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

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.Fil: Mayer, René E.. Vienna University of Technology; Austria. Interuniversity Cooperation Centre for Water and Health; AustriaFil: Reischer, Georg. Vienna University of Technology; AustriaFil: Ixenmaier, Simone K.. Vienna University of Technology; Austria. Interuniversity Cooperation Centre for Water and Health; AustriaFil: Derx, Julia. Vienna University of Technology; AustriaFil: Blaschke, Alfred Paul. Vienna University of Technology; AustriaFil: Ebdon, James E.. University of Brighton; Reino UnidoFil: Linke, Rita. Vienna University of Technology; Austria. Interuniversity Cooperation Centre Water And Health; AustriaFil: Egle, Lukas. Vienna University of Technology; AustriaFil: Ahmed, Warish. Csiro Land And Water; AustraliaFil: Blanch, Anicet R.. Universidad de Barcelona; EspañaFil: Byamukama, Denis. Makerere University; UgandaFil: Savill, Marion. Affordable Water Limited;Fil: Mushi, Douglas. Sokoine University Of Agriculture; TanzaniaFil: Cristobal, Hector Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; ArgentinaFil: Edge, Thomas A.. Canada Centre for Inland Waters. Environment and Climate Change Canada; CanadáFil: Schade, Margit A.. Bavarian Environment Agency; AlemaniaFil: Aslan, Asli. Georgia Southern University; Estados UnidosFil: Brooks, Yolanda M.. Michigan State University; Estados UnidosFil: Sommer, Regina. Interuniversity Cooperation Centre Water And Health; Austria. Medizinische Universitat Wien; AustriaFil: Masago, Yoshifumi. Tohoku University; JapónFil: Sato, Maria I.. Cia. Ambiental do Estado de Sao Paulo. Departamento de Análises Ambientais; BrasilFil: Taylor, Huw D.. University of Brighton; Reino UnidoFil: Rose, Joan B.. Michigan State University; Estados UnidosFil: Wuertz, Stefan. Nanyang Technological University. Singapore Centre for Environmental Life Sciences Engineering and School of Civil and Environmental Engineering; SingapurFil: Shanks, Orin. U.S. Environmental Protection Agency; Estados UnidosFil: Piringer, Harald. Vrvis Research Center; AustriaFil: Mach, Robert L.. Vienna University of Technology; AustriaFil: Savio, Domenico. Karl Landsteiner University of Health Sciences; AustriaFil: Zessner, Matthias. Vienna University of Technology; AustriaFil: Farnleitner, Andreas. Vienna University of Technology; Austria. Interuniversity Cooperation Centre Water And Health; Austria. Karl Landsteiner University of Health Sciences; Austri

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases

Habitat Fragmentation can Modulate Drought Effects on the Plant-soil-microbial System in Mediterranean Holm Oak (Quercus ilex) Forests

© 2015, Springer Science+Business Media New York. Ecological transformations derived from habitat fragmentation have led to increased threats to above-ground biodiversity. However, the impacts of forest fragmentation on soils and their microbial communities are not well understood. We examined the effects of contrasting fragment sizes on the structure and functioning of soil microbial communities from holm oak forest patches in two bioclimatically different regions of Spain. We used a microcosm approach to simulate the annual summer drought cycle and first autumn rainfall (rewetting), evaluating the functional response of a plant-soil-microbial system. Forest fragment size had a significant effect on physicochemical characteristics and microbial functioning of soils, although the diversity and structure of microbial communities were not affected. The response of our plant-soil-microbial systems to drought was strongly modulated by the bioclimatic conditions and the fragment size from where the soils were obtained. Decreasing fragment size modulated the effects of drought by improving local environmental conditions with higher water and nutrient availability. However, this modulation was stronger for plant-soil-microbial systems built with soils from the northern region (colder and wetter) than for those built with soils from the southern region (warmer and drier) suggesting that the responsiveness of the soil-plant-microbial system to habitat fragmentation was strongly dependent on both the physicochemical characteristics of soils and the historical adaptation of soil microbial communities to specific bioclimatic conditions. This interaction challenges our understanding of future global change scenarios in Mediterranean ecosystems involving drier conditions and increased frequency of forest fragmentation

Promoting interdisciplinary education − the Vienna Doctoral Programme on Water Resource Systems

The Vienna Doctoral Programme on Water Resource Systems (DK-WRS) is a programme that aims to educate students in interdisciplinary water science through cutting edge research at an international level. It is funded by the Austrian Science Fund and designed to run over a period of 12 yr during which 80 doctoral students are anticipated to graduate. This paper reports on our experiences of setting up and implementing the Programme. We identify three challenges: integrating the disciplines, maintaining depth in an interdisciplinary programme, and teaching subjects remote to each student's core expertise. To address these challenges we adopt a number of approaches. We use three levels of instruments to foster integration across the disciplines: joint groups (e.g. a joint study programme), joint science questions (e.g. developed in annual symposia), and joint study sites. To maintain depth we apply a system of quality control including regular feedback sessions, theses by journal publications and international study exchange. For simultaneously teaching students from civil and environmental engineering, biology, geology, chemistry, mathematics we use visually explicit teaching, learning by doing, extra mentoring and by cross relating associated subjects. Our initial assessment of the Programme shows some very positive outcomes. Joint science questions formed between students from various disciplines indicate integration is being achieved. The number of successful publications in top journals suggests that depth is maintained. Positive feedback from the students on the variety and clarity of the courses indicates the teaching strategy is working well. Our experiences have shown that implementing and running an interdisciplinary doctoral programme has its challenges and is demanding in terms of time and human resources but seeing interactions progress and watching people grow and develop their way of thinking in an interdisciplinary environment is a valuable reward

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2,andCowM3)reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only).Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associatedCowM2qPCR method combined with either the BacR orRum2Bacruminantassociated methods are most suitable for implementation

Hypothesis-Driven Approach for the Identification of Fecal Pollution Sources in Water Resources

Water resource management must strive to link catchment information with water quality monitoring. The present study attempted this for the field of microbial fecal source tracking (MST). A fecal pollution source profile based on catchment data (e.g., prevalence of fecal sources) was used to formulate a hypothesis about the dominant sources of pollution in an Austrian mountainous karst spring catchment. This allowed a statistical definition of methodical requirements necessary for an informed choice of MST methods. The hypothesis was tested in a 17-month investigation of spring water quality. The study followed a nested sampling design in order to cover the hydrological and pollution dynamics of the spring and to assess effects such as differential persistence between parameters. Genetic markers for the potential fecal sources as well as microbiological, hydrological, and chemo−physical parameters were measured. The hypothesis that ruminant animals were the dominant sources of fecal pollution in the catchment was clearly confirmed. It was also shown that the concentration of ruminant markers in feces was equally distributed in different ruminant source groups. The developed approach provides a tool for careful decision-making in MST study design and might be applied on various types of catchments and pollution situations