CICEET Progress Report for the period 3/15/05 Through 9/15/05

Project Title: Field-testing Targeted Sampling and Enterococcus faecalis to Identify Human Fecal Contamination in Three National Estuarine Research Reserves
Principal Investigator(s): Peter Hartel, and Ernesto Otero
Additional Investigator(s): Carolyn Belcher (Statistician, University of Georgia­Brunswick), Jared Fisher (Laboratory Worker, University of Georgia­Athens), Keith Gates (Associate Director, University of Georgia­Brunswick), Lisa Gentit (Research Technician II/Research Technician III, University of Georgia­Brunswick), Sarah Hemmings (Graduate Research Assistant, University of Georgia­Athens), Jennifer McDonald (Research Technician III/Research Professional II, University of Georgia­Brunswick), Beth O’Hara (Student Worker, University of New Hampshire), Karen Payne (Assistant Research Scientist, University of Georgia­Athens), Katy Austin Smith (Research Coordinator II/Research Professional II, University of Georgia­Brunswick), Yaritza Rivera­Torres (Graduate Research Assistant, University of Puerto Rico), and Karen Rodgers (Research Coordinator II, University of Georgia­Athens).

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Tables

Table 1

Table 2

Progress on Tasks
With one exception, the primary objective has been completed and additional research on related objectives is continuing. The exception was the sampling of the Sapelo Island NERR in Georgia. The additional research on related objectives was work by Tamara Bryant and Yaritza Rivera­Torres to determine the presence of fecal enterococci on marine plants. Tamara conducted her research in secondary habitats (e.g., wrack) on New Hampshire’s ocean bathing beaches, while Yaritza conducted her research on sea grasses.

We expected to submit three manuscripts to refereed journals: one on targeted sampling during calm and stormy conditions (this manuscript involved research at St. Andrews Park and Sea Island in Georgia), one on regrowth and desiccation of fecal enterococci (this manuscript involved research at all three NERRs), and one on combining targeted sampling and fluorometry (this manuscript involved research in Puerto Rico and Georgia estuarine waters).

Because the overarching (but unstated) objective of the grant was to develop inexpensive methods of bacterial source tracking (BST), fluorometry was added as an objective to the project and was to be combined with targeted sampling and Ent. faecalis speciation during the last Georgia sampling. Fluorometry detects optical brighteners from laundry and dishwashing detergents in water, and therefore is a chemical method to detect human fecal contamination. This research was to be conducted at the Sapelo Island NERR and was to complete our obligation to sample in this location.

In our last semi-annual report, we wrote not only about the success of using Ent. faecalis speciation as an inexpensive BST method, but also about our preliminary success with combining targeted sampling and fluorometry. These methods were combined for the first time at a non-NERR site, Sea Island, Georgia. Although fluorometry was successful in detecting human fecal contamination, our research also suggested that high amounts of organic matter, typical of Georgia’s estuarine waters, interfered with the fluorometric signal. Therefore, we needed to test the method at another Georgia location with high levels of organic matter. Our control would be marine waters with low organic matter. Although both Puerto Rican and New Hampshire’s estuarine waters met this criterion, we decided to test Puerto Rican waters because fecal contamination was more severe there. Therefore, we sent three technicians (Karen Rodgers, Katy [neé Austin] Smith, and Lisa Gentit) and one graduate student (Sarah Hemmings) to work with Ernesto Otero and Yaritza Rivera-Torres in Puerto Rico to conduct fluorometry during late January­early February, the driest time of the year. The sampling was successfully completed and demonstrated that fluorometry had promise as an inexpensive BST method. We do not report these results here because they were included in our 2005 Semi-Annual Report.

We then conducted targeted sampling with Ent. faecalis speciation and fluorometry in the second Georgia location. We selected St. Simons Island as our test location because it contained a creek, Postell Creek, that was purported to be contaminated with sewage from a lift station at its headwaters, and the creek had visibly high amounts of organic matter. The sampling was completed this summer. The results were confounded (see both Difficulties and Preliminary Results sections), and we decided that the results required confirmation with another BST method, fecal sterols. Because this confirmation with fecal sterols was beyond the original objective of the grant and we were still obligated to test the Sapelo NERR, Sarah Hemmings applied for and received a Graduate Student NERR Fellowship to confirm the sampling of the Sapelo Island NERR with fluorometry and detection of fecal sterols. It is for this reason that the Sapelo Island NERR sampling will not completed under the auspices of this grant; it will be completed under the auspices of the Graduate Student NERR Fellowship.

One assumption for a fecal bacterium to be useful as an indicator is that it does not persist or regrow in the environment. However, in our 2005 Semi-Annual Report, we reported that although numbers of fecal enterococci generally decreased with increased length of drying, many fecal enterococci survived desiccation and regrew in rewetted sediment. This survival and regrowth violated the assumption for a fecal bacterial indicator. Yaritza Rivera-Torres and Tamara Bryant continued research on this topic using marine plants. However, because of unforeseen circumstances, Tamara has left the project and research on survival of fecal enterococci in wrack will not be completed. This research is supplementary to the grant, therefore its loss, while significant, does not affect the overall objective. In contrast, Yaritza will continue her research on growth and survival of fecal enterococci on sea grasses, and she is scheduled to complete it in May 2006.

Finally, we will post our research on the NEMO network, and this posting will satisfy our obligation to disseminate our research to a wider audience.

Accomplishments to Date
Four abstracts and two proceedings have been published (see Dissemination, Publications). The two proceedings were published in the Proceedings of the Georgia Water Resources Conference, which was be held on April 26-28, 2005, in Athens, Georgia. The first proceedings summarizes our research on the survival and regrowth of fecal enterococci in sediments, while the second summarizes our research on combining targeted sampling and bacterial source tracking during calm and stormy conditions near Sea Island, Georgia. One of proceedings has been converted into a manuscript and has been submitted to the Journal of Environmental Quality for review. The second proceedings required more statistical analysis, which is now complete. Work on this manuscript continues and it will be submitted to the Journal of Environmental Quality before the end of the year. In addition to these publications, CICEET research has been highlighted in five invited presentations by the PI/PD.

We expect to write two more abstracts for presentation at the American Society for Microbiology meeting in Orlando, FL (May 21-25, 2006). These abstracts are due in early December 2005.

Difficulties
Combining targeted sampling and fluorometry at St. Simons Island in Georgia gave conflicting results, and it became necessary to confirm the fluorometric values with an additional BST test. The additional BST test we selected was the detection of fecal sterols. In the case of fecal sterols, their formation varies in the intestines of warm-blooded animals depending on the animal’s diet, intestinal microorganisms, and host metabolism. Humans often eat animal-based foods containing cholesterol, which is preferentially reduced to coprostanol in the intestine and cholestanol in the environment (Figure 1A). However, because cattle are herbivores, they eat plants containing plant sterols, not animal-based cholesterol. In the cow intestine, plant-derived sitosterol (24-ethylcholesterol) is reduced preferentially to stigmasterol, and stigmastanol in the environment (Figure 1B). By determining the ratios of these intestinal stanols (e.g., coprostanol:stigmasterol), it is possible to distinguish among different sources of feces (e.g., Gilpin et al., 2003). Although detection of fecal sterols has been successfully combined with fluorometry, the method has never been combined with targeted sampling. For us, the main problem with fecal sterols is that it requires a sophisticated instrument, a gas chromatograph/mass spectrometer (GC/MS), to detect them. Fortunately, such an instrument was available to us at the Marine Extension Service Laboratory in Brunswick, GA, and we have spent the last few months learning how to use the instrument to detect fecal sterols.

Project Objectives for Next Reporting Period

Objectives
to field-test targeted sampling with Ent. faecalis to identify human fecal contamination in three NERRs.

Tasks to Meet Objectives
With the exception of the Sapelo Island NERR (now scheduled to be sampled under the auspices of a Graduate NERR Fellowship awarded to Sarah Hemmings), our primary objective has been met. We have asked for a 90-day, no-cost extension in order to complete our second manuscript. Therefore, we will submit our Final Report on 15 December 2005.

Work Plan for Next Reporting Period
Yaritza Rivera­Torres is continuing her research and is expected to finish her research by May 2006. By December, we expect to submit the second of the three planned manuscripts, “Regrowth and desiccation of fecal enterococci in estuarine sediments,” to the Journal of Environmental Quality. As already noted, the statistics for this manuscript are now complete, and a portion of the manuscript is already written because it was published as a proceedings.

Fluorometry is new to the project, and will be combined with targeted sampling and Ent. faecalis speciation during the last Georgia sampling this fall. In this research, the results from the Sapelo NERR will be confirmed with the detection of fecal sterols. This research will complete our obligation to sample in this location. The detection of fecal sterols was possible because the Marine Extension Service in Brunswick has a GC/MS available for use. However, this research is still progressing and submission of our third manuscript to a refereed journal is likely to be delayed until early 2006.

Anticipated Success in Meeting Project Objectives
Except for the sampling of the Sapelo Island NERR, which will be combined with the NERR Graduate Fellowship awarded to Sarah Hemmings, we will meet the overall objective of the project.

Overall Project Timeline Update
With the exception of sampling the Sapelo Island NERR, the proposed samplings are complete.

Preliminary Data
Summary of Georgia Research
Introduction
St. Simons is one of Georgia’s barrier islands. Since April 2004, when the Georgia Environmental Protection Division switched from fecal coliforms to fecal enterococci as an indicator of fecal contamination, the beach has had chronic beach advisories with respect to fecal contamination. Our objective was to identify the sources of fecal contamination at St. Simons during calm and stormy weather conditions using targeted sampling and three inexpensive BST methods, Enterococcus speciation, the detection of the esp gene, and fluorometry. Sampling was always conducted on an ebbing tide because these sampling conditions yield the highest enterococcal numbers (Boehm and Weisberg, 2005).

Method
On 3 July 2005, we conducted a targeted sampling of St. Simons Island. A total of 66 samples was collected. The conditions were warm and sunny, but the area had received heavy rain the previous week. In addition to the beach and storm drains along the southern portion of the island, Postell Creek was also sampled. Several sites were Georgia Department of Natural Resources beach monitoring sites.

At each sampling point, the location coordinates were taken with a GPS device (Model GPSMAP 175, Garmin International Inc., Olathe, KS). Locations of each sampling site were converted to ArcView 3.2 shapefiles and were incorporated into a GIS database. At five selected sampling points, salinity, pH, temperature, and dissolved oxygen were recorded with a Hydrolab Quanta (Austin, TX).

Water samples were collected in 120-mL (4 oz.) Whirl-Pak bags (Nasco, Modesto, CA). The samples were placed on ice and processed within 6 hours using the Enterolert™ System (IDEXX Laboratories, Westbrook, ME). Samples were diluted with sterile distilled water to 10^-1 in sterile manufacturer-supplied polystyrene bottles. A package of powdered Enterolert reagent was added to each bottle. After the reagent was dissolved in the sample, the contents of each bottle were added to a Quanti-tray, a sterile disposable panel containing 97 wells. Each Quanti-tray was mechanically sealed. The sealing distributed the sample uniformly into the wells. Each Quanti-tray was incubated for 24 h at 41+0.5 °C. To be counted with the Enterolert system, cells had to fluoresce wells under a 365-nm UV light (Model EA-160, Spectronics Corp., Westbury, NY). The number of positive wells was converted to a Most Probable Number (MPN) value based on the dilution factor and manufacturer-supplied MPN tables.

Fluorometric values were determined in the laboratory with discrete samples using a field fluorometer (Model 10-AU-005, Turner Designs, Sunnyvale, CA) set to detect long wavelength optical brighteners.

To obtain enterococcal isolates for Ent. faecalis numbers as well as Ent. faecium isolates for detection of the esp gene, positive Quanti-tray wells from three locations were labeled with an acetate marker: a) upper Postell Creek, b) Beachview and Mallory Streets, and c) a duck pond and the Lord Avenue drainage ditch, which exits via the Virginia Avenue storm drain to the ocean (Figure 2). The back of the Quanti-tray was surface-disinfected with 70% ethanol, and each well was punctured with a separate sterile pipette tip. A 10-µL portion was removed from each well with the pipetter, and the portion was spotted into one well of a 96-well microtiter plate containing Enterococcosel agar (Becton Dickinson, Sparks, MD). The plates were incubated for 24 hours at 35 °C. Wells positive for esculin hydrolysis (black color) were struck onto 5-cm plates containing brain heart infusion agar with 6.5% NaCl. Plates were incubated in Ziploc bags (DowBrands, Indianapolis, IN) at 35 °C. After 48 hours, colonies on the plates (positive growth) were subjected to a catalase test with 8.82 M H₂0₂ to ensure that each isolate was catalase negative. Quanti-tray wells containing bacteria that conformed to this USEPA definition of fecal enterococci (hydrolyzed esculin, grew on brain heart infusion agar with 6.5% NaCl, and were catalase negative; USEPA, 2002) were recorded as positive for fecal enterococci, and the wells were counted towards the MPN.

All confirmed fecal enterococci from the Quanti-tray wells were speciated according to a modified Manero and Blanch (1999) protocol. The protocol was modified to identify only two fecal enterococcal species, Ent. faecalis and Ent. faecium. The remaining enterococcal species were recorded as “other enterococci.” To speciate the fecal enterococci, each isolate was randomly picked with a sterile plastic stab from a separate plate containing brain heart infusion agar with 6.5% NaCl. Each isolate was suspended in 125 µL of saline-phosphate buffer contained in a well of a 96-well microtiter plate. Two wells of the 96-well plate were reserved for American Type Culture Collection (ATCC) controls, Ent. faecalis ATCC #19433, and Ent. faecium ATCC #19434, and two wells were reserved for randomly placed uninoculated controls. Each isolate was inoculated with a sterile polypropylene replicator (Sigma Chemical Co., St. Louis, MO) into separate microtiter plates containing arginine hydrolysis medium with and without arginine, and carbon utilization media containing arabinose, ribose, and mannitol (Wheeler et al., 2002). Plates were incubated at 37 °C and reactions were recorded after 72 h.

Enterococcus faecium isolates from the three locations were analyzed to detect the presence of the esp gene. Isolates were spotted on a 0.45-µm membrane contained on a 5-cm Petri plate with mEI agar (Becton-Dickinson). The three plates were incubated at 41+0.5 °C for 24 hours and were sent by overnight mail to Biological Consulting Service of North Florida (Gainesville, FL) for analysis.

Certain limits were set to identify sites of concern. For fluorometry, any site with an optical density >100 is considered positive (Hagedorn et al., 2003), and this limit was the one chosen here. For fecal enterococci, the federal limit for a single grab sample is 104 fecal enterococci per 100 mL (USEPA, 2002) and this limit was the one chosen for the study.

Results
Little differences were observed among the five site locations with respect to pH, temperature, and dissolved oxygen (Table 1). In contrast, salinity in Postell Creek was much lower (an average of 0.3 ppt) than the salinity of the surf zone (an average 19.4 ppt). All samples obtained from the surf zone of St. Simons beach (Sites #6-14) contained <52 fecal enterococci per 100 mL (Figure 3A). Similarly, all beach samples had fluorometric values <100. Therefore, the beach of St. Simons Island did not have a problem with fecal contamination, human or otherwise, during this targeted sampling.

Sites #1-5, #15-17, and #64-66 were either storm drains or ponds leading to storm drains. With the exception of Site #1, all were sites of concern for fluorometry, and, with the exception of the storm drain at Site #2 and the retention pond at Site #64, all were sites of concern for fecal enterococci as well. The storm drain at Site #1 is on a section of beach rarely visited by beachgoers. The storm drain at Site #2 had low numbers of fecal enterococci (41 per 100 mL), but had the highest fluorometric value (662) of all the sites sampled. This storm drain carried runoff from local residences and businesses, including Laundromats. Site #4 is a duck pond that discharges into a drainage ditch (Site #3). The percentage of Ent. faecalis from this ditch was 33%, and the esp gene was not detected (Table 2). Site #5 is a storm drain located at Beachview and Mallory Streets, and contained the highest number of fecal enterococci (19,683 per 100 mL) among all the sites sampled (Figure 3A). The percentage of Ent. faecalis from this ditch was only 20%, but the esp gene was detected among the Ent. faecium isolates from this location (Table 2). Sites #15-16 and #63 are storm drains responsible for runoff from local residential areas, whereas Sites #65-66 drain Site #64, a retention pond.

In contrast to the low numbers for fluorometry and fecal enterococci observed in the surf zone of the beach, and similar to the high numbers for fluorometry and fecal enterococci observed in the island’s ponds and drainage ditches, high numbers for fluorometry and fecal enterococci were also observed in Postell Creek. In lower Postell Creek (Sites #18-33; Figure 3A), fecal enterococcal numbers declined with distance from Site #32 (1,259 fecal enterococci per 100 mL) to Site #18, the mouth of Postell Creek (31 fecal enterococci per 100 mL) with few exceptions (Sites #24 and #19-21). The major exception, Site #21, drains the section of Postell Creek where high counts of fecal enterococci from storm drains at Sites #15-17 are located. Like fecal enterococci, fluorometric values also declined with distance from Site #32 (320 fluorometric units) to Site #18 (65 fluorometric units).

In upper Postell Creek (Figure 3B), all sites were of concern for fluorometry. Fluorometric values were consistently high, and ranged between 298 (Sample #62) and 349 units (Sample #58), with the exception of Sites #56 (190 units) and Site #60 (407 units). It is noteworthy that Site #56 is near the storm drain at Site #63, which has a similar fluorometric reading (191 units). All sites were of concern for fecal enterococci, and after declining from Site #32 to 388 fecal enterococci per 100 mL, fecal enterococcal counts increased again and averaged >1250 fecal enterococci per 100 mL between Site #46 and Site #55 before falling again. Extremely high counts were observed again at Site #60 (5,794 fecal enterococci per 100 mL) and this location is also the same one with the highest fluorometric value (407 units) in Postell Creek. It is noteworthy that Site #62, the location of a lift station for residences and business located on the eastern side of the island, had a relatively low fluorometric value (190 units), and count of fecal enterococci (158 per 100 mL). When a total of 176 fecal enterococci were obtained from Sites 33, 37, 45, 50, 56, and 59, the percentage of Ent. faecalis was 56% and the esp gene was not detected (Table 2).

Discussion
The surf zone of St. Simons Island had low fluorometric values and low numbers of fecal enterococci, and therefore fecal contamination from human or nonhuman sources was not a concern. This conclusion did not extend to the vast majority of sampling sites involving storm drains and Postell Creek. With few exceptions, all these sampling sites had in excess of 104 fecal enterococci per 100 mL and were, by USEPA definition (USEPA, 2002), contaminated with excessive fecal bacteria. In particular, Site #5, with 19,863 fecal enterococci per 100 mL, was of particular concern and deserves more attention.

For Sites #1, #2, and #3, there was no contradiction in BST technology and potential sources (or not) of fecal contamination. For Site #1, a storm pipe located on a section of beach rarely visited by beachgoers, the relatively low numbers of fecal enterococci (213 per 100 mL) and no fluorescence suggests the source is likely to be nonhuman. For Site #2, the extremely high fluorometric value (662 units) but low numbers of fecal enterococci suggests the presence of optical brighteners in gray water or some other type of fluorescing agent, like diesel fuel. It is unlikely that the agent is solely organic matter because this value exceeds the highest fluorometric signal that we have ever observed with organic matter (Hartel, unpublished). This site deserves more attention for possible gray water connections to the storm water drain. For Site #3, a storm drain emptying a duck pond (Site #4), the source is likely to be birds because the nature of the site, the high percentage of Ent. faecalis (>30%), and the inability to detect the esp gene. A high percentage of Ent. faecalis is associated with both birds and humans (Kuntz et al., 2005), but the absence of the esp gene, associated 97% of the time with humans (Scott et al., 2005), suggests that humans were not a source.

The sources of fecal contamination for the remaining storm drains and sites on Postell Creek were unclear because of the contradictions in BST technology. The source of fecal contamination to the storm drain at Site #5 (Beachview and Mallory) was likely to be humans. Fluorometric values were high (173) and the esp gene was detected, however this detection was not supported by high percentages of Ent. faecalis (20%). If humans were a source, then the percentage of Ent. faecalis should have exceeded 30%. Low percentages of Ent. faecalis (15 and 23%) have been observed in the primary effluent of two of 32 Delaware wastewater treatment plants (Hartel, unpublished), so this result is possible but unusual. This site needs confirmation with another BST source.

The source of fecal contamination to Postell Creek was also contradictory, but likely to be birds. The percentage of Ent. faecalis was high (56%) and the esp gene was not detected. However, the fluorometric values were consistently high in the upper portion of the creek and then decreased with distance from Site #33. High fluorometric numbers are normally associated with human fecal contamination, but it is possible that other fluorescent sources are involved. This site needs confirmation with another BST source.

Given the contradictory results of the BST methods, additional research is required. First, it is important to determine the degree to which organic matter, particularly in Postell Creek, affects the fluorometric signal. Second, it is important to develop an additional BST test to provide some clarity to the results. Because of the availability of a GC/MS at the Marine Extension Laboratory in Brunswick, the most reasonable test is the detection of fecal sterols. These tests are currently under development.

Summary of Puerto Rico Research
Introduction
Our previous research suggested problems with the basic assumptions of BST and fecal enterococcal survival (Hartel et al., 2004). Fecal enterococci appear to survive and even grow on desiccated and rewetted sediments in temperate zones coasts affected by low and high tides (Desmarais et al., 2002). Other researchers report that these bacteria are capable of surviving on marine plants, especially sun-dried algae (Whitman et al., 2003). This enterococcal survival may limit their utility as indicators of existing fecal material sources.

In addition, our data suggest that sediment resuspension may increase false positive numbers in water samples. After confirmation, the percentage of false-positive Enterolert wells ranged up to 99.9% in water and sediment samples from La Parguera, Jobos Bay National Estuarine Research Reserve, and Mayagüez, Puerto Rico. In contrast, the upper percentage of false positive Enterolert wells from Georgia and New Hampshire locations was small (Hartel et al., 2004).

Methodology
Enterococcus faecalis and Ent. faecium were inoculated into triplicate translucent plastic enclosures over sea grasses at a final cell density of 10⁵ to 10⁷ cells L^-1. Previous work at this study site showed negligible enterococcal numbers. Water and leaf samples were collected before and after inoculation for a period up to 48 h. Aliquots of water were removed, formalin-fixed, and bacterial cells counted by epifluorescence with 4,6-diamino-2-phenylindole stain (DAPI; Sherr et al., 2001). Also, enterococci were estimated from sub-aliquots using MPN methodology.

Results
Direct counts suggested that numbers of Ent. faecium increased 6-fold and Ent. faecalis numbers doubled in 48 h (Figure 4). In contrast, MPN counts of Ent. faecium and Ent. faecalis decreased, and in the case of Ent. faecalis, decreased to below the limit of detection. The discrepancy between the two methods may be the presence of a viable-but-not-culturable state in the MPN medium, or the inability of Enterococcosel broth to recover such cells.

Dissemination
Publications (abstracts):
Hartel, P. G., K. Rodgers, S. N. J. Hemmings, J. L. McDonald, K. Gates, S. H. Jones, T. L. Bryant, B. O’Hara, E. Otero, and Y. Rivera­Torres. 2004. Desiccation as a mechanism of fecal enterococcal survival and regrowth for bacterial source tracking. Abstr. Annu. Meet. Am. Soc. Microbiol.

Feng, Y, P. G. Hartel, S. Deng, K. Rodgers, J. Fisher, and B. Liu. 2004. Survival of Enterococcus species and subspecies in sediment for bacterial source tracking. Abstr. Annu. Meet. Am. Soc. Microbiol.

McDonald, J., P. G. Hartel, L. Gentit, K. W. Gates, K. Rodgers, J. Fisher, K. Austin, K. A. Payne, S. N. J. Hemmings, and C. Belcher. 2004. Combining targeted sampling and bacterial source tracking during calm versus windy or stormy beach conditions. EPA National Beaches Conference, 13-15 October, San Diego, CA.

Rodgers, K., P. G. Hartel, J. McDonald, L. Gentit, K. W. Gates, J. Fisher, K. Austin, K. A. Payne, S. N. J. Hemmings, S. H. Jones, T. L. Bryant, B. O’Hara, E. Otero, Y. Rivera­Torres, Y. Feng, S. Deng, and B. Liu. 2004. Survival and regrowth of fecal enterococci in moist and desiccated sediments. EPA National Beaches Conference, 13-15 October, San Diego, CA.

Publications (proceedings):
Hartel, P. G., K. Rodgers, J. A. Fisher, J. L. McDonald, L. C. Gentit, E. Otero, Y. Rivera­Torres, T. L. Bryant, and S. H. Jones. 2004. Survival and regrowth of fecal enterococci in desiccated and rewetted sediments. In K. Hatcher (ed.) Proceedings of the 2005 Georgia Water Resources Conference, April 25-27, Athens, GA.

McDonald, J. L., P. G. Hartel, L. C. Gentit, K. W. Gates, K. Rodgers, J. A. Fisher, K. L. Austin, K. A. Payne, S. N. J. Hemmings, and C. N. Belcher. 2004. Combining targeted sampling and bacterial source tracking during calm and stormy conditions. In K. Hatcher (ed.) Proceedings of the 2005 Georgia Water Resources Conference, April 25-27, Athens, GA.

Publications (journal article, in review)
McDonald, J. L., P. G. Hartel, L. C. Gentit, K. W. Gates, K. Rodgers, J. A. Fisher, K. A. Smith, K. A. Payne, and C. N. Belcher. Identifying sources of fecal contamination inexpensively with targeted sampling and bacterial source tracking. J. Environ. Qual.

Conferences:
Two posters were presented at the American Society for Microbiology, 23-27 May 2004, in New Orleans, LA.

Two posters were presented at the EPA National Beaches Conference, 13-15 October 2004, in San Diego, CA.

Two proceedings were presented at the Georgia Water Resources Conference, 25-27 April 2005, in Athens, GA.

Outreach Activities:
CICEET research has been highlighted at the following invited presentations given by the PI/PD:
1) Northern Gulf of Mexico Program, 12 November 2004, Biloxi, MS.
2) Southern California Coastal Water Research Project, 22 November 2004, Westminster, CA.
3) Iowa Fifth Annual Water Monitoring Conference, 14 Jan 2005, Ames, IA (keynote speaker).
4) USEPA Region 6 Bacterial Source Tracking Workshop, 27 Jan 2005, Dallas, TX.
5) Microbial Source Tracking Workshop, sponsored by the Water Environment Research Foundation, 16 Feb 2005, San Antonio, TX.

Expenditures
As already noted in our 2005 Semi-Annual Report, four personnel were sent to Puerto Rico to help with the sampling there. Because of our success with fluorometry that eliminated the need for ribotyping, the expenses set aside for this purpose were diverted for travel. Otherwise, all expenditures were within the range anticipated for the research. In addition, Dr. Steve Weisberg, Director of the Southern California Coastal Water Research Project (SCCWRP) asked that the PI/PD present his data to his group, and CICEET graciously funded this visit.

References
Boehm, A. B., and S. B. Weisberg. 2005. Tidal forcing of enterococci at marine recreational beaches at fortnightly and semi-diurnal frequencies. Environ. Sci. Technol. 39:5575-5583.

Desmarais, T. R., H. M. Solo-Gabriele, and C. J. Palmer. 2002. Influence of soil on fecal indicator organism in a tidally influence subtropical environment. Appl. Environ. Microbiol. 68: 1165-1172.

Gilpin, B., T. James, F. Nourozi, D. Saunders, P. Scholes, and M. Savill. 2003. The use of chemical and molecular microbial indicators for faecal source identification. Water Sci. Technol. 47:39-43.

Hagedorn, C., R. B. Reneau, M. Saluta, and A. Chapman. 2003. Impact of onsite wastewater systems on water quality in coastal regions. Virginia Coastal Resources Management Program Memorandum of Agreement 50312­01­13­PT. Virginia Department of Conservation and Recreation, Virginia Department of Health, 1/28/02­6/30/03.

Hartel, P. G., K. Rodgers, J. A. Fisher, J. L. McDonald, L. C. Gentit, E. Otero, Y. Rivera­Torres, T. L. Bryant, and S. H. Jones. 2004. Survival and regrowth of fecal enterococci in desiccated and rewetted sediments. In K. Hatcher (ed.) Proceedings of the 2005 Georgia Water Resources Conference, April 25-27, Athens, GA.

Kuntz, R. L., P. G. Hartel, K. Rodgers, and W. I. Segars. 2004. Presence of Enterococcus faecalis in broiler litter and wild bird feces for bacterial source tracking. Water Res. 38:3551-3557.

Leeming, R., A. Ball, N. Ashbolt, and P. Nichols. 1996. Using fecal sterols from humans and animals to distinguish faecal pollution in receiving waters. Water Res. 30:2893-2900.

Manero, A., and A. R. Blanch. 1999. Identification of Enterococcus spp. with a biochemical key. Appl. Environ. Microbiol. 65: 4425-4430.

Scott, T. M., T. M. Jenkins, J. Lukasik, and J. B. Rose. 2005. Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution. Environ. Sci. Technol. 39:283-287.

Sherr, B., E. Sher, and P. del Giorgio. 2001. Enumeration of total and highly active bacteria. In J. H. Paul (ed.) Marine microbiology. Meth. Microbiol. 30. Academic Press, NY.

USEPA (U.S. Environmental Protection Agency). 2002. Implementation guidance for ambient water quality criteria for bacteria. EPA-823-B-02-003. U. S. Government Printing Office, Washington, D.C.

Wheeler, A. L., P. G. Hartel, D. G. Godfrey, J. L. Hill, and W. I. Segars. 2002. Potential of Enterococcus faecalis as a human fecal indicator for microbial source tracking. J. Environ. Qual. 31:1286-1293.

Whitman, R.L., D.A. Shively, H. Pawlik, M. B. Nevers, and M.N. Byappannahalli. 2003. Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan. Appl. Environ. Microbiol. 69:4714-4719.