UNS: Persistence of Molecular Markers Used in Fecal Source Tracking: Metagenomic-Based Assessment of Microbial and Human DNA

1511825布朗随着相对较新的分子生物学工具的出现，环境中粪便污染的鉴定越来越多地利用快速分子检测来鉴定粪便指示菌的DNA序列或从宿主特有的细胞中鉴定DNA，如粪便中的线粒体DNA(MtDNA)。然而，未知因素仍然存在，主要是关于依赖于培养物和不依赖于培养物化验之间的动力学关系，因为自污染以来的时间信息在监测中作为粪便病原体存活的替代指标至关重要，因此公共卫生风险。这项拟议的工作将阐明目前和新的旨在识别环境水中粪便污染的分子分析方法的优点和局限性。为了对这些问题提供新的见解，研究小组将开展关于降解人类线粒体DNA和人类特定微生物DNA靶标的基础工作，同时还将开展一套常用的培养和定量聚合酶链式反应方法，用于粪便来源追踪应用，使用基于实验室的中微体，这些中微体很好地模拟天然地表水和沉积物。将在间隔体中添加粪便污染源(健康和腹泻粪便、初级污水、分离物)，并将使用先进的元基因组学技术跟踪肠道微生物群落、原生微生物群落和病原体(如果存在)的命运(6个月)。元基因组学方法可以以前所未有的准确性和敏感度跟踪微生物群落和病原体的变化。因此，时间元基因组数据集将为目前用于粪便污染监测的基准生物标志物(粪便指示菌测试)和建议的生物标志物(例如人类mtDNA)的有效性提供理想的参考，揭示相对动力学模型和潜在的新的、强大的粪便来源追踪靶标。此外，该项目有可能为粪便污染确定更多有希望的生物标志物，从而改进水质管理人员使用的风险评估工具。因此，这些研究也可能导致新的先进水质监测技术的发展。这项研究的结果将通过与当地合作伙伴Chattahoochee River Keeper的联合研讨会和佐治亚州博览会上名为微生物宠物动物园的互动展览向公众广泛传播。

1511825BrownWith the relatively new molecular biological tools, the identification of fecal contamination in the environment increasingly makes use of rapid molecular assays to identify DNA sequences specific to fecal indicator bacteria or DNA from host-specific cells such as mitochondrial DNA (mtDNA) shed in stools. Unknowns persist, however, primarily regarding the kinetic relationships between culture-dependent and culture-independent assays, since time-since-contamination information is critical in surveillance as a proxy for fecal pathogen survival and therefore public health risk. The proposed work will elucidate the strengths and limitations of current and novel molecular assays intended to identify fecal contamination in environmental waters.To provide new insights into these issues, the research team will undertake fundamental work on degradation of human mtDNA and human-specific microbial DNA-based targets in parallel with a suite of commonly used culture and qPCR methods for fecal source-tracking applications using laboratory-based mesocosms that simulate well natural surface water and sediments. Mesocosms will be spiked with sources of fecal contamination (healthy and diarrheal stools, primary effluent, isolates) and the fate of the gut microbial community, autochthonous microbial community, and pathogens (when present) will be followed over time (6 months) with advanced metagenomics techniques. Metagenomic approaches can track changes to microbial communities and pathogens with unprecedented accuracy and sensitivity. Hence, the temporal metagenomic datasets will provide an ideal reference to benchmark and compare the efficacy of currently used (fecal indicator bacteria test) and proposed (e.g., human mtDNA) biomarkers for fecal pollution monitoring, revealing comparative kinetic models and potentially new, robust targets for fecal source tracking. Further, the project has the potential to identify promising additional biomarkers for fecal contamination, resulting in improvements in risk assessment tools used by water quality managers. Hence, these studies will likely lead to the development of novel advanced techniques for water quality monitoring as well. Results of this research will be broadly disseminated to the public via a joint workshop with a local partner, Chattahoochee River Keeper, and an interactive exhibit called the Microbe Petting Zoo at the Georgia State Fair.