Source: UNIV OF MINNESOTA submitted to NRP
BASIC AND APPLIED ASPECTS OF BACTERIAL SOURCE TRACKING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0205625
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
S-1022
Project Start Date
Oct 1, 2005
Project End Date
Sep 30, 2010
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
SOIL, WATER AND CLIMATE
Non Technical Summary
Genomic and genetic technologies will be developed and used to determine potential sources of fecal pollution in national waterways. These studies will be done to reduce costs and increase speed of current detection methodologies.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1120399110070%
1330399110030%
Knowledge Area
133 - Pollution Prevention and Mitigation; 112 - Watershed Protection and Management;

Subject Of Investigation
0399 - Watersheds and river basins, general;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
1. To combine targeted sampling with fluorometry and detection of fecal sterils as BST methods to detect fecal contamination in national waterways.
Project Methods
In these studies we will use a modified rep-PCR DNA fingerprinting technique for determination of sources of fecal contamination in waterways. We will also develop new genomics tools, to be used in conjunction with high throughput screening, to define sources of fecal bacteria in waterways. This data will be coupled with fluorometry data to detect sources of fecal contamination in national waterways.

Progress 10/01/05 to 09/30/10

Outputs
OUTPUTS: The contamination of waterways with fecal bacteria is a threat to public health in the US and other counties. While Escherichia coli and enterococci are widely used as an indicators of fecal contamination of waterways, many laboratory and field studies now indicate that these bacteria may become "naturalized" to soils. We have examined survival and growth of naturalized soil E. coli in temperate soil in northern Minnesota and on the macrophytic alga, Cladophora, in Lake Michigan. Both plate count and qPCR analyses indicated that E. coli survives for months in field soil and laboratory experiments showed that soil temperature, moisture, nutrients, and the presence of other soil organisms influenced growth and survival of E. coli in soil. We also showed that while Cladophora-borne E. coli populations in Lake Michigan fluctuate daily and seasonally, there are distinct populations that are stable over time. We also showed that despite large numbers of E. coli in the environment, very few strains carry pathogen-related genes. Taken together, these results call into question the use of E. coli as a fecal indicator bacterium and suggest that other microorganisms that survive similar to pathogens may be better suited for this role. PARTICIPANTS: John A. Ferguson, Satoshi Ishii, Murulee Byappanahalli, Richard Sawdey, Dawn Shively, John Ferguson, Brian Badgley, Mathew Hamilton, Richard Whitman, Hor-Gil Hur, Tatsuya Unno and Michael J. Sadowsky. Please note that Hor-Gil Hur, Valerie Harwood, Greg Kleinheinz and Michael J. Sadowsky directed the research of all participants. This is part of CREEES Regional Project S-1022 - Basic and Applied Aspects of Bacterial Source Tracking: participants of this project include; Dulal Borthakur, Cindy Cisar, Shiping Deng, Yucheng Feng, Terry J. Gentry, Alexandria Graves, Charles Hagedorn, Peter Hartel, Michael Jenkins, John Kaneene, Cindy Nakatsu, Michael Sadowsky, Mary Savin, Alan Sexstone, and Janice Thies TARGET AUDIENCES: See http://nimss.umd.edu/homepages/member.cfmtrackID=6536 for scope of project and targets. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Since E. coli can grow in soils, and survive for long periods of time our results indicate that E. coli is likely not to be a good choice as a fecal indicator bacterium. The association of this bacterium with soils and other non-host organisms, including the alga Cladophora raises serious implications for beach water quality and public health, especially in tropical and subtropical environments were soil warming enhances E. coli growth during the recreational season.

Publications

  • Han, D., T. Unno1, J. Jang, K. Lim, S.-N. Lee, G. Ko, M. J. Sadowsky, and H.-G. Hur. 2010. The occurrence of virulence traits among high-level aminoglycosides resistant Enterococcus isolates obtained from feces of humans, animals, and birds in South Korea. Int. J. Food Microbiol. 144:387-392.
  • Hamilton, M.H., A. Z. Hadi,, J. F. Griffith, Satoshi Ishii, and M. J. Sadowsky. 2010. Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA. Water Res. 44:5463-5473.
  • Unno, T., J. Jang, D. Han, J. Ha Kim, M. J. Sadowsky, O.-S. Kim, J. Chun and H.-G. Hur. 2010. Use of barcoded pyrosequencing and shared OTUs to determine sources of fecal bacteria in watersheds. Environ. Sci. Tech. 44:7777-7782.
  • Unno, T., D. Han, J. Jang, S.-N. Lee, J. H. Kim, G. Ko, B. G. Kim, J.-H. Ahn, R. A. Kanaly, M. J. Sadowsky, and H.-G. Hur. 2010. High diversity and abundance of antibiotic resistant Escherichia coli isolated from humans and farm animal hosts in jeonnam province, South Korea. Sci. Total Environ. 408:3499-3506
  • Ishii, S., Tao. Yan, H. Vu, D. Hansen, R. E. Hicks, and M. J. Sadowsky. 2010. Factors controlling long-term survival and growth of naturalized Escherichia coli in temperate field soils. Microbes and Environ. 25: 8-14.
  • Vanden Heuvel,. A, C. McDermott, R. Pillsbury, T. Sandrin, J. Kinzelman, J. Ferguson, M. Sadowsky, M. Byappanahalli, R. Whitman, and G. T. Kleinheinz. 2010. The Green Alga, Cladophora, Promotes Escherichia coli Growth and Contamination of Recreational Waters in Lake Michigan. J. Environ. Qual. 39:333-344.
  • Weidhaas, J., T. MacBeth, R. Olsen, M. J. Sadowsky, D. Norat, and V. Harwood. 2010. Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay. J. Appl. Microbiol. J Appl Microbiol. 109:334-347.
  • Badgley, B. D., J. Ferguson, A.Vanden Heuvel, G. T. Kleinheinz, C. M. McDermott, T. R. Sandrin, J. Kinzelman, E. Junion, M. N. Byappanahalli, R. L. Whitman, and M. J. Sadowsky. 2010. Multi-scale temporal and spatial variation in genotypic composition of Cladophora-borne E. coli populations in Lake Michigan. Water Res. doi:10.1016 j.watres.2010.08.041


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The contamination of waterways with fecal bacteria is and remains a persistent threat to public health in the US and elsewhere in the world. While Escherichia coli is widely used as an indicator of fecal contamination of waterways, recent laboratory and field studies suggest that this bacterium may become "naturalized" to soils. We have examined survival and growth of naturalized soil E. coli in temperate soil in northern Minnesota using a spontaneously-occurring, antibiotic resistant, E. coli strain, KS7-NR, that was added to a field site at 103 cells g-1 soil. Survival and growth was followed every 4 days during June-August, and monthly in September and October. The density of E. coli in soil was determined by using colony counts on agar plates supplemented with antibiotics, and by using quantitative PCR (qPCR) with strain-specific primers. Both plate count and qPCR analyses indicated that E. coli KS7-NR survived for more than 2 months in the field soil. Laboratory experiments showed that soil temperature, moisture, nutrients, and the presence of other soil organisms influenced growth and survival of E. coli KS7-NR in soil. Fluctuating temperature cycles greatly enhanced the growth and survival of E. coli in soil. PARTICIPANTS: Participants: Pimpak Heepngoen,, Kannika Sajjaphan, John A. Ferguson, Satoshi Ishii, Murulee Byappanahalli, Richard Sawdey, Dawn Shively, John Ferguson, Richard Whitman, and Michael J. Sadowsky. Please note that Richard Whitman, and Michael J. Sadowsky directed the research of all participants. This is part of CREEES Regional Project S-1022 - Basic and Applied Aspects of Bacterial Source Tracking: participants of this project include; Dulal Borthakur, Cindy Cisar, Shiping Deng, Yucheng Feng, Terry J. Gentry, Alexandria Graves, Charles Hagedorn, Peter Hartel, Michael Jenkins, John Kaneene, Cindy Nakatsu, Michael Sadowsky, Mary Savin, Alan Sexstone, and Janice Thies TARGET AUDIENCES: Target Audience: See http://nimss.umd.edu/homepages/member.cfmtrackID=6536 for scope of project and targets. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Since E. coli can grow in soils, and survive for long periods of time our results indicate that E. coli is likely not to be a good choice as a fecal indicator bacterium. The association of this bacterium with soils and other non-host organisms, including the alga Cladophora raises serious implications for beach water quality and public health, especially in tropical and subtropical environments were soil warming enhances E. coli growth during the recreational season.

Publications

  • Ishii, S., Tao. Yan, H. Vu1, D. Hansen, R. E. Hicks, and M. J. Sadowsky. 2009. Factors controlling long-term survival and growth of naturalized Escherichia coli in temperate field soils. Microbes and Environ. In Press.
  • Unno, T., D. Han, J. Jang, S.-N. Lee, G. Go, H.Y. Choi, J.H. Kim, M.J. Sadowsky, and H.-G. Hur. 2009. Absence of Escherichia coli phylogenetic group B2 strains in humans and domesticated animals from Jeonam Province, Korea. Appl. Environ. Microbiol. 75:5659-5666.
  • Hansen, D., S. Ishii, M. J. Sadowsky and R. E. Hicks. 2009. Escherichia coli populations in great lakes waterfowl exhibit spatial stability and temporal shifting. Appl Environ Microbiol. 75: 1546-1551.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The contamination of waterways with fecal bacteria remains a persistent threat to public health. The bacterial pathogens Shigella, Salmonella, Campylobacter, and shiga toxin-producing E. coli were recently found to be associated with the alga Cladophora growing in southern Lake Michigan. We investigated the population structure and genetic relatedness of Cladophora-, water-, and sediment-borne Salmonella isolates from Lake Michigan and adjacent watersheds from 2005-2007. While the Salmonella isolates associated with Cladophora exhibited a high degree of genetic relatedness, they were not all genetically identical. Spatial and temporal relationships were evident in the populations examined, with tight clustering of the isolates both by year and location. These findings suggest that the relationship between Salmonella and Cladophora is likely casual and is related to the predominant Salmonella genotype surviving in the environment during a given season. PARTICIPANTS: Participants: Pimpak Heepngoen,, Kannika Sajjaphan, John A. Ferguson, Satoshi Ishii, Murulee Byappanahalli, Richard Sawdey, Dawn Shively, John Ferguson, Richard Whitman, and Michael J. Sadowsky. Please note that Richard Whitman, and Michael J. Sadowsky directed the research of all pariciipants. This is part of CREEES Regional Project S-1022 - Basic and Applied Aspects of Bacterial Source Tracking: participants of this project include; Dulal Borthakur, Cindy Cisar, Shiping Deng, Yucheng Feng, Terry J. Gentry, Alexandria Graves, Charles Hagedorn, Peter Hartel, Michael Jenkins, John Kaneene, Cindy Nakatsu, Michael Sadowsky, Mary Savin, Alan Sexstone, and Janice Thies TARGET AUDIENCES: See http://nimss.umd.edu/homepages/member.cfmtrackID=6536 for scope of project and targets. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Results from these studies indicate that Cladophora is likely an important reservoir for Salmonella and other enteric bacterial pathogens in Lake Michigan beachsheds. The association of this pathogen with Cladophora raises serious implications for beach water quality and public health, especially in the Great Lakes where Cladophora accumulations are common during the recreational season.

Publications

  • Heepngoen, Pimpak, Kannika Sajjaphan, John A. Ferguson, and Michael J. Sadowsky. 2008. Genetic and Physiological Characterization of oxytetracycline-resistant bacteria from giant prawn farms. J. Microbiol. Biotechnol. 18:199-206.
  • Ishii, S., and M. J. Sadowsky. 2008. Escherichia coli in the Environment: Implications for Water Quality and Human Health. Microbes and Environments. 23:101-108.
  • Byappanahalli, M. N., R. Sawdey, S. Ishii, D. A. Shively, J. A. Ferguson, R. L. Whitman, and M. J. Sadowsky. 2008. Seasonal stability of Cladophora-associated Salmonella in Lake Michigan watersheds. Water Res. doi:10.1016/j.watres.2008.11.012.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: The contamination of waterways with fecal bacteria is, and remains, a persistent threat to public health. The identification of the sources of fecal contamination is necessary to develop efficient abatement strategies and needed for the determination of total maximum daily loads (TMDLs). We have used a genomic suppression DNA subtractive hybridization technique to enrich for host source-specific DNA markers for E. coli originating from pigs and chickens. We have tested 8 probes designed to detect swine E. coli, together they detect ~ 65% of the tested swine. The marker genes, however, also cross-reacted with about 8% of tested E. coli from Turkeys. We are currently developing a high throughput, automated, end-point PCR technique to use these marker genes and those developed for Bacteroides to provide a quantitative, cost-effective, and accurate library-independent method to determine sources of genetically diverse fecal bacteria for use in source tracking studies. PARTICIPANTS: The participants in this project constitute a multidisciplinary team consisting of University faculty, postdocs, and staff scientists. The participants are part of a Multistate Research Project, S-1022, entitled "Basic and Applied Aspects of Bacterial Source Tracking". TARGET AUDIENCES: Results from these studies are targeted to research scientists interested in bacterial source tracking, local, state, and federal government regulatory agencies, and citizen groups interested in determining sources of fecal pollution resulting in beach closures and contamination of waterways.

Impacts
Results from these studies are useful in determining sources of fecal bacteria in the environment. The tools and techniques developed through these studies will ultimately be used to assign health risks associated with fecal contamination of waterways, to develop effective abatement strategies to control run-off, and for TMDL determinations.

Publications

  • Ishii, S., K. P. Meyer, and M. J. Sadowsky. 2007. Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains Isolated from Diverse Human and Animal Sources. Appl. Environ. Microbiol. 73: 5703-5710.
  • Ksoll, W, B., S. Ishii S, M. J. Sadowsky, and R. E. Hicks. 2007. Presence and sources of fecal coliform bacteria in epilithic periphyton communities of Lake Superior. Appl. Environ. Microbiol. 73:3771-3778.
  • Byappanahalli, M. N., R. L. Whitman, D. A. Shively, J. Ferguson, S. Ishii, and M. J. Sadowsky. 2007. Population structure of Cladophora-borne Escherichia coli in nearshore water of Lake Michigan. Water Res. 41:3649-3654.
  • Ishii, S., D. L. Hansen, R. E. Hicks, and M. J. Sadowsky. 2007. Beach sand and sediments are temporal sinks and sources of Escherichia coli in Lake Superior. Environ. Sci. Technol. 41:2203-2209.


Progress 01/01/06 to 12/31/06

Outputs
The contamination of waterways with fecal material remains a persistent threat to public health. The identification of the sources of fecal contamination is a vital component for abatement strategies and in the determination of total maximum daily loads. We used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for E. coli originating from locally-isolated geese. Seven markers were identified and when used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested, and cross-hybridized, on average, with 5% of the human E. coli strains, and less than 10% with strains obtained from other animal hosts. We developed a high throughput, automated, macroarray screening procedure, and showed these markers provide a quantitative, cost-effective, and accurate library-independent method to determine sources of genetically diverse E. coli for use in source tracking studies.

Impacts
Results from these studies are useful in determining sources of fecal bacteria in the environment. Results from these studies will ultimately used to assign health risks associated with fecal contamination of waterways and to develop effective abatement strategies to control run-off.

Publications

  • Ishii S, W.B. Ksoll, R. E. Hicks, and M. J. Sadowsky. 2006. Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds. Appl. Environ. Microbiol. 72:612-621.
  • Ishii, S., T. Yan, D. A. Shively, M. N. Byappanahalli, R. L. Whitman, and M. J. Sadowsky. 2006. Cladophora (Chlorophyta) harbors human bacterial pathogens in nearshore water of Lake Michigan. Appl. Environ. Microbiol. 72:4545-4553.
  • Hamilton, M. J., T. Yan, and M. J. Sadowsky. 2006. Development of goose- and duck-specific DNA markers to determine sources of Escherichia coli in waterways. Appl. Environ. Microbiol. 72:4012-4019.
  • Yan, T., M. Hamilton, and M. J. Sadowsky. 2006. High throughput and quantitative procedure for determining sources of Escherichia coli in waterways using host-specific DNA marker genes. Appl. Environ. Microbiol. In press. Online version - AEM.01395-06v1
  • Yan, T., and M. J. Sadowsky. 2006. Determining sources of fecal bacteria in waterways. Environ. Monit. Assess. In press: Online version - DOI 10.1007/s10661-006-9426-z.