Brief Summary of Minutes of Annual Meeting

May 29 (Tuesday), Annual Project and Business Meeting Chuck Hagedorn opened the meeting (9:00am) and addressed a few housekeeping details as well as reminded members that annual reports should be submitted to him, as he is serving as the Chair for this year and is responsible for submitting the annual report to the project director within 60 days. Chuck reminded all in attendance that the reason for meeting in Puerto Rico (PR) was to conduct a project by working with samples that would be collected as part of the meeting in such a way that a journal publication would result. There were no administrators present. Peter Hartel began technical reports with an overview of fluorometry and targeted sampling that included an update of how Peter had combined these two techniques at different locations in Georgia. In an effort to develop an inexpensive method of detecting human fecal contamination in a freshwater creek during baseflow and stormflow conditions, Peter combined targeted sampling with fluorometry. Peter described their work in Potato Creek (a freshwater creek in GA) that had three reaches identified as containing high numbers of fecal bacteria, and these reaches were sampled by targeted sampling and fluorometry. Targeted sampling quickly and inexpensively identified humans, cattle, and dogs as the major sources of fecal contamination in the first, second, and third reaches, respectively. Fluorometric values were consistent with these identifications, but high fluorometric values were sometimes observed in areas with no fecal contamination. One likely cause of these false-positive signals was fluorescence from organic matter. This was the first study of this relatively inexpensive methodology combination in freshwater during both baseflow and stormflow conditions. Peter then led a discussion of what we hoped to accomplish in Puerto Rico, and what his prior experiences in Puerto Rico had been. Prior to the S-1022 meeting Peter and Jenny McDonald met with Dr. Ernesto Otero, Departamento de Ciencias Marinas, Laboratorios de Isla Magueyes, Universidad de Puerto Rico, Mayagüez. Ernesto had agreed to be our contact and assist in locating sampling sites as well as allowing us to use his lab at Isla Magueyes. Peter and Jenny had visited Ernestos lab to be certain that it was suitable for our use. The plans that Peter described included locating a couple of streams in Mayaguez (a city of 300,000 people) that could be sampled from the beach where the streams entered the ocean, and then sampled at locations in urban settings in Mayaguez. It was expected that such streams would have Escherichia coli (E. coli) populations of at least 1,000 colony forming units (CFU) per 100 mL. The purpose behind the targeted sampling of two streams in Mayaqüez was to compare and contrast the usefulness of several different bacterial source tracking (BST) methods that are either in use currently or under development. It was hoped that each of the participants would using different, yet complementary, BST methods to identify sources and types of bacteria (and viruses) contaminating the two streams. Use of the same samples for all analytical work across the different laboratories will reduce intra- and inter-sample variability and employing a variety of techniques in the various laboratories should result in better characterization of the streams. The final analysis of the data sets should yield recommendations on which approaches provided the more robust means of characterizing the biological quality of the streams. Following Peters update, the discussion centered on what we could physically accomplish, once samples had been collected, in the short period of time that we would be in PR. Based on the materials that had been brought or sent to PR, it would be possible to filter samples through Nucleopore filters, which could then be frozen for transport and the DNA extracted later. For both E. coli and Enterococcus, the water samples could be tested for enumeration with IDEXX Colilert and Enterolert Quanti-trays, respectively. Each member present was then asked what types of cultures they might like once we returned to the states and to consider what they might do with them. Both E. coli and Enterococcus isolates could be recovered from the Quanti-trays, and DNA could be recovered from the frozen filters. Continuing the technical reports, Alan Sexstone described his work on decentralized systems, which serve approximately 25% of the U.S. population, resulting in septic tank effluent being the most frequently reported source of groundwater contamination by pathogens. Where standard septic systems cannot be installed, there is a need for alternative and cost-effective on-site wastewater treatment systems. Subsurface flow constructed wetlands and aerobic treatment units (ATUs) currently are used as alternative on-site treatment systems in WV. They are permitted and regulated by the West Virginia Department of Environmental Protection (WVDEP), Division of Water and Waste Management (DWWM) and classified as experimental treatment systems due to limited information on operations and problems with existing installations. Alans plans include plan monitoring 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. Previously, Alan had conducted microbial enumerations, BOD5 and TSS on influent and effluent samples obtained from two ATUs equipped with devices for telemetric monitoring in real-time. The WVDEP average monthly discharge limitations for alternative systems (30 mg L-1 BOD5, 30 mg L-1 TSS, and 200 CFU 100 ml-1 fecal coliforms) sometimes were exceeded. Plans include using chemical source tracking methods (fluorometry, fecal sterols) to discriminate human and non-human fecal contamination in water and sediments obtained from the two systems. Yucheng Feng described her project using bacterial source tracking in the Catoma Creek watershed (AL). Water samples were collected monthly at six locations in the watershed during the year. E. coli concentrations in the water samples ranged from 7 to 8550 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 1000 E. coli isolates have been obtained 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, and her lab is in the process of obtaining the rep-PCR DNA fingerprints of all the E. coli isolates. To verify the presence of human contamination in the watershed, water samples were also analyzed by LC/MS/MS for nine chemicals commonly used by humans (e.g., caffeine and metabolites, triclosan, and deet). All samples contained at least one chemical at ppb levels. Seven of the nine chemicals were detected in one water sample collected at a city site, but this type of analysis will be limited due to the analytical costs of over $400/sample. Mike Sadowsky described his high throughput automated virulence gene profiling system. While several genotypic techniques have been used to determine potential sources of fecal bacteria impacting waterways and beaches, they do not allow for the rapid analysis of a large number of samples in a relatively short period of time. Mike report that gene probes identified in previous research done in MN were useful for the development of a high-throughput and quantitative macroarray hybridization system to determine numbers of E. coli bacteria originating from geese/ducks. The procedure that was developed, using a QBot robot for picking and arraying of colonies, allowed simultaneous analysis of up to 20,736 E. coli colonies from water samples, with minimal time and human input. Statistically significant results were obtained by analyzing 700 E. coli colonies per water sample, allowing for the analysis of approximately 30 sites per macroarray. Macroarray hybridization studies done on E. coli collected from water samples obtained from two urban Minnesota lakes and one rural South Carolina lake indicated that geese/ducks contributed up to 51% of the fecal bacteria in the urban lake water samples, and the level was below the detection limit in the rural lake water sample. This technique, coupled with the use of other host source-specific gene probes, holds great promise as a new quantitative microbial source tracking tool to rapidly determine the origins of E. coli in waterways and on beaches. Alex Graves described her work on evaluating the effects of lagoon treatment on the persistence of antibiotic resistant enteric bacteria isolated from swine feces. Both cool season and warm season samples were collected from a swine farm in NC and each season included three nursery swine fecal samples, three nursery swine lagoon liquid samples, four finishing swine fecal samples, three finishing swine lagoon liquid samples, and four soil samples from both nursery and finishing swine spray field. A total of 4032 E. coli isolates and 4896 Enterococcus isolates were obtained from the samples. The antibiotic resistance profiles of the isolates were determined using a set of antibiotics at various concentrations. All isolates displayed multiple antibiotic resistance, and for the isolates from the same source, the resistance patterns were similar for the antibiotics within the same antibiotic family. Percentages of resistant isolates were greater in nursery fecal samples than in finishing fecal samples for majority of antibiotic tests. For nursery samples, percentages of antibiotic resistant isolates decreased after lagoon treatment for majority of antibiotic tests. For finishing samples, no such trend was obvious. The results indicated that antibiotic resistant isolates still persist in the lagoon liquid, which may cause potential risk to human and environmental health. Because antibiotic resistance may affect later therapeutic and subtherapeutic value of these antibiotics, management strategies of agricultural antibiotic use may need to be improved. Terry Gentry described efforts 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 in TX. For the grazing studies, three small (1-2 acres) watershed sites on the Welder Wildlife Refuge will be 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. Similar studies will also be done at two other locations, a private ranch and the Riesel Experiment Station. 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. For the land application of manures project, a total of four sites will be selected for the evaluation of BMPs. The four fields will consist of three manured (wastewater, vacuum manure, dry manure) fields (corn, hay, and pasture) and one inorganic fertilized hay field. The manured fields will have buffer strips, one managed and one unmanaged. The inorganically fertilized field will not have a buffer strip. Each field will be set up for edge of field monitoring using ISCO samplers. Each field will be bermed to force runoff to run through a single outlet. This water will be split into the managed and unmanaged filter strip. An ISCO sampler 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). 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 (G.D. Di Giovanni). 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 (G.D. Di Giovanni). Janice Thies described the development of a highly discriminatory two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) method to facilitate molecular microbial community analysis. This system has been tested in soils containing naturally high concentrations of Zn and Cd in NY; and community analysis using 2D-PAGE was compared with three other community analysis methods: denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphisms (T-RFLP), and automated ribosomal intergenic spacer analysis (ARISA) in order to determine the consistency of each method as well as to compare their ability to resolve polymerase chain reaction (PCR) amplicons into the highest number of individual, operational taxonomic units (OTUs). Janice reported that the 2D-PAGE of ITS regions resulted in a three-fold increase in the number of detected OTUs in comparison to any other method. The resolution of the 2D-PAGE method was ten times higher than DGGE. Resulting gels can be compared using standard spot-matching software typically used in protein gel analysis. In addition, incorporating the use of fluorescent end-labeling and laser gel scanning improved the sensitivity of the method, and Janice believes that other marker genes beside the ITS regions may be used in this system. The methods utility for bacterial source tracking remains to be seen. Sue Hagedorn, editor of Environmental Detection News, described how the newsletter will move to journal format in 2007 to provide a format for publication of case studies. The Journal of Environmental Detection (JED) will be an international peer-reviewed on-line journal. It will welcome articles in English focusing on watershed-level case studies for both biological and non-biological pollution sources. Papers should be fully documented and should normally be between 3000 and 6000 words. Submission of the article implies that it has not been published previously, and that if accepted, will not be published elsewhere in the same form, in English or in any other language, without the written consent of the publisher. All papers should be submitted electronically to: hagedors@vt.edu. It was hoped that the combined work of all who want to be involved in collecting and analyzing the PR samples would be authors on a paper that would be published in JED. Chuck Hagedorn reported on work at two public beaches (Anderson and Hilton) in Newport News, VA, that were frequently closed to swimming in 2004 due to high Enterococcus spp. counts that exceeded the regulatory standard. The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners) were used in the summer of 2004 to determine the origins of fecal pollution at the two beaches. Both MST methods detected substantial human-origin pollution at the two beaches, in locations producing consistently high levels of Enterococcus spp. Joint investigations with municipal officials in 2005 led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of the mitigation efforts was confirmed during the summer of 2006 (no closures at either beach), and ARA, fluorometry, and pulsed-field gel electrophoresis (PFGE) all indicated the absence of human origin pollution in the summer of 2006. The last item of business was to elect the chair for 2008. Alan Sexstone was nominated and elected by unanimous decision. Alan will host the 2008 S-1022 meeting, probably in Morgantown, WV. The business meeting was adjourned at 4:15pm on 5/29/07. The research part of the annual meeting began at 8:00am on 5/30/07, with Graves, Hartel, Hagedorn, and Sexstone, accompanied by Annie Hassall (lab supervisor for Hagedorn) and Jennifer McDonald (lab supervisor for Hartel) participating in the stream sampling with the assistance and guidance of Ernesto Otero. Two urban streams on the southern end on Mayagüez were selected (one stream was named Sabalo and the other was Savat) where samples could be collected as the streams flowed into the ocean, and then at two locations upstream in urban neighborhoods. Samples were collected in Whirl-pak bags and placed on ice in coolers. The morning was spent finding sampling locations and collecting the samples, then the samples were transported to Oteros lab for analysis that afternoon and evening. As it was desirable to sample streams where the water was expected to be highly contaminated (sewage and industrial discharges), safe practices were observed in collecting and handling the samples. The two most expendable members of the sampling team (Hartel and Sexstone) actually walked the streams to obtain the samples. The afternoon and evening of 5/30/07 was spent processing the samples, including filtering with a variety of different filters for various purposes, processing Enterolert and Colilert trays, and recording fluorometry results for all samples. Everyone participated in these activities. Total DNA was extracted from all water samples in order to assess them for the presence of marker genes for key pathogens. A 20 mL portion of each water sample was filtered through both 0.22 and 0.45 µm filters and all filters were placed in sterile plastic bags, placed on ice and transported to Cornell University the following day. Rocks with adhering biofilms were also sampled from the stream bed in both streams in order to detect the presence of bacterial pathogen populations persisting in epilithic biofilms. Rocks were placed in sterile plastic bags, placed on ice and transported to Cornell University the following day. Most of 5/31/07 was spent reading and recording results after overnight incubation and preparing materials for shipping back to various investigators labs. On 6/1/07 the last results were recorded for those materials needing 48hr incubation, and materials were collected and transported to the post office and shipped to the appropriate labs. With the shipping, this concluded the research part of the 2007 annual meeting for S-1022. Respectively submitted, Charles Hagedorn, 2007 Chair

See attached Annual Report