Development of host-specific metagenomic markers for microbial source tracking using a novel metagenomic approach

Fecal contamination of source waters is an important issue to the drinking water industry. Improper disposal of animal waste, leaky septic tanks, storm runoff, and wildlife can all be responsible for spreading enteric pathogens into source waters. As a result, methods that can pinpoint fecal pollution sources in natural waters are needed to assist in the development and evaluation of adequate management practices targeting pollution control. In the last decade, several methods have been developed to identify fecal sources, collectively known as microbial source tracking (MST) methods. Early studies focused on the use of methods that rely on generating library dependent databases. More recently library independent PCR-based approaches have become more popular among MST practitioners as they do not rely on the development of large culture-based databases. One potential concern associated with the use of library independent approaches relates to the development of host-specific assays using sequencing information from genes not involved in host-microbial interactions. To address this issue, our research group has applied a novel method called genome fragment enrichment (GFE) to select for genomic regions that differ between different fecal metagenomes. We have shown that the vast majority of the selected fragments are indeed specific to the fecal community in question suggesting that this is an efficient way of enriching for community specific DNA regions. Sequences from the enriched fragments were used to develop host-specific PCR assays. Thus far, we have successfully developed several assays specific to human, cow, and chicken fecal communities. Additionally, several assays are capable of detecting multiple avian species, further suggesting that similar gut environments can select for similar host-specific populations. The latter finding is relevant as it shows that is possible to develop assays targeting multiple species. Signals from metagenomic-based assays were detected in water samples demonstrating their potential as source tracking tools. A brief summary of the history and limitations of current MST tools and the discussion of results with metagenomic markers will be the main topics of this manuscript. The need for a risk assessment tool-box that includes assays targeting indicators, source identifiers and pathogens will also be discussed.