From the pasture to the water: multiparametric laboratory experiments to determine microbial release from feces

To ensure the supply of clean water, we need tools to accurately predict where microorganisms of fecal origin come from, how they move in the environment and where they go to. To date, however, there have been few studies that have looked at bacterial overland transport (BOT). The current state of knowledge is mainly based on data from point sources (sewage treatment plants), whereas little is known about diffuse fecal sources from wildlife and livestock. The aim of this study is therefore to investigate the influence of the type of fecal matter (cow and red deer) as well as storage time and conditions (temperature and moisture) on resuspension and re-mobilization of (genetic) fecal indicators and/or pathogens. For this purpose standardized fecal samples from cow and deer were prepared in the laboratory and stored for different lengths of time (0 to 120 days) under diverse climatic conditions reflecting seasons. Fecal samples were then used in shaking experiments in which the samples were covered with water in Erlenmeyer flasks and placed in a shaking incubator. Different rainfall intensities were simulated by different shaking speeds (60 rpm and 85 rpm) and the effect of the rainfall duration was simulated by the duration of shaking (10 min and 60 min). Cultivation-based methods were used to determine fecal indicator organisms (FIB) such as E. coli, enterococci and Clostridium perfringens spores as well as somatic coliphages in the water. A panel of different qPCR-based DNA and/or RNA markers will then be used to determine host-associated genetic markers (qPCR). This multifactorial experimental approach provides the first quantitative estimates of the persistence and mobility of microbial target organisms in standardized fecal pellets from cattle and deer. The chosen multi-parametric and multi-method approach allows 1) comparison of culture-based with qPCR-based results and 2) comparison of RNA vs. DNA targets. NGS (next generation sequencing) data allows to draw conclusions on intestinal microbial persistence and to evaluate whether they reflect the mobilized load, an important information for the subsequent modelling approach. To summarize, the present study represents the first holistic quantitative approach to determine bacterial overland transport. The state-of-the-art combination of quantitative, microbiological and molecular methods and parameters will provide the scientific basis for accurate prediction of BOT.