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DEVELOPMENT OF HOST-SPECIFIC METAGENOMIC MARKERS FOR MICROBIAL SOURCE TRACKING USING A NOVEL METAGENOMIC APPROACH
Citation:
SANTO-DOMINGO, J. W., J. LU, O. C. SHANKS, R. LAMENDELLA, C. A. KELTY, AND D. B. OERTHER. DEVELOPMENT OF HOST-SPECIFIC METAGENOMIC MARKERS FOR MICROBIAL SOURCE TRACKING USING A NOVEL METAGENOMIC APPROACH. Presented at Disinfection 2007 - Current Practices and Future Trends in Disinfection: Water, Wastewater Stormwater, Water Reuse and Biosolids, Pittsburgh, PA, February 04 - 07, 2007.
Impact/Purpose:
To inform the public
Description:
Fecal contamination of source water has always been an important issue to the drinking water industry. Improper disposal of animal waste, leaky septic tanks, storm runoff, and the abundance of wildlife in natural water systems can all be responsible for the spread of 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, we applied a novel method called genome fragment enrichment (GFE) against fecal DNA extracts. GFE is based on the use of genomic hybridization to select for genomic regions that differ between fecal metagenomes. Genomic hybridizations were performed to enrich for human, cattle, and chicken specific DNA fragments. Between 300 and 540 enriched metagenomic sequences were generated for each of the three potential fecal sources. Bacteriodales-like sequences dominated the pool of potential markers in the human and cattle experiments, while Clostridium-like sequences were more predominant in the chicken metagenomic enrichments. Most of the enriched sequences are likely to be part of membrane associated proteins, suggesting that they are involved in host-microbial associations. A small number of sequences were then selected to develop host-specific PCR assays. The human and cattle-specific assays showed a high degree of host- specificity, host distribution and geo graphic stability. Several of the chicken-specific assays generated positive signals when challenged against fecal DNA extracts from chicken, turkey, seagull, and geese suggesting the conservation of some genetic markers among different avian species. Positive signals were also obtained when host-specific PCR assays were tested against DNA extracts from fecally contaminated surface waters. The results from these studies suggest that competitive hybridization is a rapid and viable approach for the identification of unique genomic regions that could be used as potential genetic markers to detect fecal sources of pollution. This approach does not rely on the availability of sequence information and can potentially generate hundreds of host specific markers by simultaneously targeting different functional genes. The need for integrating a multi-level approach to environmental monitoring, risk assessment, and risk management for evaluating microbial water quality in source waters will also be discussed in this seminar.