verifying the results by comparing measured concentrations of the informed choice of parameters (i.e. chemical tracers, C. perfringensspores, and host-associated genetic microbial source tracking (MST) markers) in the river, and by multi-parametric correlation analysis. We tested the approach at a study area in Vienna, Austria. The daily produced microbial particle numbers according to the probabilistic estimates indicated that, for the dry weather scenario, the discharge of treated wastewater (WWTP) was the primary contributor to fecal pollution. For the wet weather scenario, 80-99 % of the daily produced FIBs and pathogens resulted from combined sewer overflows (CSOs) according to the probabilistic estimates. When testing our hypothesis in the river, the measured concentrations of the human genetic fecal marker were log10 4 higher than for selected animal genetic fecal markers. Our analyses showed for the first-time statistical relationships between C. perfringens spores (used as conservative microbial tracer for communal sewage) and a human genetic fecal marker (i.e. HF183/BacR287) with the reference pathogen Giardia in river water (Spearman rank correlation: 0.78-0.83, p < 0.05. The developed approach facilitates urban water safety management and provides a robust basis for microbial fate and transport models and microbial infection risk assessment.

Have genetic targets for faecal pollution diagnostics and source tracking revolutionized water quality analysis yet?

The impacts of nucleic acid-based methods - such as PCR and sequencing - to detect and analyze indicators, genetic markers or molecular signatures of microbial faecal pollution in health-related water quality research were assessed by rigorous literature analysis. A wide range of application areas and study designs has been identified since the first application more than 30 years ago (>1100 publications). Given the consistency of methods and assessment types, we suggest defining this emerging part of science as a new discipline: genetic faecal pollution diagnostics (GFPD) in health-related microbial water quality analysis. Undoubtedly, GFPD has already revolutionized faecal pollution detection (i.e., traditional or alternative general faecal indicator/marker analysis) and microbial source tracking (i.e., host-associated faecal indicator/marker analysis), the current core applications. GFPD is also expanding to many other research areas, including infection and health risk assessment, evaluation of microbial water treatment, and support of wastewater surveillance. In addition, storage of DNA extracts allows for biobanking, which opens up new perspectives. The tools of GFPD can be combined with cultivation-based standardized faecal indicator enumeration, pathogen detection, and various environmental data types, in an integrated data analysis approach. This comprehensive meta-analysis provides the scientific status quo of this field, including trend analyses and literature statistics, outlining identified application areas, and discusses the benefits and challenges of nucleic acid-based analysis in GFPD.

Rapid and sensitive quantification of Vibrio cholerae and Vibrio mimicus cells in water samples by use of catalyzed reporter deposition fluorescence in situ hybridization combined with solid-phase cytometry.

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 10(1) cells ml(-1) in the large saline lake Neusiedler See to 5.6 × 10(4) cells ml(-1) in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.

Holy springs and holy water: underestimated sources of illness?

Use of holy springs and holy water is inherent in religious activities. Holy spring water is also used extensively for personal drinking water, although not assessed according to drinking water standards. Holy water in churches and chapels may cause infections via wetting of lips and sprinkling on persons. Our aim was to assess the microbiological and chemical water quality of holy springs and holy water in churches and hospital chapels. Of the holy springs investigated, only 14% met the microbiological and chemical requirements of national drinking water regulations. Considering results from sanitary inspections of the water catchments, no spring was assessed as a reliable drinking water source. All holy water samples from churches and hospital chapels showed extremely high concentrations of HPC; fecal indicators, Pseudomonas aeruginosa and Staphylococcus aureus occurred only in the most frequently visited churches. We conclude that it is highly necessary to include holy springs in programs for assessment and management of water quality. Public awareness has to be raised to perceive holy springs as potential sources of illness. Holy water can be another source of infection, especially in hospital chapels and frequently visited churches. Recommendations are made for proper water quality management of both water types.

Transport and removal of spores of Bacillus subtilis in an alluvial gravel aquifer at varying flow rates and implications for setback distances.

To guarantee proper protection from fecally transmitted pathogen infections, drinking water wells should have a sufficiently large setback distance from potential sources of contamination, e.g. a nearby river. The aim of this study was to provide insight in regards to microbial contamination of groundwater under different flow velocities, which can vary over time due to changes in river stage, season or pumping rate. The effects of these changes, and how they affect removal parameters, are not completely understood. In this study, field tracer tests were carried out in a sandy gravel aquifer near Vienna, Austria to evaluate the ability of subsurface media to attenuate Bacillus subtilis spores, used as a surrogate for Cryptosporidium and Campylobacter. The hydraulic gradient between injection and extraction was controlled by changing the pumping rate (1, 10 l/s) of a pumping well at the test site, building upon previously published work in which tracer tests with a 5 l/s pumping rate were carried out. Attachment and detachment rate coefficients were determined using a HYDRUS-3D model and ranged from 0.12 to 0.76 and 0-0.0013 h-1, respectively. Setback distances were calculated based on the 60-day travel time, as well as a quantitative microbial risk assessment (QMRA) approach, which showed similar results at this site; around 700 m at the highest pumping rate. Removal rates (λ) in the field tests ranged from 0.2 to 0.3 log/m, with lower pumping rates leading to higher removal. It was shown that scale must be taken into consideration when determining λ for the calculation of safe setback distances.

Corrigendum: Genetic microbial source tracking support QMRA modeling for a riverine wetland drinking water resource.

[This corrects the article DOI: 10.3389/fmicb.2021.668778.].

Applicability of solid-phase cytometry and epifluorescence microscopy for rapid assessment of the microbiological quality of dialysis water.

BACKGROUND: Currently, the gold standard to assess the microbiological quality of dialysis water is the determination of heterotrophic plate counts (HPC). The long waiting time of the HPC method and the fact that most bacteria are not culturable on agar plates provokes the search for rapid alternative methods for monitoring the microbiological quality of dialysis water. METHODS: We tested the applicability of total viable counts (TVC) and total direct counts (TDC), determined via solid-phase cytometry and epifluorescence microscopy (EFM), in comparison to the standard HPC determination method in 113 samples from 13 dialysis water treatment units (59 drinking water and 54 dialysis water samples). Additionally, for a set of dialysis water samples (n = 22) endotoxin concentrations were also determined. RESULTS: TVC showed high correlation with HPC and results were of comparable magnitude for most investigated dialysis water samples [median: 3 cells/colony forming units (CFU) 100 mL(-1)]. However, in one dialysis water sample, HPC values (5800 CFU 100 mL(-1)) were >35-fold lower than TVC values (2.05 × 10(5) cells 100 mL(-1)) indicating severe limits of the HPC method to assess the microbiological quality of dialysis water. For drinking water, TVC (median: 4.8 × 10(4) cells 100 mL(-1)) was on average one order of magnitude higher than HPC (median: 2.5 × 10(3) cells 100 mL(-1)). TDCs (median dialysis water: 1.1 × 10(4) cells 100 mL(-1) and median drinking water: 4.9 × 10(6) cells 100 mL(-1)) were always several orders of magnitude higher than HPC or TVC. CONCLUSIONS: We propose that the TVC/solid-phase cytometry approach is a reliable and rapid alternative to the culture-dependent approach for assessment of the microbiological quality of dialysis water, especially when fast results are needed. TDC determined via EFM lacks sensitivity and reliability for assessing microbial concentrations in low-cell dialysis water samples since the limits of detection and quantification are high.