MICROBIOLOGY RESEARCH AT SOUTHERN CALIFORNIA COASTAL WATER RESEARCH PROJECT (SCCWRP)

1. MICROBIOLOGY RESEARCH ATSOUTHERN CALIFORNIA COASTAL WATER RESEARCH PROJECT(SCCWRP) Stephen B. WeisbergExecutive Director

2. WHAT IS SCCWRP? • Joint Powers Agency founded in 1969 • Initiated to address regional monitoring and research needs • Cumulative regional assessments • Methods development • Data integration • Member organizations include city, county, state, and federal agencies • Unique combination of regulators and regulated

3. MEMBER ORGANIZATIONS • City of San Diego • City of Los Angeles • Ventura County Flood Control District • Los Angeles County Department of Public Works • Los Angeles County Sanitation Districts • Orange County Sanitation District • San Diego Regional Water Quality Board • Santa Ana Regional Water Quality Board • Los Angeles Regional Water Quality Board • State Water Resources Control Board • U.S Environmental Protection Agency

4. SOME DIFFICULTIES WITH BACTERIAL MONITORING SYSTEMS • Laboratory processing is slow • You should not have been swimming yesterday • Relationship to health risk is not well established • Santa Monica Bay Epidemiology study is the only one to evaluate urban runoff • Poor relationship between bacteria and pathogens • Hard to fix if we don’t know the source • Difficult to define appropriate clean-up levels • Background is not zero

5. SOME DIFFICULTIES WITH BACTERIAL MONITORING SYSTEMS • Laboratory processing is slow • You should not have been swimming yesterday • Relationship to health risk is not well established • Santa Monica Bay Epidemiology study is the only one to evaluate urban runoff • Poor relationship between bacteria and pathogens • Hard to fix problems if we don’t know the source • Difficult to define appropriate clean-up levels • Background is not zero

6. SCCWRP’s MICROBIOLOGICAL RESEARCH • Rapid microbiological measurement methods development • Mission Bay epidemiology study • Microbiological source tracking evaluation • Natural sources loading

7. NEED FOR RAPID MEASUREMENT METHODS • Present methods are slow • Culture-based • Take 24-96 hours • Slow speed compromises the warning system • Exposure occurs during sample processing • Most events last less than 24 hours • Slow speed also compromises upstream tracking • Need a “Geiger counter”

8. Surface Proteins Small molecules ATP Nucleic Acids Molecules released into medium Things to Measure in a Bacterium

9. CONCENTRATION TECHNIQUES: IMMUNOMAGNETIC SEPARATION (IMS) Magnetic Beads Antibody Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y = Magnetic Bead Y = Antibody

10. MIX BEADS WITH ENVIRONMENTAL SAMPLE Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y silica Clay Algae Bacterium Clay Clay Algae

11. EXPOSE MIXTURE TO MAGNET SYSTEM FOR SEPARATION (E.G. MILTENYI, IMMUNICON OR DYNAL) Add Buffer & Dye Place tube in field Collect Bacteria 30 min. Bacteria move to tube walls Remove Non-magnetic Debris S N N S

12. FLOW CYTOMETRY: OPTICS Labeled bacterium Scatter Detector Scatter signal Fluorescence Detector Laser excitation Fluorescence signal High performance filters

13. “Near Real Time Microbial Analysis”

14. APPROACH • Adapt technology from other industries • Drinking water • Hospital testing • Food service • Counter – bioterrorism • Partner with specialists in these areas • Held a workshop in Monterey in May • Shared developments to date • Defined method evaluation protocols • Will conduct blind testing this summer

15. MISSION BAY EPIDEMIOLOGY STUDY • Is there a health risk of swimming in Mission Bay? • Comparison of swimmers and non-swimmers • Can we relate to bacterial indicator concentration? • Comparison among swimmers at different times and locations • Can we relate health risk to non-traditional microbiological indicators? • Virus • Phage • Bacteroides

16. SAMPLING DESIGN • Six beaches • 32 sampling days • Between 2 and 5 sampling sites per beach • Dependent on beach length and swimmer density • Sampled hourly from 12:30 to 3:30 • Total and fecal coliforms by MF • Enterococcus by Idexx • Single beach composite at 12:30 • Phage, virus, Bacteroides • Total and fecal by Idexx • Enterococcus by MF

17. ENROLLMENT – ACTUAL VS. GOAL

18. DEMOGRAPHICS OF STUDY POPULATIONRACE AND GENDER

19. PRIOR LARGE RECREATIONAL WATER STUDIES

20. SCCWRP’s MICROBIOLOGICAL RESEARCH • Rapid microbiological measurement methods development • Mission Bay epidemiology study • Microbiological source tracking evaluation • Natural sources loading

21. MICROBIOLOGICAL SOURCE TRACKING • MST tools potentially allow managers to discriminate between human and non-human sources • Some even discriminate among non-human sources • There are more than a dozen proposed MST methods • Most have had limited testing • No marine testing • Which methods are most cost-effective? • How reliable are the results?

22. STUDY DESIGN • Characterize five sources • Human (direct samples) • Human (sewage influent) • Dog • Cow • Seagull • Place these sources in combination into “blind” water matrix samples • Three matrices • Freshwater • Saltwater • Freshwater with humic acid addition

23. EXAMPLE TEST MATRIX

26. STUDY CONCLUSIONS • No method predicted source material perfectly • Genetic techniques did best • Identified dominant sources in about 75% of samples • Phenotypic techniques produced many false positives • Host-specific PCR showed the most promise • Not yet quantitative • Only developed for two sources so far • Viral measures accurately identified sewage, but did not identify individual human sources

27. NATURAL LOADING PROJECT • Determine properties of waterbodies in undeveloped watersheds • Measure a wide array of constituents • Bacteria • Metals • Nutrients • Provide perspective for various activities • 303D listing • Model calibration • TMDL development

28. SAMPLING DESIGN • 12 sampling sites • Stratified based on geology and land cover • Dry season sampling • Twice per year (fall and spring) • Two years (2004-2005) • Wet season sampling • Two – three storms at each site • Multiple samples over duration of the storm • Sampled burned vs. unburned catchments twice this winter

29. Questions or Comments? Steve Weisberg – Southern Ca. Coastal Water Research Project 714-372-9203 stevew@sccwrp.org

30. SOME TOUGH ISSUES • Measuring low concentrations • Many detectors are based on 1 ml sample • State standard is 104 bacteria per 100 ml • Dead or alive? • Do our standards apply when measuring fragments? • Intercalibration tests are necessary • How do we use real-time information in a patchy environment? • Rapid processing allows adaptive sampling • Lesser processing cost allows more samples • Capital equipment and training costs • Many technologies require up-front investment • Some require specialized training • Per sample cost is typically lower

31. REPORTED ILLNESSES

32. COMPARISON TO THRESHOLDS

33. POTENTIAL TOOLSNATURAL SOURCES IDENTIFICATION • Microbial source tracking – bacteria • Iron-normalization – metals • Chiral chemistry – organics • Compound specific istotope analysis - organics

34. IRON NORMALIZATION

35. CHIRAL ENVIRONMENTAL CHEMISTRYENANTIOMERS: MIRROR IMAGES The Ultimate in Pollutant Speciation R-(-) o,p-DDT S-(+) o,p-DDT

36. COMPOUND SPECIFIC ISOTOPE ANALYSIS • Elements occur in various isotope ratios (e.g. 12C/13C) • IR’s are affected by many natural and anthropogenic processes • Can be extremely effective for source identification

37. OUR APPROACH • Held a workshop jointly with USEPA to develop method evaluation protocols • Defined four phases of testing • Sequentially increasing level of complexity • Conducted an evaluation test last year • 22 research labs • Tested 12 different MST methods

38. EXAMPLE MST METHODS • Phenotypic • Antibiotic resistance profiling • Carbon source utilization • Genotypic • Ribotyping • Pulsed field gel electrophoresis • Polymerase chain reaction • Library-independent genotypic • Terminal restriction fragment length polymorphism • Host-specific PCR • Toxin genes • Direct pathogen measurement • Adenovirus • Enterovirus • Phage

39. EVALUATION CRITERIA • Distinguish presence/absence of human source • Correctly identify all sources • Accurately determine relative contribution from each source • Stability across matrices