Source: EAST CAROLINA UNIVERSITY submitted to NRP
INTEGRATED WATERSHED-BASED MOLECULAR AND HYDROLOGIC MONITORING TECHNIQUES TO ASSESS PATHOGEN LOADING IN ESTUARINE ENVIRONMENTS AND IMPROVE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0198337
Grant No.
2003-51130-03139
Cumulative Award Amt.
$549,526.00
Proposal No.
2004-05747
Multistate No.
(N/A)
Project Start Date
Oct 1, 2004
Project End Date
Sep 30, 2008
Grant Year
2005
Program Code
[110.D]- (N/A)
Recipient Organization
EAST CAROLINA UNIVERSITY
EAST 5TH STREET
GREENVILLE,NC 27858
Performing Department
(N/A)
Non Technical Summary
A. Increasing pathogen contamination of esturaine waters are of concern. B. Complexity of loading relationships make it difficult to determine the sources of the pathogens. C. Limited resources require that better methods be developed to determine sources andpathways in order to mitigate. A. To determine sources and pathways for pathogen loading in estuarine waters from a variety of land use types. B. Evaluate a variety of methods for determing those sources --MAR, ribotyping, PFGEhydrologic, watershed assessment. Determinie confounding factors, improved approaches, efficacy of each.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1120320107050%
1120320115020%
1124010104030%
Knowledge Area
112 - Watershed Protection and Management;

Subject Of Investigation
0320 - Watersheds; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1070 - Ecology; 1150 - Toxicology;
Goals / Objectives
Objectives: The first objective of this project is to further the knowledge base with regards to sources and transport vectors of fecal bacterial contamination. Four watersheds in the coastal zone with various land uses-livestock swine production, residential/ industrial, row-crop agriculture, and forestry -- will be the study sites. Secondly, this project will broaden the DNA and MAR source libraries and further refine and evaluate MAR and genotyping techniques with regards to identifying bacterial sources and pathogen densities relative to E. coli and enterococci indicators. Objective 2a will be to quantify residual levels for antibiotics in the environment and model their fate by testing both the scat and water samples, and by measuring degradation rates and partitioning between dissolved and particulate phases. By determining the antimicrobial residue levels in the respective environment, we will be able to understand the chemical hazard that may be associated with the representative environmental sources. In addition, we will determine correlation between the chemical residue levels and presence and level of multi-drug resistant strains. Objective 2b will be to identify and partially quantify active, metabolizing pathogenic bacteria in each water and scat sample using stable isotope probing and DGGE. Furthermore the active organisms will be analyzed using PFGE to determine the type of organism and specific antimicrobial resistance genes and related determinants (such as integrons) will be tested using polymerase chain reaction (PCR) etc. Third, this enhanced level of pathogen identification information will analyzed relative to the indicator data to investigate densities of harmful pathogen and antimicrobial resistant strains in estuarine waters relative to the density of the indicator data. This particular data set will be used to develop the source loading rates, identify potential transport vectors to explore management options to improve the NC Shellfish Sanitation Program. For the various land uses of study, the costs of these strategies will be assessed within the context of their known efficacy for mitigating bacterial contamination. Fourth, training materials for local government officials, planners, and watershed managers will be developed and delivered. Additionally, this information will be used to supplement the curriculum and provide study opportunities for students enrolled at the Duke Marine Lab, as well as the College of Design, School of Veterinary Medicine and Applied Environmental Microbiology programs at NC State University.
Project Methods
Methods: Methods to be employed in this project are divided into three major areas of effort: bacteria source tracking, review and development of management strategies, and dissemination of educational material. Bacterial Source Tracking: A multi-tiered, tool-box approach will be used for the bacterial source tracking effort to differentiate sources and quantify loads of fecal coliform bacteria including: 1) watershed and land use assessment; 2) storm event and grab sample hydrologic monitoring; 3) bacterial store / scat sample library characterization; 4) identification of residual antibiotics in the sample set; 5) identification and partial quantification of the metabolically active population of bacteria in sample sets; 6) isolation of E. coli and enterococci bacteria; followed by 7) Multiple Antibiotic Resistance; 8) Ribotyping; and 9) PFGE analyses.

Progress 10/01/05 to 10/01/06

Outputs
PD Hyman's lab has been conducting DNA-based SIP experiments. Focus since last report is on identifying appropriate feeding strategies for 13C substrates, isolation of DNA from attached rather than suspended bacteria and steps to improve the reproducibility of the final molecular analyses of 13C-labeled DNA. Appropriate feeding strategies is perhaps the most significant issue as the SIP signal intensity (ie detectable heavy DNA) is strongly influenced by the population size in test sample and extent of exposure to labeled substrates which can label different or unrepresentative populations. A further complication is that this strategy labels secondary organisms that either utilize metabolites generated by primary degraders (i.e. excreted metabolites or cell debris obtained from dead cells) or are artificially revived as a consequence of the extended feeding procedure. These complications obviously devalue the data that can be obtained from SIP when it is targeted at identifying metabolically active organisms within water samples. We still anticipate we will be able to provide SIP analyses of background water and storm-impacted water samples before the end of the funding period. PD Line reports monitoring of rainfall and runoff from 8 storm events at the Bailey pig farm waste application site was conducted. A composite sample from 3 of the storms was recovered and sent to the various labs for analysis. A grab sample of baseflow was also collected on 12/4/06 and sent to the various labs for analysis. Monitoring of discharge and sample collection at the three other sites continued. PD Gebreyes transferred to Ohio State University. Sample and assessment continued uninterrupted. National forest site is continuing to show high levels of Salmonella infestation with 246 of 295 samples positive (83% prevalence). Antimicrobial susceptibility showed (237) were susceptible to all the 12 antimicrobials, 9 isolates showed resistance to streptomycin. PD Shea has established degradation rates of antibiotics across a range of environmental conditions. This will add to the growing database on this issue. They are also going to be able to demonstrate antibiotic exposures at various site types, and data from this study will be combined with that from another USDA study and an EPA funded project. There have been no detectable levels of antimicrobial in these sites. They are trying quantitative description of site type influence on exposures. They have tested applicability of a simple surface water fate model to predict antibiotic exposure downstream. Two publications expected in approx. 3months. PD White is continuing ribotyping analyses begun under the USDA CSREES grant #2001-51130-11458. Bionumerics will be used for matching analyses. Expect all matching to be completed this summer.

Impacts
Improved understanding of pathogen density in natural coastal waters, the

Publications

  • No publications reported this period


Progress 10/01/04 to 09/30/05

Outputs
Four sites have been instrumented and have been collecting discharge data continuously throughout the period.The highest levels of fecal coliform were found in storm samples from the Open Grounds site followed by the residential site. The Hog Farm site had the lowest level of fecal coliform in storm samples. The average level of fecal coliform in baseflow samples was lowest for the Croatan site. Unexpectedly, the fecal coliform levels in baseflow samples from the Open Grounds site were the highest. Hyman is focused on the developing the technique of stable isotope probing for identifying metabolically active organisms within complex microbial communities. This novel technique relies on extensive stable isotope (13C or 15N) incorporation into actively growing and dividing bacteria. The mastery of several independent techniques and steps are, in sequence, the introduction and metabolism of stable isotope sources, isolation of total DNA from microbial communities, density gradient centrifugation to separate heavy and light-labeled DNA and analysis of PCR-amplified 16s rRNA sequences derived from purified labeled DNA. Our activities have focused mainly on the last three techniques. We have had greater success using terminal restriction fragment length polymorphism (T-RFLP) analyses for these analyses. The remaining part of the technique we have not yet addressed or optimized is the exposure of samples to 13C (or 15N)-labeled sources. A key issue in SIP is the need for metabolically active cells to metabolize and grow on isotopically labeled sources. Consequently it is important to know how rapidly and extensively 13C-labeled sources are metabolized over the next few months. The Gebreyes lab has been actively involved in monitoring a pig farm in Bailey, NC and processing for Salmonella isolation and characterization from the various sites in the project. To date, thirty-two samples have been processed and, from these samples, a total of 186 isolates have been recovered. Twenty-one of the thirty-two samples (65.6%) were positive for Salmonella. Interestingly, none of the samples (base flow or storm) from the pig farm site have been positive for Salmonella, whereas 100% of the storm samples from both Open Grounds Farm and Jumping Run Creek were positive. The frequency of Salmonella recovery in storm event at Pettiford Creek was 86% (n=7). For the base flows, the frequency was as follows: Jumping and Run Creek (50%, n=4), Open Grounds Farm (25%, n=4) and Pettiford Creek (25%, n=4). The Shea lab has been analyzing environmental and scat samples for antibiotics and has nearly completed the laboratory studies to estimate antibiotic degradation rates and partitioning between dissolved and particulate phases. The environmental analyses has consistently shown non-detectable or very low (parts per trillion) concentrations of antibiotics. The laboratory studies indicate relatively low variability in half-lives and partitioning among replicates within a given site, but high site-to-site variability

Impacts
Improved understanding of pathogen density in natural coastal waters, the effectiveness of the currently touted MST technologies for detecting them, and thereby improved understanding of management strategies .

Publications

  • No publications reported this period