CICEET Progress Report for the period 9/15/04 Through 3/15/05

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, Ernesto Otero
Additional Investigator(s): Katy Austin, Carolyn Belcher, Jared Fisher, Keith Gates, Lisa Gentit, Sarah Hemmings, Jennifer McDonald, Beth O’Hara, Karen Payne, Yaritza Rivera­Torres, and Karen Rodgers.
Project Start Date:

Figures

Figure 1

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Tables

Table 1

Tasks to meet objectives In the first six months of the second year, the objectives required us to: a) have another coordination meeting, b) conduct one targeted sampling for fecal enterococci in one contaminated river or bay near or in the appropriate NERR in Georgia, New Hampshire, and Puerto Rico, and c) sample the sediment from these contaminated rivers or bays. Again, our previous results suggested that if we found a high percentage of Ent. faecalis (>30%) during baseflow conditions, then human or wild bird fecal contamination was likely. Therefore, each sampling required us to isolate Enterococcus faecalis. We would use ribotyping, a DNA-based method of bacterial source tracking, to distinguish between human and bird sources as necessary.

Progress on Tasks
The coordination meeting was not held. We have been working to advance inexpensive bacterial source tracking (BST) methods and in our annual report wrote about success of combining fluorometry with targeted sampling for the first time at a non-NERR site, Sea Island, Georgia. Fluorometry detects optical brighteners in waters and thus is a chemical method designed to detect human fecal contamination. Although fluorometry appeared to work well, the high amount of organic matter in Georgia’s water interfered with the fluorometric signal. We needed to test waters with low organic matter during baseflow conditions. Therefore, instead of a coordination meeting, we sent four persons, Karen Rodgers, Katy Austin, Lisa Gentit, and Sarah Hemmings to work with Ernesto Otero and Yaritza Rivera-Torres in Puerto Rico in order to conduct fluorometry during late January­early February, the driest time of the year (Figure 1). These samplings are now complete.

In our annual report, we also mentioned that we had stopped our targeted sampling because we had discovered a serious error with our isolation methodology. Also, it appeared that sediment was much more important to numbers of fecal enterococci than we had previously thought. The methodological problem was solved and a new sediment sampling protocol was designed. As a result of this new protocol, our results now include two Most Probable Numbers (MPNs) for each sediment assay, one “original,” and one “corrected.” The “corrected” MPN represents the actual number of fecal enterococci. The desiccation experiment was restarted at all three locations. In this experiment, sediment was dried for 2, 30, and 60 days. At each sampling, the sediment was wetted for 1 hour (to determine survival) and for 24 hours (to determine any regrowth). Speciation of the fecal enterococci for Ent. faecalis continued as normal. To study the effects of seasonal changes on the sediment, two samplings were conducted at each site, one during the winter and one during the summer. These experiments are now complete. By virtue of the additional fluorometric research we conducted, the Puerto Rico samplings are now finished, while sampling is continuing in Georgia and New Hampshire.

Finally, a targeted sampling on St. Andrews Park during stormy conditions (not just windy) was conducted on an ebbing spring tide, which is considered a worst-case scenario.

Accomplishments
Four abstracts have been published (see Dissemination, Publications), and two proceedings manuscript have been submitted and accepted. The two proceedings manuscripts will be published in the Proceedings of the Georgia Water Resources Conference, which will 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. Both proceedings are in the process of being converted to manuscripts for submission to the Journal of Environmental Quality. Both manuscripts should be submitted by the final report in September 2005. In addition to these publications, CICEET research has been highlighted in five invited presentations by the PI/PD.

Difficulties
Fecal enterococci are bacteria widely used as indicators of fecal contamination in marine and estuarine waters. One requirement for a fecal bacterium to be useful as an indicator is that it not persist or regrow in the environment. However, our continuing problems with high numbers of fecal enterococci in sediment suggested that these bacteria are doing just that. We conducted experiments with fecal enterococci to determine their ability to survive desiccation and to regrow in marine and estuarine sediments from Georgia, New Hampshire, and Puerto Rico after 0, 2, 30, and 60 days. Although numbers of fecal enterococci generally decreased with increased length of drying, many fecal enterococci survived desiccation and regrew in rewetted sediment, violating the assumption that fecal bacteria not persist or regrow in the environment. These experiments are now complete, and because there is currently no better alternative to fecal enterococci as fecal indicator bacteria, we are recommending that care should be taken not to disturb the sediment when sampling water for fecal contamination, or if the sediment is already disturbed (e.g., windy days or runoff conditions), then the influence of sediment should be considered.

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
Our objective required us to conduct a second targeted sampling for fecal enterococci in a second contaminated river or bay in Georgia and New Hampshire. Our results were to be summarized in a second-year, annual report due September 2005.

Work Plan for Next Reporting Period
Tamara Bryant and Yaritza Rivera­Torres are both working on the presence of fecal enterococci in marine plants. Tamara is working on the presence of fecal enterococci in secondary habitats (e.g., wrack) on New Hampshire’s ocean bathing beaches, while Yaritza is conducting microcosm research on how five different fecal enterococcal species survive on turtle grass (Thalassia testudinum). The status of their research will be summarized in our final report due in September 2005.

We expect to submit three manuscripts to refereed journals by September 2005: one on targeted sampling during calm and stormy conditions (this is the St. Andrews Park and Sea Island work), one on regrowth and desiccation of fecal enterococci (presented in the Preliminary Data), and one on combining targeted sampling and fluorometry (initial Puerto Rico studies presented in the Preliminary Data).

Fluorometry is new to the project, and will be combined with targeted sampling and Ent. faecalis speciation during the last Georgia sampling this summer. This research will be conducted at the Sapelo Island NERR and complete our obligation to sample in this location. We will be able to conduct fluorometric research at this location because Sarah Hemmings was just awarded a NERR Graduate Fellowship to do this. In addition, we hope to include fecal sterols in this research since the Marine Extension Service in Brunswick has a GC-MS available.

Anticipated Success in Meeting Project Objectives
The methodological problems of sediment sampling in the first year meant a delay in conducting the two planned targeted samplings at each location. These problems have been resolved and the sampling is back on track. Our success with fluorometry has meant that there is no need to use ribotyping to distinguish between human and bird sources, and ribotyping will not be used in the project. The overall project objective will be met.

Overall Project Timeline Update
Puerto Rico has completed its sampling, and Georgia and New Hampshire are on track to complete their sampling by September 2005 as scheduled.

Preliminary Data
Targeted sampling of St. Andrew’s Park during Stormy Conditions
In the annual report, we combined targeted sampling and enterococcal speciation to identify sources of fecal contamination to St. Andrews Park on Jekyll Island, Georgia. Sampling was conducted during baseflow (calm) conditions, but not during stormflow (runoff) conditions. We tried to sample during stormflow conditions, but the anticipated rain did not occur, and sampling was conducted only during windy conditions (26 to 32 km hour^-1). This sampling was fortuitous as it identified sediment disturbance by wind as a mechanism whereby fecal enterococcal counts increased. However, we continued to monitor the weather and were able to sample St. Andrews Park under stormy conditions on an ebbing spring tide on 24 Feb 2005. Fecal enterococcal counts in Beach Creek ranged from 148 (mouth of the creek) to 428 (uppermost portion of the creek) per 100 mL. Counts from northern limit of St. Andrews Park to the southern tip of Jekyll Island were: 182, 249, 134, 158, 173, 109, 63, 110, 134, 74, 120, 41, 10, 10, 10, and 10 fecal enterococci per 100 mL. With the exception of one location above Beach Creek (109 fecal enterococci per 100 mL), counts in the sound around the park were low, generally 10 fecal enterococci per 100 mL. Therefore, our original suggestion that wildlife in Beach Creek were likely to be responsible for high numbers of fecal enterococci was correct.

Summary of desiccation and regrowth research
Fecal enterococci are bacteria widely used as indicators of fecal contamination in marine and estuarine waters. One assumption is that fecal enterococci, indeed all fecal indicator bacteria, do not persist or regrow in the environment (Clesceri et al., 1998). Sediments have long been known as a reservoir for fecal bacteria (Stephenson and Rychert, 1982). For example, when cattle have access to streams, their fecal bacteria survive for long periods in the sediment (Howell et al., 1996). This discovery is unsurprising because clay particles and organic matter often protect the bacteria from adverse environmental conditions (Robert and Chenu, 1992).

We were concerned about the survival and potential regrowth of fecal enterococci in sediments during conditions of desiccation and rewetting because of our interest in bacterial source tracking. Bacterial source tracking is based on the principle that specific markers or strains of bacteria are associated with specific animal species. Therefore, it may be possible to match fecal contamination in environmental waters to specific animal species. However, if these bacteria survive and regrow in sediment, then this survival and regrowth may affect bacterial source tracking because the bacteria may represent long past sources of fecal contamination.

Few studies have been reported on the survival or regrowth of fecal bacteria in desiccated sediments. Fecal enterococci can regrow when sterile sediment is added to nonsterile sediment and the wetting and drying of the tidal cycle is simulated (Desmarais et al., 2002). Furthermore, soil bacteria regrow in rewetted soils as survivors dine on the deceased (Birch, 1958). Also, enterococci are known to survive desiccation for >11 weeks on surfaces associated with farm buildings (Bale et al., 1993).

Given the potential for fecal enterococci to survive and regrow in dried sediments, we conducted experiments to determine the extent to which fecal enterococci survive and regrow in marine and estuarine sediments from Georgia, New Hampshire, and Puerto Rico. These locations were selected because of their differences in latitude and differences (or lack of differences) in annual seasonal temperature. Also, clays protect bacteria from environmental conditions (and presumably from the selective properties of culture medium). This protection may yield a large number of false positive isolates. For this reason, all fecal enterococci isolates were confirmed according to the USEPA definition (2002).

Methods
Sediment samples were collected at low tide, once during warm weather conditions and once during cold weather conditions, at three locations: Academy Creek, located near Brunswick, Georgia; Bunker Creek, located in the Great Bay of New Hampshire; and Chun-Chin Creek, located in the Jobos Bay National Estuarine Research Reserve in south central Puerto Rico.

Surface sediment samples (uppermost few millimeters) were collected with an ethanol-disinfected spoon and were placed into sterile polypropylene bottles. Bottles were placed on ice and the sediment was processed within 6 hours. Sediment samples were allowed to resettle for 1 hour, after which the overlying water was removed aseptically. The sediment was analyzed for organic carbon and texture (sand, silt, and clay) by standard methods. The percentages of organic carbon and clay were 6.2 and 10.6% for Academy Creek, Georgia; 3.5 and 32.4% for Bunker Creek, New Hampshire; and 6.4 and 4.2% for Chun-Chin Creek, Puerto Rico.

For microbiological analysis, a 20-mL portion of sediment was placed in a 10-cm pre-weighed Petri dish and the weight recorded. The top of the Petri dish was removed and the sediment was allowed to air-dry at room temperature (20 to 22 °C) for 0, 2, 30, and 60 days. Each sampling was done in triplicate. In addition, triplicate 20-mL portions of sediment were placed in preweighed aluminum dishes and the dry weight determined gravimetrically after drying at 95 °C for 24 hours.

After 0, 2, 30, and 60 days, triplicate samples of sediment in the Petri dishes were rewetted with sterile distilled water to their original weight. The dishes sat for 1 hour before half the sediment was processed for fecal enterococci with the Enterolert System (IDEXX Laboratories, Westbrook, ME). The Most-Probable-Number (MPN) was determined as described by Hartel et al. (2004). The three sediment samples were then left covered at room temperature for 24 hours and the sampling was repeated with the remaining half of the sediment. In this manner, any regrowth could be recorded. Because the presence of sediment might affect the accuracy of the Enterolert system, the content of each positive (fluorescing) Quanti-tray well was confirmed for the presence of fecal enterococci as described by Hartel et al. (2004). To be recorded as positive, at least one isolate from a positive Quanti-tray well had to conform to the USEPA (2002) definition of fecal enterococci: be able to hydrolyze esculin, be able to grow on brain heart infusion agar with 6.5% NaCl, and be catalase negative. Wells containing at least one isolate that conformed to this definition were counted towards the MPN. The results were expressed on a per gram dry weight basis.

Results
All moist sediments had large numbers of fecal enterococci (see Table 1). The corrected MPNs of fecal enterococci g^-1 dry weight of moist sediment varied from an average of 8,172 in Academy Creek (Georgia) to an average of 167 in Bunker Creek (New Hampshire) to an average of 166 in Chun-Chin Creek (Puerto Rico).

With a few exceptions, numbers of fecal enterococci decreased with increased length of drying. After 60 days, Georgia and New Hampshire sediments still contained between 63 and 1,200 fecal enterococci g^-1 dry weight of sediment, whereas Puerto Rico sediment had low numbers of fecal enterococci (between 2 and 17).

Fecal enterococci regrew in some rewetted sediments but not in others. For example, in the Academy Creek sediment from Georgia, fecal enterococci increased from 1,202 to 28,840 g^-1 dry weight after 60 days (December 2003). In contrast, in the Bunker Creek sediment from New Hampshire, fecal enterococci decreased from 302 to 53 g^-1 dry weight after 60 days (March 2004).

The number of false positive Enterolert wells (wells that fluoresced but contained no fecal enterococci) was highly variable and resulted in decreases between the original and corrected MPNs ranging from 0 to >99.9%. This variability was observed in sediments from Georgia, New Hampshire, and Puerto Rico regardless of whether the sediment was moist, dried and rewetted for 1 hour (survival), or dried and rewetted for 24 hours (regrowth). The greatest decreases between original and corrected counts were observed in Puerto Rico sediments (average >99.6%).

Discussion
There were large numbers of fecal enterococci in moist sediments. These results are similar to many other studies that suggest sediments are reservoirs to large numbers of fecal bacteria (e.g., Howell et al., 1996). Disturbing this sediment may affect fecal counts. Therefore, care should be taken not to disturb the sediment when sampling, or if the sediment is already disturbed (e.g., windy days or runoff conditions), then the influence of sediment should be considered.

Fecal enterococci survived desiccation and sometimes regrew in sediment after rewetting. The most reasonable explanation for this survival and regrowth is the ability of the fecal enterococci to tolerate the high salt concentrations in the sediment. Fecal enterococci can tolerate 6.5% NaCl (USEPA, 2002).

Fecal enterococcal survival was poorest in Puerto Rican sediment, likely because of soil texture. Puerto Rican sediment contained a higher percentage of sand (46.9%) than sediments from New Hampshire (7.2%) or Georgia (9.1%). Soils with a high percentage of sand dry faster and have poorer bacterial survival than soils with a high percentage of clay (Hartel and Alexander, 1986).

According to the definition in Standard Methods for the Examination of Water and Wastewater (Clesceri et al., 1998), fecal indicator bacteria should not persist in the environment. Our research suggests that fecal enterococci violate this criterion. Furthermore, survival and regrowth affect bacterial source tracking results because the bacteria may represent a source of long past fecal contamination. These results reaffirm that an ideal fecal indicator bacterium does not exist, and care should be taken in interpreting fecal enterococcal data.

There was a serious methodological problem with the Enterolert system because false positive wells affected the MPN results. The greatest problem was observed in Puerto Rico sediment. Why there was so much variability among the sediments is unclear. Nevertheless, these results suggest that the Enterolert system should be used with caution in waters containing high amounts of sediment.

Summary of Puerto Rico research
Sampling was conducted from 31 January to 5 February 2005, at three locations on the west coast of Puerto Rico: La Parguera, Boqueron, and Mayagüez Bay. Sampling of the Jobos Bay NERR was completed in the last six-month cycle and will be presented in the final report after statistical analysis. The weather for all sampling dates was dry, with the exception of one brief rain event that did not result in any surface runoff. The ambient temperature ranged from 21 to 27° C.

The first targeted sampling site was La Parguera (Figure 2). Thirty water samples from various locations along the coast were tested for the presence of Escherichia coli. In addition to collecting fluorometric data, we also collected data on water salinity, turbidity, temperature, dissolved oxygen (DO), and pH. The Most-Probable-Numbers (MPN) ranged from 31 to 3,873 E. coli per 100 mL. Four “hotspots” (Sites #5, 10, 17, and 25) were resampled the next day. Additional water samples for Ent. faecalis were also obtained.

Site #5 (309 E. coli per 100 mL) was the outfall of a wastewater treatment plant. Upon resampling, MPNs ranged from 31 E. coli per 100 mL at a site furthest from the wastewater treatment plant to 214 E. coli per 100 mL at the outfall. The corresponding MPN for fecal enterococci was 457 per 100 mL. Field fluorometric readings at Site #5 ranged from 15 to 28; the outfall reading was 22. There was concern that bubbles disturbed the readings so the readings were redone with a second fluorometer in the laboratory. This instrument was calibrated identically to the field fluorometer except it was set for discrete samples. Fluorometric readings ranged from 16 to 121; the outfall reading was 121. Why the field and the lab data did not match well was unclear.

Site #10 (275 E. coli per 100 mL) was located near some houses and boats. Upon resampling, the counts ranged from 74 to 512 E. coli per 100 mL. The fecal enterococci MPN was <10 per 100 mL. The fluorometric readings ranged from 8 to 9 in the field and from 11 to 12 in the lab. Therefore, the readings from the field and the lab matched reasonably well.

Site #17 (3,873 E. coli per 100 mL) had the highest count of the four hotspots. Upon resampling, counts ranged from 98 to 309 E. coli per 100 mL of water. Therefore, this hotspot fits a “transient” profile (not a persistent source) and does not required BST. The fecal enterococcal count was 20. Field fluorometric data ranged from 6 to 13, while lab fluorometric data ranged from 9 to 10. Again, the fluorometric readings from the field and the lab matched reasonably well.

Site #25 (253 E. coli per 100 mL) was around a nautical club where many boats were docked. Upon resampling, counts ranged from 10 to 216 E. coli per 100 mL. The fecal enterococcal count was 10. Field and lab fluorometry matched well, ranging from 7 to 9 in the field, and from 10 to 12 in the lab.

The DO, salinity, water temperature, and pH remained relatively constant. The DO ranged from 5.4 to 7.5 and averaged 6.6 + 0.5. The salinity ranged from 35.5 to 36.2 and averaged 36 + 0.1. The temperature ranged from 26.2 to 27.5 °C, while the pH ranged from 8.1 to 8.2.

The second targeted sampling site was of Boqueron Bay, a popular tourist site thought to have fecal contamination (Figure 3). Thirty water samples were obtained as well as the standard water data. Field fluorometric data was unstable and the fluorometer was re-calibrated. Overall, Boqueron Bay was relatively uncontaminated. Of the 30 samples, only five had counts over 200 E. coli per 100 mL, and only one site with a count >300 E. coli per 100 mL. Because the boat was unavailable, we did not conduct any additional samplings. The DO ranged from 3.7 to 7.0 and averaged 5.6 + 0.8. The salinity ranged from 31.5 to 36.1 and averaged 35.0 +1.4. The temperature ranged from 24.2 to 26.9 °C, and the pH ranged from 7.3 to 8.2.

The third targeted sampling site was Mayagüez Bay. In terms of potential sources of fecal bacteria, this bay contains some industry (notably a tuna packing plant and an animal feed plant) and a large wastewater treatment plant servicing the city of Mayagüez. The Yagüez River flows through the city into the bay, and a previous sampling had found the river to be grossly contaminated. The initial sampling was conducted with a boat around the bay as close to the shore as possible, given the depth and roughness of the water, and the sampling continued on foot with the testing of the streams that ran into the bay.

The open water samples had counts ranging from <10 to 4,106 E. coli per 100 mL. Samples taken at the mouths of the creeks ranged from 74 to >24,192 E. coli per 100 mL, while counts of fecal enterococci ranged from 52 to 1,515 per 100 mL. Fluorometric readings collected by boat ranged from 5 to 17. Readings for sites sampled on foot were generally higher, ranging from 8 to 91.

Counts for the second sampling were not as high as they were for the first sampling and ranged from 200 to >24,192 E. coli per 100 mL. However, while the first sampling contained four sites over the limit, the second sampling contained only one. Nevertheless, all the streams entering Mayagüez Bay can be considered contaminated. Two sites on the Yaguez River were sampled intensively for enterococcal speciation and fluorometry. One, a pipe near a textile mill, had counts of 9,139 and 14,830 E. coli per 100 mL; fecal enterococcal counts were 1,437, 1,060, 143, and 310 per 100 mL. Counts upstream of the mill were only 256 E. coli per 100 mL and 243 fecal enterococci per 100 mL. Of 76 enterococci isolated from the pipe effluent, 15 were Enterococcus faecalis, 21 were Enterococcus faecium, 21 were Enterococcus gallinarum, and 19 were other enterococci. The other site was one near the mouth of the Yagüez River. Of 147 enterococci obtained from this location, 7 were Ent. faecalis, 91 were Ent. faecium, 37 were Ent. gallinarum, and 12 were other enterococci. The isolates have recently been sent to BCS of North Florida to test them for the presence of human virulence factor.

The DO of Mayagüez Bay ranged from 1.7 to 7.0 and averaged 6.0 + 1.0. The salinity ranged from 0.18 in a freshwater creek feeding the bay to 36.18 in the bay. The temperature ranged from 22.5 to 25.9 °C and the pH ranged from 7.6 to 8.3.

The Puerto Rico data are currently being analyzed statistically and a full report of the results will be completed by the final report in September. Again, these data will be combined with fluorometry in Georgia to complete the manuscript.

Dissemination
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 (accepted).

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 (accepted).

Workshops: None

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 will be presented at the Georgia Water Resources Conference, 25-27 April 2005, in Athens, GA.

Training: None

Manuals, Protocols: None

Outreach:
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.

Contact with End Users: None

Expenditures
As already noted, 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 work accomplished in the last six months. This sampling was successful and sampling in Puerto Rico is now complete.
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
Bale, M. J., M. Hinton, J. E. Beringer, and P. M. Bennett. 1993. The survival of bacteria exposed to desiccation on surfaces associated with farm buildings. J. Appl. Bacteriol. 75:519-528.

Birch, H.F. 1958. The effect of soil drying on humus decomposition and nitrogen availability. Plant Soil 10: 9-31.

Clesceri, L. S., A. E. Greenberg, and A. D. Eaton. 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DC.

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.

Hartel, P. G., and M. Alexander. 1986. Role of extracellular polysaccharide production and clays in the desiccation tolerance of cowpea bradyrhizobia. Soil Sci. Soc. Am. J. 50: 1193-1198.

Hartel, P., K. Gates, K. Payne, J. McDonald, K. Rodgers, J. Fisher, S. Hemmings, and L. Gentit. 2004. Targeted sampling of St. Andrews Park on Jekyll Island to determine sources of fecal contamination. Dept. of Natural Resources. 9 p.

Howell, J. M., M. S. Coyne, and P. L. Cornelius. 1996. Effect of sediment particle size and temperature on fecal bacteria mortality rates and the fecal coliform/fecal streptococci ratio. J. Environ. Qual. 25:1216-1220.

McDonald, J., J. Nelson, C. Belcher, K. Gates, and K. Austin. 2003. Georgia estuarine and littoral sampling study to investigate the relationship among three analytical methods used to determine the numbers of enterococci in coastal waters. DNR Report.

Robert, M., and C. Chenu. 1992. Interactions between soil minerals and microorganisms. P. 307-404. In G. Stotzky and J.-M. Bollag (ed.) Soil biochemistry. Vol. 7. Marcel Dekker, New York.

Stephenson, G.R., and R.C. Rychert. 1982. Bottom sediment: A reservoir of Escherichia coli in rangeland streams. J. Range Manage. 35:119-123.

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.