ABSTRACTBOOK Photo credit: Wiener Wildnis 20th Symposium on Health-Related Water Microbiology (HRWM) Vienna, Austria (15-20 September 2019) www.hrwm.eu ORAL PRESENTATIONS SESSION 1 – METAGENOME, MICROBIOME AND COMMUNITY ANALYSIS 01 Quantitative Viral Metagenomic Method Combining Long and Short Read Sequencing Kathryn Langenfeld, Melissa Duhaime, Krista Wigginton University of Michigan Metagenomics are allowing unprecedented insight into microbial communities. Next generation sequencing (NGS) has led to many discoveries about viruses, but three notable challenges exist in applying NGS to study the viromes of complex matrices. Despite being the most highly abundant members of most communities, viral nucleic acids are a small portion of the total nucleic acids. Efficient viral enrichment methods are needed. Viral isolation and enrichment methods are not equally efficient. Optimized methods are needed to quantitatively compare viral representation across samples. Viral genomes are mosaic with a high mutation rate and, therefore, difficult to assemble with short reads produced with Illumina sequencing technologies. Supplemental sequencing technologies need to be explored. To address these challenges, our research advances NGS methods with a quantitative sequencing approach on the isolated virus fraction from wastewater with a combination of long and short read sequencing. Wastewater viruses are concentrated with tangential and dead-end ultrafiltration 575-fold in influent and 1,150-fold in effluent. The viruses are purified with chloroform to lyse cells and DNase to degrade extra-viral DNA. The concentration and purification protocol recovered approximately half of the viral DNA while removing 4-logs of 16S rRNA. For quantitative comparisons, 86 dsDNA and 5 ssDNA internal standards were spiked into samples at 1% of the total DNA concentration prior to simultaneously sequencing ssDNA and dsDNA on Illumina NovaSeq. The internal standards were used to determine sequencing efficiency with respect to GC content. The Illumina sequencing of the dsDNA phages was complemented by long-read sequencing on the Oxford Nanopore GridION. Short and long read sequencing data was de novoco- assembled to identify phages in influent and effluent. The absolute abundance of contigs was determined by mapping the short reads to the contigs and converting the number of reads aligning to contigs to a concentration using the sequencing efficiency. 02 Factors shaping the intestinal microbiome of vertebrate faecal sources: unravelling the role of diet and host phylogeny Georg H. Reischer (A, B), Nicholas D. Youngblut (C), William Walters (C), Nathalie Schuster (A), Chris Walzer (D), Gabrielle Stalder (D), Ruth E. Ley (C), Andreas H. Farnleitner (A, B, E) (A) TU Wien, Institute of Chemical, Environmental and Bioscience Engineering, Research Group for Environmental Microbiology and Molecular Diagnostics 166/5/3, 1060 Vienna, Austria, (B) ICC Interuniversity Cooperation Centre Water & Health, Vienna, Austria, (C) Department of Microbiome Science, Max Planck Institute for Developmental Biology, Max Planck Ring 5, 72076 Tübingen, Germany, (D) Research Institute of Wildlife Ecology, University of Veterinary Medicine, 1160 Vienna, Austria, (E) Research Division Water Quality and Health, Karl Landsteiner University for Health Sciences, 3500 Krems an der Donau, Austria High-throughput sequencing is allowing unprecedented insight into microbial community structure. Understanding the composition and dynamics of intestinal microbiota is an essential basis for assessment of faecal pollution in the environment and for the development of molecular diagnostic tools. According to previous studies community assembly in the vertebrate gut seems to be modulated by multiple factors such as diet or host phylogeny though there is disagreement as to the relative contribution of these drivers. To resolve this, we compiled and analysed a large and highly diverse animal 16S rRNA faecal microbiome dataset, which in contrast to previous studies, comprises 80 % wild animals. It included data from 213 samples from 128 different species of Mammalia, Aves, Reptilia, Amphibia, and Actinopterygii. Using bioinformatic tools we managed to decouple the effects of host evolutionary history and diet on gut microbiome diversity and could show that each factor 2 modulates different aspects of diversity. Moreover, we found particular microbial taxa associated with host phylogeny and, to a lesser extent, with diet. Interestingly, the signal of cophylogeny was much stronger in Mammalia compared to non-mammalian hosts. We hypothesize that this difference might be due to the more sophisticated digestive physiology of mammals, especially in the group of the fore- gut fermenting ruminants. Another factor might be more pronounced vertical transmission of microbiota between generations in mammals during birth and nursing. In the context of faecal pollution characterisation our findings suggest that there is a gradient from low to high host- association of intestinal populations from fish and amphibians to reptiles and birds to mammals showing the highest level of coevolution. Taken together with the strong influence of diet on abundance patterns and the presence of transient and opportunistic populations in the investigated microbiota this study highlights future challenges and opportunities in molecular faecal pollution detection and source tracking. 03 Impacts of anthropogenic activities on the health related microbes in a river ecosystem Yaohui Bai, Jinsong Liang, Kailingli Liao, Chen Zhao, Jiuhui Qu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Anthropogenic activities (e.g. wastewater discharge and pesticide use) have a great impact on the biotic properties of natural aquatic ecosystems, especially the microbial diversity and function. A number of reports have documented the impacts of anthropogenic activities on the variations in the microbial community, but directly using microbial community indices to track the pollution source and discern the levels of anthropogenic activities is still limited. Here, we integrated flow cytometry, metagenomic sequencing and chemical analyses to investigate the composition, and function of the health related microbes (antibiotic resistant bacteria and pathogen) in three areas along a gradient of anthropogenic disturbance (less-disturbed mountain area, wastewater-discharged urban area, and pesticide-using agricultural area) in a river ecosystem. Multiple statistical methods were used to explore the causal relationships between the changes in environmental factors and the health related microbial variation. Results showed that anthropogenic activities (wastewater discharge and agricultural fertilizer) facilitated the production of potentially harmful bacteria, affected the distribution of dominant pathogen species, and accelerated the horizontal gene transfer of antibiotic resistant genes (ARGs). ARGs abundance persistently increased from the upstream (mountain area) to midstream (urban area), and to downstream (agriculture area). By co-occurrence network analysis and source track technique, wastewater treatment plant was identified as the main sources of ARGs. Pseudomonas, identified as the most abundant host of ARGs, changed obviously among three areas with significantly higher aminoglycoside, beta-lactam, sulfonamide and quinolone in wastewater- discharged urban area. Finally, we also identified the types and abundance of pathogens carrying the ARGs, which differently distributed in three areas. 04 Assessing the spatial and temporal variability of bacterial communities in two Bardenpho wastewater treatment systems via Illumina MiSeq sequencing Samendra Sherchan, Jia Xue, Bradley Schmitz, Ian Pepper, Charles Gerba Tulane University, University of Arizona Wastewater treatment plants (WWTPs) serve an important function by reducing organic loads in wastewater before discharge into the environment. Such facilities treat influent from a mixture of sources containing household sewage, storm water runoff, industrial effluent, and reclaimed water. Next generation sequencing provides new insights into the diversity and ecophysiology of bacteria communities throughout wastewater treatment plants (WWTP), as well as the fate of pathogens in wastewater treatment system. In the present study, we investigated the bacterial communities and human-associated Bacteroidales (HF183) marker in two WWTPs in North America that utilize Bardenpho treatment processes. Although, most pathogens were eliminated during wastewater 3 treatment, some pathogenic bacteria were still observed in final effluents. The HF183 genetic marker demonstrated significant reductions between influent and post-Bardenpho treated samples in each WWTP, which coincided with changes in bacteria relative abundances and community compositions. Consistent with previous studies, the major phyla in wastewater samples were predominantly comprised by Proteobacteria (with Gammaproteobacteria and Alphaproteobacteria among the top two classes), Actinobacteria, Bacteroidetes, and Firmicutes. Dominant genera were often members of Proteobacteria and Firmicutes, including several pathogens of public health concern, such as Pseudomonas, Serratia, Streptococcus, Mycobacterium and Arcobacter. Pearson correlations were calculated to observe the seasonal variation of relative abundances of gene sequences at different levels based on the monthly average temperature. These findings profile how changes in bacterial