Brief Summary of Minutes of Annual Meeting

MINUTES OF THE MULTISTATE PROJECT S-1022 BASIC AND APPLIED ASPECTS OF BACTERIAL SOURCE TRACKING ANNUAL MEETING West Virginia University Morgantown, West Virginia May 20 and 21, 2008 Project Participants: Jonathan Edelson (Oklahoma State University), Yucheng Feng (Auburn University), Alexandria Graves (North Carolina State University), Peter Hartel (University of Georgia), Annie Hassall (Virginia Tech), Mike Sadowsky (University of Minnesota), Alan Sexstone, Susan Spiker, and Rachel Wiechman (West Virginia University), and Janice Thies (Cornell University) Guest Participants: Gary Bissonnette (Professor, West Virginia University); Emily Markle, (graduates student, West Virginia University) May 20 (Tuesday), Annual Project and Business Meeting At 9:00 AM Alan Sexstone opened the meeting with a discussion of the proposed agenda. Participants were reminded that electronic copies of their annual reports should be submitted to Alan in a timely manner, in order to submit the annual report to the project director within 60 days. Participants were notified that due to a last minute conflict, Terry Gentry (Texas A&M) was unable to attend the meeting as planned, but had submitted his annual report and had volunteered to host the annual meeting in 2009. This offer was accepted immediately, with the proviso that Terry consider hosting the meeting in the area of South Padre Island. In separate discussions it was established that Peter Hartel would host the 2010 meeting, probably in Savannah, GA; and will coordinate submission of the final project report. Jonathan Edelson introduced himself as the new project administrator. He provided an update on the Farm Bill, reorganizations within CSREES, and changes in NRI funding. Participants asked for an explanation of differential funding among multistate participants by different institutions, i.e. some received a real dollar budget for supplies, students, and/or travel while others received no money and supported multistate research efforts through their other grants. Jonathan indicated that there was no set formula and that Experiment Station Directors had flexibility on how they allocated funds. He pointed out the requirement that a minimum percentage of Hatch funds coming to Experiment Stations be received from multistate projects, and thus our participation in such projects should be viewed positively and supported by administrators. That prompted a discussion of ghost participants, i.e. those individuals who are listed on projects but seldom participate in meetings or submit annual reports. Participants discussed enforcement of requirements for continued membership in S-1022 as adopted at the 2006 annual meeting. Those current project members failing to comply with participation requirements of S-1022 should expect to be dropped from the project. Peter Hartel began the technical reports with an overview of fluorometry and various studies he has performed to assess and optimize the method. Fluorometry identifies human fecal contamination by detecting optical brighteners, primarily from laundry detergents, in environmental waters. One curious result from the 2007 S-1022 Puerto Rico (PR) study was the occasional lack of correlation observed between very high fluorescence readings (which should be indicative of a sewage outfall) and numbers of fecal coliforms. The most plausible explanation proposed for for these data was the practice of piping of gray water separately to an outfall in order to avoid overloading of septic systems. Other fluorometry issues that remain to be investigated include the contribution of high bacterial numbers to background fluorescence, and the interference of turbidity from the presence of silts and clays. It was proposed that these studies be performed following the meeting, since Peter brought his portable fluorometer and other accessories to WV for Sexstones use. Peter discussed the outcomes of last years multistate collaborative work in Puerto Rico. The purpose behind the targeted sampling of two streams in Mayaquez, PR was to compare and contrast several different bacterial source tracking (BST) methods in waters low in background dissolved organic matter. Methods employed included fluorometry, enumeration of fecal coliforms and enterococci, antibiotic resistance analysis (ARA) and rep PCR patterns of bacterial isolates, and molecular detection and/or comparison of specific bacterial groups or communities. Peter currently is assembling available data from last years work, and coordinating preparation of a publication targeted for an October, 2008 submission to the Journal of Environmental Detection. Participants discussed the current state of data and unfinished analyses. It was determined via email that Terry Gentry had frozen sample filters that if necessary could be provided for completion of some molecular analyses. Mike Sadowsky summarized several ongoing areas of research relevant to environmental sources and sinks of E. coli, several of which were published during 2007. He briefly reported on 1) the occurrence of indigenous soil E. coli 2) a high throughput and quantitative procedure for determining sources of E. coli in waterways using host-specific DNA marker genes 3) on phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains isolated from diverse human and animal sources, and 4) on population structure of Cladophora-borne Escherichia coli in near-shore water of Lake Michigan. Other projects summarized by Mike included1) results demonstrating that E. coli are more abundant in benthic fish (compared with pelagic fish), which may vector of E. coli from other sources, rather than serve as a new source of E. coli contamination in aquatic environments 2) the presence (up to 1.4 × 105 CFU cm2) and sources (68 to 99% from waterfowl) of fecal coliform bacteria in epilithic periphyton communities of Lake Superior 3) Results suggesting that wave action may influence the early colonization and homogeneous distribution of E. coli in beach sand and the subsequent release of sand or sediment-borne E. coli into lake water, indicating that sand and sediment serve as temporal sources and sinks of human and waterfowl-derived E. coli that contribute to beach closures. Yucheng Feng described the continuation of her work using bacterial source tracking in the Catoma Creek watershed (AL). This was the last year of sample collection on this project. Water samples were collected monthly at six locations in the watershed. E. coli concentrations in the water samples ranged from 5 to 3650 CFU/100 ml of water. Total phosphorus concentrations in the water samples were consistently higher than the EPA region 9 reference level of 0.036 mg/L. More than 2500 E. coli isolates have been obtained from water samples for source identification. The size of the E. coli rep PCR DNA fingerprint library has been increased to about 1000 E. coli isolates for nine source groups. The results showed that humans, domestic animals, and wildlife all contributed to the fecal contamination in the watershed. The presence of human signatures was verified by LC/MS analysis of nine chemicals commonly used by humans. Alex Graves described her work to determine whether malfunctioning on-site wastewater systems are contributing to bacterial loading in North River, Carteret Co. (NC). Septic tank effluents from 11 homes were collected , E. coli isolates recovered, then analyzed by antibiotic resistance analysis. These human isolates will be used along with ARA patterns generated from various animal to assign to source unknown E. coli isolates recovered from the North River. Thirty-four sampling sites have been identified for the project. Alex reported analytical problems with her fluorometer, which she hopes to use in this study. Peter Hartel volunteered to send his portable fluorometer once others complete their work with the instrument. Alex also is investigating land application of swine lagoon effluent as a potential source of antibiotic resistant genes in surface water. To date, 75 E. coli strains from swine manure, lagoon effluent and nearby ground and surface waters have been recovered for the purpose of evaluating both presence of antibiotic resistance genes and phenotypic expression of resistance. Bacterial isolates (1200) recovered from swine manure, cattle manure, human septic, wildlife, and pets have/ will be analyzed by antibiotic resistance analysis to serve as a library database. To date, 624 unknown E. coli isolates have been recovered from surface water, groundwater and riparian waters. When database development is complete, these unknown source isolates will be compared against the database for fecal source identification. Annie Hassall attended the meeting and reported for the Hagadorn lab. Samples collected from streams in Mayaguez, Puerto Rico were processed using Idexx trays for enumeration of fecal bacteria. Trays shipped on ice to Hagedorns lab were used to obtain isolates of Enterococcus and E. coli. These isolates currently are available to S-1022 participants for further characterization. The Biolog identification procedure was performed on 400 Enterococcus isolates to determine species composition. E. faecalis and E. faecium in high concentrations can be indicative of the presence of human fecal contamination, while E. gallinarum is an indicator of the presence of fecal contamination from dogs and/or birds. Results obtained confirmed that human isolates were more prevalent downstream of known sewage outfalls. Enterococcus species identification could complement other BST protocols and could be an adjunct method when isolation and cultivation of enterococci is required for other purposes such as monitoring. Janice Thies reported on the development and use of a quantitative real-time polymerase chain reaction protocol (Q-PCR) for the detection and enumeration of Escherichia coli genomes in both water and epilithic biofilms samples obtained from streams in Mayaguez, Puerto Rico. A probe designed against the intergenic transcribed spacer (ITS) regions of the E. coli genome was found to detect less than 100 genomes per sample while being insensitive to the presence of other enteric bacteria. Peaks in E. coli abundance in both water and epilithic biofilm samples ranged from 2-4 orders of magnitude higher at point sources over samples located either upstream or downstream. In samples of stream water collected at the sewage outlets, E. coli abundances ranged from 6x105 to 8x106 genomes per sample. E. coli were present at abundances ranging from 102 to 3x103 in samples collected upstream of sewage culverts, where downstream concentrations increased to ³ 4x103. Detection of low genome numbers in epilithic communities was more problematic, however sewage outlet samples reliably yielded high quantities of biofilm resident E. coli (>105 genomes sample-1). The ability to identify fecal contamination point sources using the Q-PCR method was straightforward and should be applicable to targeted management to control primary sources of fecal contamination. Rachel Wiechman and Susan Spiker (graduate students) reported for the Sexstone lab. The Davis College of Agriculture at West Virginia University maintains a certified organic farm to evaluate efficacy of defined organic production systems for both plants and animals. Rachel and Susan have compared patterns of antibiotic resistance in 600 E coli. isolates from organic and conventionally raised chickens and sheep, and from soil they graze on. Compared with organic chickens, more conventional chickens isolates were resistant to ampicillin and cephalothin. All chickens harbored isolates that were resistant to rifampin, streptomycin and neomycin. Conventional and organic sheep isolates were primarily resistant to rifampin, as were isolates from baseline soil samples. Following eight weeks of grazing by organic chickens, isolates from paddock soils became significantly resistant to tetracycline. Preliminary BOX A1R rep-PCR fingerprints were obtained from the E. coli isolates and subjected to cluster analysis. Data supported a separate baseline soil and organic chicken cluster. Soil isolates obtained following exposure to organic chickens clustered more closely with chickens than with baseline soils. An additional 1000 isolates from soils and pasture-raised beef cattle currently are being analyzed. Future work includes utilization of additional antibiotic sensitivities, improved rep PCR analyses, and modifications of the clustering technique employed (Feng, Sadowsky, and Thies all offered constructive criticism, provided pertinent suggestions to improve the experimental work and data analyses, and graciously offered their help with various technical problems). The business meeting was adjourned at 4:15 PM on 5/20/08. Most of the group continued to meet informally on Tuesday evening during dinner. May 21 (Wednesday), Meeting and S-1022 Collaborative Research in Sexstone Lab Beginning at 9:00 AM on 05/21/08 Alan Sexstone and Peter Hartel worked with Rachel Wiechman and Susan Seese to perform various fluorometry analyses. Feng, Graves, Sadowsky, observed and offered comments and suggestions prior to leaving for the airport at midday. Thies joined the fun later in the day. Previously, Sexstone had located an orphaned fluorometer which Sadowsky helped to install and set up prior to his departure. As part of the current multi-state project, Sexstones team is monitoring on-site systems discharging from individual households into Paradise Lake (39o 30' 6.52" N, 79o 54' 23.35" W). The study site is a ~30 ha impoundment surrounded by residential housing with no centralized wastewater collection. The lake also supports a large population of non-migrating Canadian geese, and grass-eating carp. Twenty four water samples were collected from the shoreline around the lake and subjected to preliminary fluorometry. None exhibited a strongly positive signal. These data were affected by ongoing heavy rainfall in the area, and demonstrated the need to sample with higher resolution around the circumference of the lake. Hartel worked with Susan and Rachel to examine potential interference of high E coli numbers on the fluorescence signal from a known source. They also designed an experiment to examine the effects of montmorillonite or kaolinite turbidity on fluorescence from a known source to be conducted following the meeting. These activities were concluded in late afternoon bringing the 2008 S-1022 meeting to a close. Respectively submitted, Alan Sexstone, 2008 Chair

Research Accomplishments: Objective 1. To combine targeted sampling with fluorometry and detection of fecal sterols as bacterial source tracking (BST) methods to detect fecal contamination in national waterways. Yucheng Fengs lab continued to use bacterial source tracking in the Catoma Creek watershed. They continued to collect water samples monthly at six locations in the watershed during the year. E. coli concentrations in the water samples during the 12-month period ranged from 5 to 3650 CFU per 100 ml of water. Total phosphorus concentrations in the water samples were consistently higher than the Ecoregion 9 reference level of 0.036 mg/L. More than 2500 E. coli isolates have been obtained from water samples for source identification. The size of the E. coli rep PCR DNA fingerprint library has been increased to about 1000 E. coli isolates for nine source groups. The results showed that humans, domestic animals, and wildlife all contributed to the fecal contamination in the watershed. The presence of human signatures was verified by LC/MS analysis of nine chemicals commonly used by humans. Host specific genetic markers from Bacteroidetes for humans and cattle will be used to provide another line of evidence. Alex Graves will perform ARA analyses on Enterococcus isolates obtained in the May 2007 sampling of the Sabelo and Saval streams in Mayaguez, Puerto Rico. She has initiated a funded project entitled Fecal Coliform Impacts, N. River Community, Carteret Co., North Carolina which will identify sources of human fecal pollution entering a stretch of the North River. Human and animal E. coli isolates are being collected to establish a reference library against which unknown isolates will be sourced. These data will help the North River Community establish a baseline of need with which to seek infrastructure improvement for sewage treatment. Chuck Hagedorns lab in conjunction with the S-1022 multistate working group conducted targeted sampling of the Sabalo and Savat streams in Mayaguez, Puerto Rico, as part of its annual meeting in May 2007. Samples were processed for detection of fecal contamination by multiple methods. Enterococcus and E. coli were enumerated in IDEXX trays which were then shipped to Hagedorns lab in order to obtain representative isolates from positive IDEXX wells. Isolates were frozen for long-term storage, and are available to S-1022 participants for futher analyses. The Biolog identification procedure was performed on 400 of the Enterococcus isolates. These isolates were from Mayaguez, Puerto Rico, 200 each from the Savat (3 sampling sites) and Sabalo (2 sampling sites) streams (Figures 1 and 2). There were clear differences in the species composition profiles between sites within both streams. For the Savat stream, locations Y and W were close to what appeared to be sewage outfalls and the species composition was mainly Ent. faecalis and Ent. faecium (locations Y and W, Figure 1). Neither of these species were found at a site located upstream from the outfalls and the species composition at this site was much more diverse than at the other two sampling sites (location V, Figure 1). For the Sabalo stream, the Enterococcus species composition was more diverse (Figure 2) that for the Savat stream (Figure 1). In the Savat stream there were two sampling sites, one above and one below what appeared to be a sewage outfall. Both sites contained Ent. faecalis and Ent. faecium (Figure 2), but otherwise resembled location V at the Savat stream where a large percentage of the species composition was Ent. mundtii and Ent. gallinarum, more indicative of birds and wildlife (Figure 1). There are probably other outfalls located above both sampling sites in the Sabalo stream, and this would account for the presence of Ent. faecalis and Ent. faecium and the similarity of the species composition at both sites. Multiple sources of fecal pollution are apparent in both streams. Peter Hartels program continues to develop inexpensive methods of BST. Hartel was asked by Georgia Environmental Protection Agency, the Georgia Department of Natural Resources, and the State Health Districts to conduct BST research to identify possible sources of human fecal contamination to six of their priority areas: 1) the beach at south end of St. Simons Island, 2) the beach at Tybee Island, 3) the beach at Jekyll Island, 4) the coast of McIntosh County, 5) the Kings Ferry portion of Ogeechee River, and 6) the Jerico River along the Liberty/Bryan County border. Combined targeted sampling with fluorometry was used to identify sources of human fecal contamination to the six priority areas. Targeted sampling works by identifying hotspots of fecal contamination through multiple samplings over ever-decreasing distances. Fluorometry identifies human fecal contamination by detecting optical brighteners, primarily from laundry detergents, in environmental waters. However, as a method, fluorometry does not work well because other compounds in environmental waters also fluoresce. Therefore, it was important to solve this problem before the six priority areas were sampled. This was accomplished by 1) adding a 436-nm emission filter to the fluorometer and 2) exposing environmental water samples to ultraviolet light to differentiate between optical brighteners and other fluorescing organic compounds. With the 436-nm emission filter in place, optical brighteners were likely present when the relative percentage difference in fluorometric value of the water before and after UV light exposure was >30% (glass cuvettes, 30 minute exposure) or >15% (polymethacrylate cuvettes, 5 minute exposure). In a blind study, the presence or absence of optical brighteners was correctly identified in 178 of 180 (99%) of the samples tested with a more expensive field fluorometer and in 175 of 180 (97%) of the samples tested with a less expensive handheld fluorometer. In the field, the method correctly identified two negative and three positive locations for human fecal contamination. Subsequently, the methods were tested on the six priority areas. None of the priority areas had problems with human fecal contamination except the beach at St. Simons Island. At the 2007 S-1022 annual meeting, the improved fluorometric method were tested in Puerto Rico because its waters there are remarkably low in interfering organic matter. Researchers from GA and VA collaborated with multistate researchers from five other member states (AL, MN, NC, TX, and WV) on field-testing the improved fluorometric method. Mike Sadowskys team has been investigating sources and sinks of E. coli in the environment. E. coli and fecal coliform bacteria were isolated from five benthic and four pelagic fish species to determine their role in the fecal contamination of recreational waters. All fish were collected from Southworth Marsh in the Duluth-Superior Harbor, a public beach that is commonly posted to minimize water contact, due to high E. coli levels. Although fecal coliforms were isolated from each fish species, they were only isolated from 66% and 72% of the individual benthic and pelagic fish, respectively. While 42% of the fecal coliforms from benthic fish were E. coli, only 4% from pelagic fish were E. coli. Cluster analysis showed different fish species harbored identical strains of E. coli and some fish had multiple E. coli strains. The potential source for 65% of the E. coli isolates obtained from fish were identified by using the HFERP DNA fingerprinting method and libraries of E. coli DNA fingerprints from warm-blooded animals and environmental isolates collected in the area. The E. coli strains whose source could be identified were most similar to strains isolated from sediments, Canada geese, mallard ducks, and wastewater. None of the fish E. coli had DNA fingerprints matching those from any water or beach sand isolates. Although our results demonstrate that E. coli are found in benthic fish, it may be more appropriate to consider these fish as a vector of E. coli from other sources, rather than a new source of E. coli contamination in aquatic environments. Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior to determine if fecal coliforms and E. coli were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 × 105 CFU cm2 by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the E. coli bacteria isolated from these periphyton communities could be identified when compared with a library of E. coli fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton E. coli strains that could be identified. Several periphyton E. coli isolates were genotypically identical (e92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified E. coli strains from periphyton were added to the known source library, then 57% to 81% of E. coli strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated E. coli rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that E. coli from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution. The Duluth Boat Club (DBC) Beach, located in the Duluth-Superior harbor of Lake Superior, is frequently closed in summer due to high counts of Escherichia coli, an indicator of fecal contamination. However, the sources of bacteria contributing to beach closure are currently unknown. The potential sources of E. coli contaminating the DBC beach were investigated by using modified rep-PCR (HFERP) DNA fingerprinting. Over 3600 E. coli strains were obtained from 55 lake water, 25 sediment, and 135 sand samples taken from five transects at the DBC beach at 11 different times during the summer through fall months. Potential sources of E. coli at this beach were determined by using a known-source DNA fingerprint library containing unique E. coli isolates from wildlife, waterfowl, and treated wastewater obtained near Duluth, MN. Amounts E. coli in the samples were enumerated by membrane filtration counting, and the presence of potentially pathogenic E. coli was determined by using multiplex PCR. E. coli counts in all samples increased during the summer and early fall (July to September). While E. coli in spring samples originated mainly from treated wastewater effluent, the percentage of E. coli from waterfowl increased from summer to fall. DNA fingerprint analyses indicated that some E. coli strains may be naturalized, and autochthonous members of the microbial community in the beach sand and sediments were examined. However, multiplex PCR results indicated that <1% of the E. coli strains at the DBC was potentially pathogenic. Results suggest that wave action may influence the early colonization and homogeneous distribution of E. coli in beach sand and the subsequent release of sand or sediment-borne E. coli into lake water. Taken together, these results indicate that sand and sediment serve as temporal sources and sinks of human and waterfowl-derived E. coli that contribute to beach closures. Other work published in 2007 included such topics as a high throughput and quantitative procedure for determining sources of Escherichia coli in waterways using host-specific DNA marker genes; phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains isolated from diverse human and animal sources, and; population structure of Cladophora-borne E. coli in nearshore water of Lake Michigan Alan Sexstone continues to evaluate the effectiveness of alternative on-site wastewater treatment systems such as subsurface flow constructed wetlands and aerobic treatment units (ATUs) currently used in West Virginia. As part of the current multi-state project, Sexstones team has begun to monitor on-site systems discharging from individual households into Paradise Lake. The study site is a ~30 ha impoundment surrounded by residential housing with no centralized wastewater collection. The lake also supports a large population of non-migrating Canadian geese, and a large population of resident grass carp. Attempts are underway to use chemical source tracking methods (fluorometry, fecal sterols) to discriminate human and non-human fecal contamination in water and sediments obtained from this system. Janice Thies and Peter Bergholz developed fluorogenic probes that can be used to detect the presence and quantify the abundance of E. coli genomes in a quantitative real-time PCR (Q-PCR) assay. The E. coli probes were designed and tested on known E. coli strains and non-target Enterobacterial genera to determine their specificity. A probe designed against the intergenic transcribed spacer (ITS) regions of the E. coli genome was found to be able to detect less than 100 genomes per sample while being insensitive to the presence of other enteric bacteria. E. coli was detected in both water and epilithic biofilm DNA extracts from both the Sablos and Saval streams in Mayagues, Puerto Rico. E. coli genome abundance was determined along the length of the two streams both up- and down-stream of sewage outlets (Figure 3). In samples of stream water collected at the sewage outlets, E. coli abundances ranged from 6x105 to 8x106 genomes per sample. E. coli were present at abundances ranging from 102 to 3x103 in samples collected upstream of the two sewage culverts on the Sabalos stream, where downstream concentrations increased to ³ 4x103. E. coli was also quantified in community DNA samples isolated from epilithic biofilms collected in both streams. Detection of E. coli genomes in biofilm DNA samples was less straightforward than detection in water, with abundances in some samples below the limit of detection in our samples (Figure 4). However, sewage outlet samples reliably yielded high quantities of biofilm resident E. coli with >105 genomes sample-1 located at the outlet in the Sabalo stream and >106 genomes sample-1 detected at the outlet in the Saval stream. Figure 4: Abundance of E. coli in epilithic biofilm communities in the A) Saval and B) Sabalo streams. Saval sites Y and W and Sabalo sites 6 and 7 include sewage outlets. Missing data points indicate E. coli were not detected in the sample (< 100 genomes sample-1). Block arrows indicate the location of the sewage outlets. Objective 2. To determine the stability of antibiotic resistance genes in fecal indicator bacteria under different environmental conditions in order to understand the movement of these genes in soils and waters receiving animal wastes, and the effect of their stability on the reliability of detection methods based on characterizing these genes. Terry Gentry is involved in two projects to evaluate the ability of different management practices to reduce the environmental contribution of E. coli and antibiotic resistant bacteria from different grazing systems and the land application of animal manures. For the grazing studies, three different sites across Texas will be tested  Welder Wildlife Refuge, Riesel Experiment Station, and a private ranch (TBD). Evaluation and demonstration of proper grazing management will primarily take place on the Welder Wildlife Refuge near Sinton, Texas. Three small (1-2 acres) watershed sites on the Welder Refuge have been refurbished and equipped to measure runoff and collect samples for three years from three different treatments - one with no grazing, one with prescribed grazing, and one with heavy grazing. These data will be compared to that collected at Riesel on (1) ungrazed native prairie and (2) grazed improved pasture over the same three year period to identify regional differences and effects of grazing. On the private ranch, effects of prescribed grazing used in conjunction with alternative water supplies and fencing will be determined over a three year period. Runoff samples will be collected from watershed sites on the Welder Refuge and Riesel using Isco samplers. Upstream and downstream grab samples are currently being collected on a periodic basis (~ every 2 weeks) from the private ranch. Water samples from all locations will be analyzed for E. coli and antibiotic resistant bacteria during runoff events or on a periodic basis for streams. Samples will also be analyzed for a ruminant Bacteroides marker. For the land application of manures project, a total of four sites have been selected for the evaluation of BMPs. The four fields consist of three manured (wastewater, vacuum manure, dry manure) fields (corn, hay, and pasture) and one inorganic fertilized hay field. The manured fields have buffer strips, one managed and one unmanaged. The inorganically fertilized field does not have a buffer strip. Each field will be set up for edge of field monitoring using Isco samplers. Each field will be bermed forcing the runoff to run through a single outlet. This water will be split into the managed and unmanaged filter strip. An Isco will be placed prior to the buffer at the edge of the field and after each buffer at the edge of that land management unit (nine monitoring sites, plus one control). Runoff from storm events will be collected by the Isco samplers. Grab samples will be collected upstream and downstream from the sites. E. coli numbers will be analyzed from each runoff sample. E. coli will be isolated from each potential source and fingerprinted using a combination of the enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction technique (ERIC-PCR) and RiboPrinting. Genetic fingerprints of E. coli isolates will be added to a developing statewide source tracking library (TAES-El Paso AREC). E. coli will be isolated from edge of field runoff samples, collected after storm events, and upstream and downstream grab samples four times per year. These isolates will be compared to the environmental library to determine the source(s) of the isolates and the relative contribution of each source to the total E. coli load. Water samples will also be analyzed for Bacteroides human and animal genetic markers. Alex Graves and her team are studying the agricultural use of antibiotics to see if this practice is partly responsible for the emergence of antibiotic-resistant organisms. Research efforts at NCSU include a project entitled Antibiotic Resistance and Water Quality: Land Application of Swine Lagoon Effluent as a Potential Source of Antibiotic Resistant Genes in Surface Water. The goal of this study is to evaluate the association of antibiotic resistance genes found in E.coli isolated from swine with the actual phenotypic expression of the resistance. Additional plans include the development of an antibiotic resistance database for E. coli isolates from a commercial swine facility and assessment of its efficacy for tracking movement of bacteria from swine confinement houses to surface waters. The specific objectives include: 1) Determine the relationship between presence of antibiotic resistance genes for tetracycline, sulfonamides, streptomycin and apramycin resistant genes found in E. coli strains from swine manure, lagoon effluent and nearby ground and surface waters with the actual phenotypic expression of the resistance. 2) Develop a database of antibiotic resistance patterns for E. coli isolated from swine manure, cattle manure, wildlife manure, human and pets, and 3) Evaluate the usefulness of this database for assessing movement (or dispersal) of E. coli from a confined swine operation to a nearby stream. Seventy-five E. coli strains from swine manure, lagoon effluent and nearby ground and surface waters have been recovered for the purpose of evaluating for antibiotic resistance genes and the phenotypic expression of the resistance. Two hundred twenty five more E. coli isolates will be recovered and analyzed as described in the proposal. To date, 912 bacterial isolates recovered from swine manure, cattle manure and human septic have been recovered and analyzed by antibiotic resistance analysis. The database will be completed with the addition of 288 isolates collected from wildlife manure and pet manure. Six hundred twenty four E. coli isolates have been recovered from surface water, groundwater and riparian waters. The 624 E.coli isolates have been analyzed by antibiotic resistance analysis. When database development is complete, the unknown source isolates collected from the environmental waters will be compared against the database for fecal source identification. Mary Savin is assessing whether low levels (<1 ppb) of antibiotics or bacteria that may be entering the environment with wastewater treatment plant effluent are altering levels of antibiotic resistance in aquatic ecosystems. Her team has been measuring E. coli and total coliform most probable numbers (MPN) in the presence and absence of antibiotics. Samples have been collected downstream of where effluent enters the stream, in the effluent, and upstream of the effluent input in Northwest Arkansas. E. coli and total coliforms were enumerated using defined substrate technology (IDEXX Laboratories, Inc., Colilert reagent). Most probable numbers of bacteria were determined in the absence and presence of ampicillin, ofloxacin, suflamethoxazole, trimethoprim, or tetracycline in July, August and September, 2007, in Mud Creek, an effluent driven Northwest Arkansas stream. Resistant populations are reported as percent of MPN measured in the same water sample in the absence of any antibiotic. Pre-liminary data analysis suggests that levels of resistance are dependent on sampling site, time, and antibiotic tested. Ofloxacin resistance was low and primarily measured in the effluent. Ofloxacin-resistant coliforms were, but E. coli were not, detected downstream. Tetracycline-resistant coliforms and E. coli sometimes increased in the effluent, but were not different or were lower by the second downstream site. Sulfamethoxazole-resistant coliforms were higher in August and September in the effluent and downstream at all three sampling times. Trimethoprim-resistant coliforms and E. coli were higher in the effluent in August and September. Alan Sexstone reported that The Davis College of Agriculture at West Virginia University maintains a certified organic farm to evaluate efficacy of defined organic production systems for both plants and animals. His group has compared patterns of antibiotic resistance in 600 E coli. isolates from organic and conventionally raised chickens and sheep, and from soil they graze on. Compared with organic chickens, more conventional chickens isolates were resistant to ampicillin and cephalothin. All chickens harbored isolates that were resistant to rifampin, streptomycin and neomycin. Conventional and organic sheep isolates were primarily resistant to rifampin, as were isolates from baseline soil samples. Following eight weeks of grazing by organic chickens, isolates from paddock soils became significantly resistant to tetracycline. Preliminary BOX A1R rep-PCR fingerprints were obtained from the E. coli isolates and subjected to cluster analysis. Data supported a separate baseline soil and organic chicken cluster. Soil isolates obtained following exposure to organic chickens clustered more closely with chickens than with baseline soils (figure 5). An additional 1000 isolates from soils and pasture-raised beef cattle currently are being analyzed. Future work includes utilization of additional antibiotic sensitivities, improved rep PCR analyses, and modifications of the clustering technique employed. Usefulness of Findings: Feng: Fecal contamination of surface water is a nationwide problem. Fecal contamination in a watershed usually comes from multiple sources, which cannot be readily determined. Identification of fecal contamination source is essential to develop effective pollution control strategies and to ensure that pollution control efforts are directed at the correct sources. Gentry: The projects described under Objective 2 will provide baseline data on the effectiveness of different management practices to reduce the numbers of E. coli and antibiotic resistant bacteria from grazing systems and the land application of dairy manures. These studies will also evaluate the usefulness of current Bacteroides markers for determining the level of fecal contamination. Graves: Objective 1. This project seeks use microbial source tracking technology to generate data on the bacterial loading from the North River Community. The North River Community is composed of 250 households within Carteret County, North Carolina and covers a stretch of 4 miles. This predominately African-American community dates back to the days of Emancipation. Unfortunately, this community lies in the 100-year flood plain bordering shellfish sensitive waters and has struggled maintaining septic systems in the poor soil and high water table. Given the historical importance of this community to both Carteret County and the State of North Carolina, it is imperative that the wastewater problems in the community be addressed in order to avoid further detriment to the problems in the community as well as surrounding surface waters. As an unincorporated community, the residents of North River need support and assistance in obtaining sewer service in the community. This study will provide scientific data to be included in future proposals for grants and/or loans for this type of infrastructure development. Objective 2. Microbial resistance to antibiotics is spreading fast; incidence of vancomycin resistance has increased from less than 1% to 17% within a span of 10 years This study is intended to evaluate the association of antibiotic resistance genes found in E.coli isolated from swine with the actual phenotypic expression of the resistance. Additionally to develop an antibiotic resistance database for E. coli isolates from a commercial swine facility and assess its efficacy for tracking movement of bacteria from swine confinement houses to surface waters. Quantitative polymerase chain reaction will provide robust, sensitive and highly discriminant data. The results of this research will provide important information regarding the role of land application of lagoon effluent in spreading of bacteria with antibiotic resistance genes to surface waters. Early diagnosis of the problem will allow for the development of improved technologies and mitigation strategies. Hagedorn: There are a few reports that indicate an apparent relationship between some Enterococcus species and different sources. For example, E. faecalis and E. faecium in high concentrations can be indicative of the presence of human fecal contamination, while E. gallinarum is an indicator of the presence of fecal contamination from dogs and/or birds. The results in figures 1 and 2 confirmed this approach and demonstrated that Enterococcus species identification can be one of several BST protocols that could be useful when characterizing sources of fecal pollution in urban watersheds, and could be an adjunct method when isolation and cultivation of enterococci is required for other purposes such as monitoring. Hartel: When combined with counts of fecal bacteria, the new fluorometric method was a simple, quick, and inexpensive way to identify human fecal contamination in environmental waters. Sadowsky: Macroarray hybridization, coupled with the use of other host source-specific gene probes described under Objective 1, holds great promise as a new quantitative microbial source tracking tool to rapidly determine the origins of E. coli in waterways and on beaches. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. Savin: Coliforms, including E. coli, were enumerated at every site and sampling time. In addition, resistant bacteria were introduced from the WWTP effluent. The proportion of E. coli resistant to an antibiotic appears to increase in effluent samples as compared to upstream samples. While E. coli numbers per 100 mL may be lower in effluent than the stream samples, about one-third of the E. coli detected in the effluent were resistant to sulfamethoxazole or trimethoprim. Data analysis is currently ongoing. However, although the effluent is introducing resistant bacteria, proportions of resistant E. coli measured at the second downstream site were often not higher than upstream. Research is continuing to try to understand the impact of wastewater treatment effluent on bacterial antibiotic resistance in stream water. Collaborative work between Dr. Savin (University of Arkansas) and Dr. Cisar (Northeastern University in Tahlequah, OK) resulted in a Northeastern University student becoming a graduate student working on this project in Dr. Savins laboratory in 2006. Sexstone: Objective 1. Application of chemical and biological microbial source tracking in the Paradise Lake impoundment may identify sewage outfalls of concern to public health. Data obtained from the past year support goals outlined in Objective 2. The presence of distinct E. coli strain among organically and conventionally reared chickens, from various experimental systems at the WVU farms in Morgantown, WV suggests that distinct strains are obtained based on the production practice employed; which in turn affects populations and persistence of E. coli in soils. Thies: Q-PCR detection of E. coli in two Puerto Rican waterways using ITS probes allowed us to quantify the abundance of E. coli over at least 6 orders of magnitude in DNA extracts from these environmental samples. This method of determining E. coli genome abundance in water is very precise, however, it does not allow us to separately quantify the total abundance of both pathogenic and non-pathogenic E. coli, as do other approved methods such as the Colilert-18TM system. This latter information is needed in order for the method to be used successfully as an indicator of water quality as the presence of environmental sources of non-pathogenic E. coli could skew perceptions about local water quality based on quantification alone. The use of Q-PCR to detect E. coli in two streams in Mayaquez, Puerto Rico, enabled us to identify point sources of fecal contamination. Peaks in E. coli abundance in both water and epilithic biofilms ranged from 2-4 orders of magnitude higher at point sources over samples located either upstream or downstream. The ability to identify the fecal contamination point sources using the Q-PCR method was straightforward in the two streams analyzed, and should permit targeted management of the stream landscape to control the primary sources of fecal contamination, although further monitoring may be warranted to ensure that fecal contamination is not merely repositioned by management efforts. Analysis of the same samples using multiple microbial detection methods in several laboratories will permit us to develop recommendations on the more robust approaches to detecting fecal contamination in our nations waterways. Work Planned for Next Year: Feng: Continue projects described under Objective 1. Gentry: Begin sample collection and analyses for E. coli, antibiotic resistant bacteria, and source tracking. Graves: Obtain unknown E. coli isolates for microbial source tracking adjacent to the North River Community. Evaluate E. coli isolates described under Objective 2 for resistance and virulence genes. Hagedorn and Hartel: The field portion of research conducted at Puerto Rico is finished, and all that is necessary is preparation of a manuscript for submission to a refereed journal. This writing will be completed during summer and fall of 2008. Sadowsky: Continue projects described under Objective 1. Savin: Work on the enumeration of the antibiotic resistant bacterial community, and detection of multiple antibiotic resistance will continue into 2008. In addition, plasmids will be extracted from water and isolates and investigated for the presence of broad host range determinants and resistance to multiple antibiotics. Sexstone: Objective 1: Monitoring of indicator bacteria and fluorometry in Paradise Lake. Objective 2: Fecal bacterial isolates obtained from organic and conventionally raised chicken and sheep, and from pasture-raised beef cattle, will be characterized to compare patterns of antibiotic resistance, rep-PCR fingerprints, and persistence in soil. Thies: Continued work on Q-PCR for Objective 1.

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. Dickerson, J.W. Jr., C. Hagedorn and A. Hassall. 2007. Detection and remediation of human-origin pollution at two public beaches in Virginia using multiple source tracking methods. Water Research 41: 3758-3770. Dickerson, J.W. Jr., J.B. Crozier, C. Hagedorn and A. Hassall. 2007. Assessment of the 16S-23S rDNA intergenic spacer region in Enterococcus spp. for microbial source tracking. In Press: Journal of Environmental Quality 36:1661-1669. Edenborn, S.L., and A.J. Sexstone. 2007. DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses culture-independent analyses. Soil Biology & Biochemistry. 39:1570-1579. Gao, H., Z.K. Yang, T.J. Gentry, L. Wu, C.W. Schadt, and J. Zhou. 2007. Microarray-based analysis of microbial community RNAs by whole community RNA amplification. Appl. Environ. Microbiol. 73:563-571. Gentry T.J., C.W. Schadt, Z. He, and J. Zhou. 2007. Chapter 83, Functional gene arrays for microbial community analysis, p. 1052-1062. In C.J. Hurst, R.L. Crawford, J.L. Garland, D.A. Lipson, A.L. Mills, and L.D. Stetzenbach (ed.). Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, D.C. Graves, A.K., C. Hagedorn, A. Brooks, R.L. Hagedorn, and E. Martin. 2007. Microbial source tracking in a rural watershed dominated by cattle. Water Research 41: 3729-3739. Hartel, P. G., K. Rodgers, G. L. Moody, S. N. J. Hemmings, J. A. Fisher, and J. L. McDonald. 2008. Combining targeted sampling and fluorometry to identify human fecal contamination in a freshwater creek. J. Water Health 6:105-116. Hartel, P. G., S. P. Myoda, K. J. Ritter, R. L. Kuntz, K. Rodgers, J. A. Entry, S. A. Ver Wey, E. C. Schröder, J. Calle, M. Lacourt, J. E. Thies, J. P. Reilly, and J. J. Fuhrmann. 2007. Geographic sharing of ribotype patterns in Enterococcus faecalis for bacterial source tracking. J. Water Health 5:539-551. Hartel, P. G., J. L. McDonald, L. C. Gentit, K. Rodgers, K. L. Smith, S. N. J. Hemmings, C. N. Belcher, R. L. Kuntz, Y. RiveraTorres, E. Otero, and E. C. Schröder. 2007. Improving fluorometry as a source tracking method to detect human fecal contamination. Estuaries Coasts 30:551-561. Hartel, Peter G., Charles Hagedorn, Jennifer L. McDonald, Jared A. Fisher, Michael A. Saluta, Jerold W. Dickerson Jr., Lisa C. Gentit, Steven L. Smith, Nehru S. Mantripragada, Kerry J. Ritter, and Carolyn N. Belcher. 2007. Exposing water samples to ultraviolet light improves fluorometry for detecting human fecal contamination. Water Research: 3629-3642. He, Z., T.J. Gentry, C.W. Schadt, L. Wu, J. Liebich, S.C. Chong, Z. Huang, W. Wu, B. Gu, P. Jardine, C. Criddle, and J. Zhou. 2007. GeoChip: A comprehensive microarray for investigating biogeochemical, ecological, and environmental processes. ISME J. 1:67-77. 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. 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. Salinas, K. A., S. L. Edenborn, A. J. Sexstone and J. B. Kotcon. 2007. Bacterial preferences of the bacterivorous soil nematode Cephalobus brevicauda (Cephalobidae): Effect of bacterial type and size. Pedobiologia. 51(1):55-64. Van Nostrand, J.D., T.V. Khijniak, T.J. Gentry, M.T. Novak, A.G. Sowder, J.-Z. Zhou, P.M. Bertsch, and P.J. Morris. 2007. Isolation and characterization of four gram-positive nickel-tolerant microorganisms from contaminated sediments. Microb. Ecol. 53:670-682. Yan, T., M. Hamilton, and M. J. Sadowsky. 2007. High throughput and quantitative procedure for determining sources of Escherichia coli in waterways using host-specific DNA marker genes. Appl. Environ. Microbiol. 73:890896. Yan, T., and M. J. Sadowsky. 2007. Determining sources of fecal bacteria in waterways. Environ. Monitor. Assess. 129:97-106 Accepted Full Length Articles (Refereed Journals): Hansen, D. L., J. J. Clark, S. Ishii, M. J. Sadowsky, and R.E. Hicks. 2007. Sources and sinks of Escherichia coli in benthic and pelagic fish. J. Great Lakes Res. In Press. Sanders, S.M., P. Srivastava, Y. Feng, J.H. Dane, J. Basile, and M.O. Barnett. 2008. Sorption of the veterinary antimicrobials sulfadimethoxine and ormetoprim in soil. J. Environ. Qual. (in press) In Print Abstracts: Akiyama T., and Savin, M. C. 2007. Antibiotic-resistant bacteria in a stream receiving wastewater treatment plant effluent. In Annual Meeting South Central Branch American Society for Microbiology, November 2-3, 2007, University of Arkansas at Little Rock, AR. Caster, S, D. Zuberer, F. Hons, T. Provin, and T. Gentry. 2007. Dissolved organic carbon and nutrients in native soil under three turfgrasses and two sand-based sports-fields. In Abstracts, ASA-CSSA-SSSA Annual Meet., New Orleans, LA. 4-8 November 2007. Hagedorn, C. 2007. An evaluation of beach remediation in Virginia using four microbial source tracking methods. Invited Plenary Presentation, XXI Congreso Nacional de Microbiologia, Sevilla, Espana, 17-20 septiembre. (paper S15.1.) Harclerode, C., T. Gentry, and J. Aitkenhead-Peterson. 2007. E.coli and nutrients in Brazos County Carter Creek subcatchments. Bacteria in Our Bayous: Water Quality Standards and Restoration Options. 15-16 October 2007. University of St. Thomas, Houston, TX. Harclerode, C., K. Wagner, E. Martin, T. Gentry, J. Aitkenhead-Peterson, L. Redmon, and S. Feagley. 2007. Microbial water source contamination. Texas Plant Protection Conference, College Station, TX. 4-5 December 2007. Hartel, P. G., C. Hagedorn, J. L. McDonald, J. A. Fisher, M. Saluta, J. Dickerson, L. C. Gentit, S. Smith, N. S. Mantripragada, K. J. Ritter, and C. N. Belcher. 2007. Exposing water samples to ultraviolet light improves fluorometry for detecting human fecal contamination. American Society for Microbiology Annual Meeting, May 21-25, Toronto, Canada. Hassall, A., C. Hagedorn, D. Barker, and P. Yeomans. 2007. Evaluation of a regional Enterococcus library with a blind challenge set. American Society for Microbiology Annual Meeting, May 21-25, Toronto, Canada. Saluta, M., C. Hagedorn, A. Hassall and Dickerson, J.W. Jr. 2007. Fluorometric detection of optical brighteners in detergents as a human-specific marker in microbial source tracking. American Society for Microbiology Annual Meeting, May 21-25, Toronto, Canada. Savin, M.C. and T. Akiyama. 2007. Antibiotic resistance in aquatic bacteria downstream from an effluent discharge. In 2007 AWRC Conference Presentation and Poster Abstracts and Bios [CD-ROM]. Arkansas Water Resources Conference, Fayetteville, AR. Toth, J.D., Z. Dou, J.D. Ferguson, C.F. Ramberg, Jr., C. Wang, S.C. Rankin, Q. Wang, and Y. Feng. 2007. Effect of veterinary pharmaceuticals on metabolic functions of native soil bacteria. Annual Meetings Abstracts [CD-ROM]. ASA, CSSA, SSSA, Madison, WI. Wade, T. R., C. Hagedorn, M. Saluta, and A. Hassall. 2007. Use of Escherichia coli for microbial source tracking in a mixed-used watershed in northern Virginia. American Society for Microbiology Annual Meeting, May 21-25, Toronto, Canada. Wagner, K.L., L. Redmon, T. Gentry, and C.A. Jones. 2007. Reducing bacterial contamination of Texas watersheds. USDA-CSREES National Water Conference, Savannah, GA. 28 January  1 February 2007. Wiechman, R.J., S. R. Seese, and A. J. Sexstone. Genetic Fingerprints of E. coli in Cattle, Sheep, Chickens, and Impacted Soils Within Conventional and Alternative Production Systems. Soil Science Society of America Annual Meetings. November 4-8. New Orleans, Louisiana. Wijesinghe R.U., Y. Feng, M.R. Owsley, C.W. Wood, and S. Ditchkoff. Evaluation of Escherichia coli rep-PCR DNA fingerprint library for bacterial source tracking. 2007. Annual Meetings Abstracts [CD-ROM]. ASA, CSSA, SSSA, Madison, WI. Books and Book Chapters: Feng, Y. 2008. Soil microbiology. In: Encyclopedia of Soil Science, W. Chesworth (ed). Springer Publishing Company, New York, NY. Proceedings: Clement Solomon, Joseph Kamalesh, and Alan Sexstone. 2007. Analysis of remote monitored water-use trends in a commercial facility. In. Proceedings of the National Onsite Wastewater Recycling Association (NOWRA) Annual Meeting. March 12-14, 2007. Baltimore, Md. (refereed proceedings, paper # XX-SPE-07-44) Feng, Y., P. G. Hartel, J. A. Fisher, K. Rodgers, B. Liu, and K.J. Ritter. 2007. Differences in survival among Enterococcus faecalis subspecies in two freshwater creek sediments. p. 603-606. In T. C. Rasmussen, G. D. Carroll, and A. P. Georgakakos (ed.) Proceedings of the 2007 Georgia Water Resources Conference, March 27-29, Athens, GA. Theses and Dissertations: Sherie Edenborn. 2007 Molecular and functional characterization of bacterial communities in organic farm and pasture soils. Ph.D. dissertation. Division of Plant & Soil Sciences,. West Virginia University. Morgantown, WV 26506. https://eidr.wvu.edu/eidr/documentdata.eIDR?documentid=5300