Microbial Source Tracking (MST) Analyses in the Sasco Brook, Lower Farm River, and Goodwives River Watersheds
Microbial Monitoring in Ambient Water
Oral Presentation
Presented by m. pascucilla
Prepared by M. Pascucilla1, M. Copper2, A. Caccone3, D. Knauf4, L. Brooks1
1 - East Shore District Health Department, 689 East Main Street, Branford, Connecticut, 06405, United States
2 - Westport Weston Health District, 180 Bayberry Lane, Westport, CT, 06880, United States
3 - Yale University, 21 Sachem Street, New Haven, CT, 06520, United States
4 - Town of Darien, , Darien, CT, 06820, United States
Contact Information: mpascucilla@esdhd.org; 203-619-1286
ABSTRACT
Abstract:
Goals of this project were to: develop a protocol to identify sources of bacteria from various sources by utilizing DNA test markers, monitor the water quality of each Connecticut watershed to accurately identify significant human and nonhuman sources of bacterial contamination, use the identified sources to develop of strategies to reduce and/or eliminate bacterial sources, while raising awareness and support for evidenced based watershed planning.
A growing body of research indicates Microbial Source Tracking (MST) using Real Time Quantitative Polymerase Chain Reaction (qPCR) may be a useful tool for identifying species contributing bacteria found in waters. A PCR water sample testing protocol and implementation process was developed and tested. Three tributaries to Long Island Sound, Saco Brook in Westport, Goodwives River in Darien, and the Lower Farm River in Branford, were selected as model watersheds to utilize qPCR to detect host specific genetic markers and identify sources of bacteria in the watersheds. Water samples were collected over a 12-month period and analyzed for traditional fecal indicator bacteria (Escherichia coli) using culture based methods. In addition, Bacteroidetes, a largely anaerobic phylum of bacteria commonly used in MST approaches, were analyzed using culture-independent qPCR. Samples were then analyzed for total Bacteroidetes, in an effort to identify host specific detection from multiple sources including human, ruminants, and other animal sources.
This study did not detect significant human contributions to the bacteria levels in the three watersheds. Furthermore, DNA markers associated with feces from poultry, dogs, and cattle were analyzed, but not found in any sample results. Ruminant markers were also used in this study as they encompass cattle and sheep, and wildlife such as deer. Detection of the ruminant marker was rare at the selected sites with no positive samples in Darien, one in Branford, and two in Westport. Finally, the two assays associated with sea gulls and general avian sources failed to pass the quality screening for successful runs. Steps taken to improve performance of both the GFC (specific for seagulls) and the GFD (associated with birds) assays failed, resulting in non-specific amplification or no amplification. The inability to pass the quality screenings means these assays are not included in the analyses.
The results indicate that there is not a detected high level of contribution of bacteria from any of the selected potential sources. While this information does not result in action-based recommendations to address the elevated E. coli levels observed at the monitored sites, it still provides a valuable starting point for future work to develop more reliable markers for birds and other potential sources of bacteria.
Microbial Monitoring in Ambient Water
Oral Presentation
Presented by m. pascucilla
Prepared by M. Pascucilla1, M. Copper2, A. Caccone3, D. Knauf4, L. Brooks1
1 - East Shore District Health Department, 689 East Main Street, Branford, Connecticut, 06405, United States
2 - Westport Weston Health District, 180 Bayberry Lane, Westport, CT, 06880, United States
3 - Yale University, 21 Sachem Street, New Haven, CT, 06520, United States
4 - Town of Darien, , Darien, CT, 06820, United States
Contact Information: mpascucilla@esdhd.org; 203-619-1286
ABSTRACT
Abstract:
Goals of this project were to: develop a protocol to identify sources of bacteria from various sources by utilizing DNA test markers, monitor the water quality of each Connecticut watershed to accurately identify significant human and nonhuman sources of bacterial contamination, use the identified sources to develop of strategies to reduce and/or eliminate bacterial sources, while raising awareness and support for evidenced based watershed planning.
A growing body of research indicates Microbial Source Tracking (MST) using Real Time Quantitative Polymerase Chain Reaction (qPCR) may be a useful tool for identifying species contributing bacteria found in waters. A PCR water sample testing protocol and implementation process was developed and tested. Three tributaries to Long Island Sound, Saco Brook in Westport, Goodwives River in Darien, and the Lower Farm River in Branford, were selected as model watersheds to utilize qPCR to detect host specific genetic markers and identify sources of bacteria in the watersheds. Water samples were collected over a 12-month period and analyzed for traditional fecal indicator bacteria (Escherichia coli) using culture based methods. In addition, Bacteroidetes, a largely anaerobic phylum of bacteria commonly used in MST approaches, were analyzed using culture-independent qPCR. Samples were then analyzed for total Bacteroidetes, in an effort to identify host specific detection from multiple sources including human, ruminants, and other animal sources.
This study did not detect significant human contributions to the bacteria levels in the three watersheds. Furthermore, DNA markers associated with feces from poultry, dogs, and cattle were analyzed, but not found in any sample results. Ruminant markers were also used in this study as they encompass cattle and sheep, and wildlife such as deer. Detection of the ruminant marker was rare at the selected sites with no positive samples in Darien, one in Branford, and two in Westport. Finally, the two assays associated with sea gulls and general avian sources failed to pass the quality screening for successful runs. Steps taken to improve performance of both the GFC (specific for seagulls) and the GFD (associated with birds) assays failed, resulting in non-specific amplification or no amplification. The inability to pass the quality screenings means these assays are not included in the analyses.
The results indicate that there is not a detected high level of contribution of bacteria from any of the selected potential sources. While this information does not result in action-based recommendations to address the elevated E. coli levels observed at the monitored sites, it still provides a valuable starting point for future work to develop more reliable markers for birds and other potential sources of bacteria.
