Bakterielle Gemeinschaft in Struktur und Zusammesetzung von tropischen Flüssen und Trinkwasser – Einblinke aus deep sequencing und korrelation zu ökologischer Treiber

The aim of the thesis is to study the bacterial communities of tropical river water and its derived and treated drinking water in correlation with environmental factors to yield a comprehensive understanding of the respective ecosystems and their driving forces. We determined physico-chemical, microbiological and trophic level variables in tropical river water with a watershed dominated by pristine forest, urban, and agricultural land use types during low and high hydrological regimes. Principal component analysis showed that land use was the most influential factor driving the tropical riverine system. This finding was supported by the fact that significant correlations among physico-chemical and microbiological variables were only possible with pooled data of all land use types rather than individual land use data. Of all variables tested only biological oxygen demand, nutrients, Clostridium perfringens and chlorophyll-a significantly discriminated sampling sites according to land use types indicating their high sensitivity towards diverse influences. Characterisation of water samples using Illumina MiSeq sequencing of the 16S rRNA gene showed that riverine microbial communities were greatly driven by watershed land-use rather than environmental or climate characteristics. Land-use promoted allochtonous bacterial sequences, eutrophication-linked bacterial sequences, and extinction and stress to indigenous taxa. Despite the considerable variation across watershed land-use types, the overall microbial richness was contributed by among-site richness rather than within-site richness. Microbial communities showed a remarkable stability over time irrespective of climatic perturbations emphasizing the possibility of being temporally predictable. Comprehensive investigation of a tropical drinking water treatment plant fed by water from the pristine location of the investigated tropical riverine system showed coherent dynamics of taxa, and microbial community shifts along the treatment barriers of the drinking water treatment plant. By sequencing 16S rRNA gene amplicons at adequate depth, a high degree of microbial diversity and overrepresentation of typical freshwater genera including Undibacterium, Novosphingobium and Cylindrospermopsis were observed. Undibacterium had a considerable contribution to the abundance of the phylum Proteobacteria and demostrated a remarkable ability to predict microbial diversity. Shifts in community structure were due to substantial elimination of bacterial taxa by sand filtration, and significant enrichment of rare abundant taxa following chlorination. Taxa coherent dynamics across treatment barriers revealed the presence of a series of discrete microbial secondary succession punctuated by treatment barriers. Based on microbial community succession data, the fate of noxious bacteria in drinking water treatment plant is potentially predictable. The outcome of this study provided critical insights into the potential factors shaping the riverine system and drinking water treatment plant in tropical environment. The fact that this is the first comprehensive study covering the critical aspects of source and drinking water in tropical environment, the results can be of great importance in designing appropriate riverine and treatment plant management approaches, and identifying suitable monitoring tools in order to boost source water quality and performance of drinking water treatment plants in tropical environment.Ziel dieser Arbeit ist das Bakterioplankton von Flusswasser und davon aufbereitetem Trinkwasser in tropischen Klimaten im Zusammenhang mit den Umweltfaktoren zu analysieren und ein weitergehendes Ökosystemverständnis davon abzuleiten. Hierzu wurde das Flusswasser dreier unterschiedlich anthropogen genutzter Bereiche eines tropischen Flusssystems (Morogoro River) durch ein breites Spektrum an physico-chemischen und mikrobiologischen Parameter für die klimatisch relevanten Perioden der Trocken- und Regenzeit untersucht. Untersucht wurden natürliche und anthropogen unbeeinflusste Bereiche im gebirgigen Oberlauf, sowie urbane und landwirtschaftlich genutzte Bereiche im Flachland, beide stromabwärts von den natürlichen Oberlaufbereichen. Statistische Analysen dieser Parameter zeigten, dass die Landnutzung der wesentlichste Einflussfaktor für die Flusswasserqualität war, mit guten Korrelationen zwischen mikrobiologischen und der Summe der physico-chemischen Parametern. Die Flusswasserproben wurden weiterhin hinsichtlich ihrer bakteriellen Biozönose mit Hilfe von Next Generation Sequencing (NGS, Illumina MiSeq) der 16S rRNA-Gene untersucht. Auch bei diesen Analysen konnten die Biozönosen entsprechend der Landnutzungstypen signifikant unterschieden werden. Die anthropogene Landnutzung bedingte das Auftreten von allochthonen Bakterien, von Eutrophierungsindikatoren, sowie die Reduzierung von Bakterien aus den natürlichen Oberlaufbereichen. Die bakteriellen Gemeinschaften des Flusssystems zeigten eine hohe zeitliche Stabilität unabhängig von klimatischen Faktoren wie Trocken- und Regenzeit. Weiterhin wurde die bakterielle Biozönose einer Trinkwasseraufbereitungsanlage analysiert, deren Wasser aus dem natürlichen Oberlaufsystems des untersuchten tropischen Flusssytems stammt. Die NGS-Analyse der bakteriellen Biozönosen, zeigte eine kohärente Dynamik im Hinblick auf einzelne wesentliche Taxa wie auch auf die gesamte Gemeinschaft in Korrespondenz zu den Behandlungsschritten. Basierend auf einer ausreichenden Sequenzierungstiefe konnte die bakterielle Diversität und die Dominanz verschiedener Süsswassergattungen wie Undibacterium, Novosphingium und Cylindrospermopsis dargestellt werden. Die Gattung Undibacterium stellte einen wesentlichen Anteil des Phylum Proteobacteria dar, und war ein Indikator für die bakterielle Diversität. Die Ergebnisse dieser Arbeit erlauben eine weitgehende Einsicht in Faktoren, die von besonderer Relevanz sind für Flusssysteme und Trinkwasseraufbereitung in tropischen Klimaten. Sie können im weiteren dazu herangezogen werden, um das Management für Flusssysteme und Trinkwasseraufbereitung zu optimieren, und geeignete Monitoringwerkzeuge zu identifizieren, insbesondere für tropische Regionen vergleichbar mit dem des studierten Morogoro-Flusssystems in Tansania

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

Journal of Water and HealthTropical 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 countriesinfo:eu-repo/semantics/publishedVersio

Whole-genome enrichment provides deep insights into Vibrio cholerae metagenome from an African river

The detection and typing of Vibrio cholerae in natural aquatic environments encounter major methodological challenges related to the fact that the bacterium is often present in environmental matrices at very low abundance in nonculturable state. This study applied, for the first time to our knowledge, a whole-genome enrichment (WGE) and next generation sequencing (NGS) approach for direct genotyping and metagenomic analysis of low abundant V. cholerae DNA (<50 genome unit/L) from natural water collected in the Morogoro river (Tanzania). The protocol is based on the use of biotinylated RNA baits for target enrichment of V. cholerae metagenomic DNA via hybridization. An enriched V. cholerae metagenome library was generated and sequenced on a Illumina MiSeq platform. Up to 1.8X107 bp (4.5x mean read depth) were found to map against V. cholerae reference genome sequences representing an increase of about 2500 times in target DNA coverage compared to theoretical calculations of performance for shotgun metagenomics. Analysis of metagenomic data revealed the presence of several V. cholerae virulence and virulence associated genes in river water including major virulence regions (e.g. CTX prophage and Vibrio pathogenicity island-1) and genetic markers of epidemic strains (e.g. O1-antigen biosynthesis gene cluster) that were not detectable by standard culture and molecular techniques. Overall, besides providing a powerful tool for direct genotyping of V. cholerae in complex environmental matrices this study provides a \u201cproof of concept\u201d on the methodological gap that might currently preclude a more comprehensive understanding of toxigenic V. cholerae emergence from natural aquatic environments

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

Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Species Including P. aeruginosa in a Cooling Tower.

Pseudomonas species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. P. aeruginosa is the species most commonly associated with human disease, causing a wide variety of infections with links to its presence in freshwater systems. Though several other Pseudomonas species are of ecological and public health importance, little knowledge exists regarding environmental abundances of these species. In the present study, an Illumina-based next-generation sequencing (NGS) approach using Pseudomonas-specific primers targeting the 16S rRNA gene was evaluated and applied to a set of freshwater samples from different environments including a cooling tower sampled monthly during 2 years. Our approach showed high in situ specificity and accuracy. NGS read counts revealed a precise quantification of P. aeruginosa and a good correlation with the absolute number of Pseudomonas genome copies in a validated genus-specific qPCR assay, demonstrating the ability of the NGS approach to determine both relative and absolute abundances of Pseudomonas species and P. aeruginosa. The characterization of Pseudomonas communities in cooling tower water allowed us to identify 43 phylotypes, with P. aeruginosa being the most abundant. A shift existed within each year from a community dominated by phylotypes belonging to P. fluorescens and P. oleovorans phylogenetic groups to a community where P. aeruginosa was highly abundant. Co-occurrence was observed between P. aeruginosa and other phylotypes of P. aeruginosa group as well as the potentially pathogenic species P. stutzeri, but not with phylotypes of the P. fluorescens group, indicating the need to further investigate the metabolic networks and ecological traits of Pseudomonas species. This study demonstrates the potential of deep sequencing as a valuable tool in environmental diagnostics and surveillance of health-related pathogens in freshwater environment

Performance assessment 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 non target 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.Fil: Reischer, Georg H.. Vienna University of Technology; Austria. InterUniversity Cooperation Centre Water & Health; AustriaFil: Ebdon, James E.. University of Brighton. School of Environment and Technology. Environment & Public Health Research Unit ; Reino UnidoFil: Bauer, Johanna M.. Vienna University of Technology; AustriaFil: Schuster, Nathalie. Vienna University of Technology; AustriaFil: Ahmed, Warish. CSIRO Land and Water; AustraliaFil: Åström, Johan. Chalmers University Of Technology; SueciaFil: Blanch, Anicet R.. Universidad de Barcelona. Departamento de Bioquimica; EspañaFil: Blöschl, Günter. Vienna University of Technology; AustriaFil: Byamukama, Denis. Makerere University. Department of Biochemistry; UgandaFil: Coakley, Tricia. University Of Kentucky; Estados UnidosFil: Ferguson, Christobel. ALS Water Sciences Group; AustraliaFil: Goshu, Goraw. Bahir Dar University. Blue Nile Water Institute. College of Agriculture and Environmental Sciences; EtiopíaFil: Ko, GwangPyo. Seoul National University; Corea del SurFil: de Roda Husman, Ana Maria. National Institute for Public Health and the Environment ; Países BajosFil: Mushi, Douglas. Sokoine University; TanzaniaFil: Poma, Hugo Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Investigación para la Industria Química (i); ArgentinaFil: Pradhan, Bandana. Tribhuvan University; NepalFil: Rajal, Verónica Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Investigación para la Industria Química (i); ArgentinaFil: Schade, Margit A.. Bavarian Environment Agency; AlemaniaFil: Sommer, Regina. InterUniversity Cooperation Centre Water & Health; Austria. Medical University of Vienna. Institute for Hygiene and Applied Immunology, Water Hygiene; AustriaFil: Taylor, Huw. University of Brighton. School of Environment and Technology. Environment & Public Health Research Unit ; Reino UnidoFil: Toth, Erika M.. Eötvös Lorand University. Department of Microbiology. Biological Institute; HungríaFil: Vrajmasu, Virgil. Veterinary State Laboratory; RumaniaFil: Wuertz, Stefan. University Of California At Davis; Estados UnidosFil: Mach, Robert L.. Vienna University of Technology; AustriaFil: Farnleitner, Andreas H.. Vienna University of Technology; Austria. InterUniversity Cooperation Centre Water & Health; Austri