Chesapeake Bay Watershed Interdisciplinary Science Partnership

1. Chesapeake Bay Watershed Interdisciplinary Science Partnership University of Maryland The MdBio Foundation Anne Arundel Community College Howard Hughes Medical Institute

2. Project Goals • Understand the importance of scientific research in improving human health • Students will • Learn about watershed ecology, sources and health consequences of bacterial contamination, and factors that affect levels of bacterial contamination (e.g., patterns of land use, rainfall) • Design simple, hypothesis-driven investigations and will test their hypotheses with water samples they collect near their schools • Compare their results to those obtained by students state-wide via a project website

3. Water Cycle

4. Cholera • Severe Bacterial Infection • Targets Small Intestine • Profuse watery diarrhea (gallons per day) • Vomiting • Leads to dehydration and electrolyte loss • 20% of body weight in 24 hrs http://commons.wikimedia.org/wiki/File:Cholera_hospital_in_Dhaka.jpg

5. Battling an Ancient Scourge, With Satellites and Sari ClothPublished: July 6th, 2011 in Impacts, Climate, Oceans & Coasts, Health, Global, United States, Mid-Atlantic “The world has seen seven global cholera outbreaks since 1817, and the current one seems to have come to stay. Rising temperatures and a stubbornly persistent, toxic bacteria strain appear to have given the disease the upper hand.” By Doug Struck, DailyClimate.org

6. Vibriocholerae • Causative agent of cholera • Symptoms caused by bacterial toxin (CTX or cholera toxin) http://dhiez.files.wordpress.com/2008/05/cholera.jpg

7. Scanning Electron Micrograph of Vibrio cholerae O1

9. Cholera Kills in Developing Nations • Primarily passed in contaminated drinking water and shellfish • Harbored in zooplankton • outbreaks often follow zooplankton blooms • Water temperature Dependent • Last outbreak in US was in 1911 • Water chlorination, ozone, UV, or cloth filter and boiling June 23, 2010; Kenya http://www.nation.co.ke/News/regional/Crisis%20feared%20as%20cholera%20outbreak%20kills%2060/-/1070/945376/-/t2lpkm/-/

10. Cholera Outbreaks http://gamapserver.who.int/mapLibrary/Files/Maps/Global_CholeraCases0709_20091008.png

11. Cholera outbreak in Haiti • December 30, 2010 • First reported case: 2nd week of October, 2010 • As of December 30, 2010: reported hospitalized cases:70,865 • Reported Deaths: 2,761 deaths • Overall fatality rate of 2.1 per cent nationwide. • The fatality rates in South- East reached to 12-13% in early December. http://ochaonline.un.org/tabid/6412/language/en-US/Default.aspx

12. Climate Change and Health Climate can have a profound influence on health, both directly and indirectly Direct effects include deaths and illnesses related to excessive heat or cold exposure Indirect effects via waterborne vector borne diseases influenced by: occurrence and geographic distribution of known vectors, e.g., mosquitoes, rodents and copepods have been implicated with diseases, such as Malaria, Dengue fever Cholera Encephalitis & Haemorrhagic fever. Hanta virus

13. Susceptibility to Cholera • People with Type O are most susceptible, AB least susceptible • Heterozygous carriers of Cystic Fibrosis gene have some protection • Malnourishment or immunocompromized 1915 http://en.wikipedia.org/wiki/Cholera

14. A Model for Cholera Epidemic • Sunlight • Precipitation • Temperature • Nutrients Phytoplankton: micro & macro-algae Zooplankton & Animal larvae Disease Outbreak Increase of V. cholerae population Phage – another player? Infectious dose is now readily available

15. Infectious Dose of Bacteria that Can Cause Cholera (Cash et al, 1974) InoculumSymptom 10,000 with antacids Mild diarrhea 1,000,000 with food Severe diarrhea >1,000,000 with water Severe diarrhea

16. Vibriospp in the Chesapeake Bay • Vibrio’s infect cuts • “hand swollen to the size of a catchers mitt” • Infected shellfish cause GI illness • Public health websites suggest to protect yourself against infection: • Avoid swimming 48 hours after any heavy rainfall • Do not swim with an open cut or wound • If you get cut while in the water, wash it thoroughly and cover with a waterproof bandage • Try not to swallow water while swimming Chesapeake Bay Foundation. 2009. Bad Water 2009

17. Fecal Bacteria “Where do the bacteria come from? There are about 180 failing septic tanks in the Severn River’s suburbanized watershed, according to the Maryland Department of the Environment (MDE). A far more significant source of bacteria in the river is pet waste, which produces an estimated 69 percent of the E. coli bacteria in Voith’s section of the Severn River, with wildlife contributing 24 percent, livestock three percent, and humans three percent, according to an April 2008 MDE analysis of pollution in the Severn River. About 41 percent of dog owners in the area admit they do not pick up after their animals most of the time, the report says“ Chesapeake Bay Foundation. 2009. Bad Water 2009

18. Our GI Microflora • There are 10-100 times more microbes in your gut than cells in your body with your DNA Source: www.nature.com

19. This is a two-way street • Benefits the bacteria • We keep them warm • We protect them from the environment • We send food • Benefits Us • Bacteria help fight off pathogens • Bacteria help us digest our food • Bacteria produce useful products for us (e.g., Vitamins) • Bacteria also interact with our body systems to help us keep balanced

20. The Operative Word is BALANCE • When pathogenic bacteria invade us, we usually get sick • When good bacteria end up in the wrong place, we sometimes get sick • When bacteria get into improper balance (“dysbiosis”), we can get sick • When our immune system gets confused about the good guys, we can get sick http://commons.wikimedia.org/wiki/File:Balanced_scales.svg

21. One Member of the GI Microflora • Enterococcusfaecalis • Part of normal flora of all mammals and birds • About 10 million Enterococci per gram of human feces. • Gram-positive cocci, facultative anaerobe • Tolerate a wide range of growth conditions including salt and oxygen Enterococcusfaecalisinfecting lung tissue. Source: Wikipedia

22. Coliforms Coliform bacteria include genera that originate in feces (e.g. Escherichia) as well as genera not of fecal origin (e.g. Enterobacter, Klebsiella, Citrobacter). Basics of fecal coliforms In general, increased levels of fecal coliforms provide a warning of failure in water treatment, a break in the integrity of the distribution system, or possible contamination with pathogens. When levels are high there may be an elevated risk of waterborne gastroenteritis. Tests for the bacteria are cheap, reliable and rapid (1-day incubation). http://en.wikipedia.org/wiki/Fecal_coliform

23. Opportunistic pathogen • Can cause: • Bladder infections • Endocarditis (infection of heart lining) • Bacteremia (bacteria in blood) • Peritonitis (infection in abdominal cavity) • Meningitis (brain infection) • Most cases are hospital-acquired (“nosocomial”) infections • Hard to treat • Naturally antibiotic resistant to penicillins • Acquired resistance to many other antibiotics

24. E. Faecalis is a Good Indicator Organism in the Environment • Stays alive but doesn’t grow in environment • So… numbers stay constant • So…counts are representative of volume of pollution sources Scanning Electron Micrograph of Enterococcusfaecalis. Sources: CDC Public Health Image Library (PHIL), Photo by Janice Haney Carr http://phil.cdc.gov/Phil/details.asp

25. How might pathogens reach our water?

26. Our Activity • Step 1- Collect water samples • Step 2- Filter water samples and culture overnight • 2 different volumes • 10 ml • 100 ml • Allows for best opportunity to get a countable plate of 20-60 colonies http://www.umesc.usgs.gov/aquatic/drug_research/capabilities.html

27. Our Activity • Step 3- (Next Day) Count Colonies Example of bacterial growth on selective media. Photo courtesy of Hornor Lab, Anne Arundel Community College, Arnold, MD.

28. Equipment Setup • Completely assembled filtration apparatus • Water samples in ice bucket • Field data sheet • Sterile 10 ml syringe • Beaker with ethanol holding forceps • Sterile paper filter

29. Sterile Technique • Forceps removed from ethanol, flamed • THEN handed to students

30. Place Filter 1 • Peel cover off filter (best done by instructor or partner) • Grab edge with sterilized forceps

31. Place Filter 2 • Place paper filter grid side up on top of metal screen • Paper must completely cover screen to get proper filtration

32. Reassemble Filtration Apparatus • Place filter funnel on top of paper filter • Clamp glassware in place

33. 10 ml Sample • Wet filter with 10 ml sterile, distilled water • Water removes static from syringe • When the water has suctioned through filter, apply 10 ml of water sample to filter

34. Wash Filter Funnel • With clean syringe, wash the sides of the funnel to get any splashes

35. Remove Filter • Unclamp filter funnel • Flame forceps • Grab edge of filter and break vacuum seal

36. Place on Plate • Hold plate tilted downward and away • Place filter at bottom edge of plate • Roll onto media to minimize bubbles • Cover and incubate 24 hrs

37. Repeat for 100 ml • Place new filter on filtration apparatus • Wet filter and suction through • Pour 100 ml into funnel • Wash sides of funnel • Place filter on media

38. After Incubation • This is what the students will see after a 24 hour incubation at 41˚C (chicken body temperature) • Left-hand plates came from Patuxent River • Right-hand plates came from Warehouse Creek off South River • Top plates are 10 ml, bottom plates are 100ml samples

39. What issues affect the Chesapeake Bay?

40. Chesapeake Bay Foundation • http://www.youtube.com/watch?feature=player_embedded&v=o7kB7-UN7m4

41. Antibiotics--Are You Drinking Them in Your Tap Water?