A unifying basis for faecal detection and source tracking

The contamination of water by faecal pollution has enormous impacts on a global scale. Estimates state that 1.1 billion people worldwide lack access to save drinking water. For over a century the microbiological water quality has been tested by cultivation of indicator bacteria. Microbial hazard- and risk assessment increasingly demand for comprehensive faecal pollution analysis including the quantification of total microbial faecal pollution and a reliable identification of its major contributing sources. However, sole application of standard faecal indicators cannot sufficiently meet these challenges. The abundant intestinal bacterial populations are very promising alternative targets. There is growing evidence that intestinal microbial communities from vertebrate hosts possess populations distinct from those of non-intestinal habitats. In addition it seems that co-evolution has led to a co- diversification of vertebrate hosts and their intestinal microbiota. However, microbial populations in intestinal and non-intestinal systems exhibit a dazzling array of diversity which could not be resolved in the past due to methodical constraints. As a consequence molecular analysis of microbial faecal pollution has been based on a fragmentary puzzle of very limited sequence information. The AIM of the proposed research programme is to establish a unifying eco-phylogenetic framework for intestinal communities from vertebrate faecal excreta and to evaluate its robustness as a future foundation for molecular bacterial faecal indicator diagnostics. The realisation of the project becomes feasible for the first time by combining state-of-the-art sequencing technologies, novel bioinformatics and a hypothesis driven study design. PHASE 1 - will establish a defined 16S-rRNA-gene ultra- deep sequencing database covering a representative sample selection from faecal excreta of endothermic (mammals and birds) and ectothermic vertebrates (fish, amphibians and reptiles) and representatively chosen non-intestinal habitats. Sampling design for intestinal communities will be guided by phylogenetic vertebrate relationships, intestinal physiology and diet. Selection of non-intestinal habitats will cover carefully chosen and well-defined soil habitats. Non-intestinal habitats will be selected to exhibit a defined gradient of faecal pollution levels covering locations with only a "pristine" background level caused by wild-life to sites with intensive agricultural fertilisation using large amounts of faecal excreta. PHASE 2 - will focus on community sequence data analysis using comparative and multivariate statistics. The hypotheses on the distinctness of intestinal vertebrate populations as well as the co-diversification of vertebrate host groups and their intestinal populations will be reviewed as a foundation of molecular detection of total faecal pollution and source allocation, respectively. Finally, the possibility will be evaluated whether the established eco-phylogenetic framework supports the design of a modular molecular bacterial faecal indication system with nested levels of specificity (i.e. markers for total faecal pollution and respective vertebrate sub-groups). In conclusion, the proposed research will provide the first scientific basis to systematically understand the occurrence of abundant intestinal bacterial populations in the environment. The eco- phylogenetic framework will be open for iterative adaptations and expansion towards the global scale supporting water quality testing for tomorrow`s generation.

Pollution of water resources through faecal input is a constant threat to global public health. The WHO estimates that over 660 million people worldwide are without access to safe drinking water, prompting the UN to make improvement of drinking water quality and sanitation one of the Sustainable Development Goals. This project set out to build a new and solid basis for the state-of-the-art detection of faecal pollution in water. Modern molecular technologies based on the detection of specific fragments of DNA (genetic markers) need a comprehensive and high-quality foundation of DNA sequence data. To that end this project collected more than 400 faecal samples from 177 different species of vertebrate animals including wild mammals, birds, reptiles, amphibians and fish. In addition wastewater was collected from 29 wastewater treatment plants from 13 countries on six continents to represent municipal wastewater as one of the most important sources of faecal pollution. It could be shown that human-associated DNA markers for faecal pollution were present in high concentrations in wastewater from all around the world. These existing markers can therefore be considered very useful tools for the global detection of human faecal pollution, irrespectively whether to be monitored in America, Europe, Asia or Australia. However animal faecal sources also play an increasingly critical role in global faecal pollution. In order to assess the performance of existing tools for molecular detection of both human and animal faecal pollution and to allow the development of future improved methods, the faecal bacterial community (microbiome) in the collected samples was investigated by a high- resolution DNA sequencing approach. In addition to sequencing data, the resulting Faecal Source sequence database (FSdb) also contained 150 categories of sample data ranging from host animal phylogeny and diet to sampling location and processing details. This deep insight into the composition of the intestinal community of the host animals allowed the testing of research questions such as the relative role of host phylogeny (Is there selective interaction of the host with its microbiome?) versus the role of diet (What can the microbiome feed on?). It was found that co-evolution of the host and its microbial guests plays a mayor role for the community composition, especially for mammals. On the other hand, diet has a strong impact on the diversity of the community with herbivores harbouring more diverse communities than carnivores. The results of this project confirm the increasing role that molecular diagnostics have in the detection and characterisation of faecal pollution. These methods have the potential to revolutionise water quality testing in the near future. Beyond the field of health-related water microbiology the identification of host-associated vs. diet- associated bacterial populations might have impacts in human and veterinary medicine (healthy gut microbiome, probiotics) and in nutrition sciences (prebiotics).