CHESAPEAKE BAY WATERSHED INTERDISCIPLINARY SCIENCE

PARTNERSHIP

University of MarylandThe MdBio Foundation

Anne Arundel Community CollegeHoward Hughes Medical Institute

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

Water Cycle

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

Battling an Ancient Scourge, With Satellites and Sari Cloth

Published: 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

Vibrio cholerae Causative agent of

cholera Symptoms caused

by bacterial toxin (CTX or cholera toxin)

http://dhiez.files.wordpress.com/2008/05/cholera.jpg

Scanning Electron Micrograph of Vibrio cholerae O1

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/-/

Cholera Outbreaks

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

 Cholera outbreak in HaitiDecember 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

Climate Change and HealthClimate can have a profound influence on health, both

directly and indirectly  Direct effects include deaths and illnesses related to excessive heat or cold exposureIndirect 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 feverCholeraEncephalitis &Haemorrhagic fever.Hanta virus

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

http://en.wikipedia.org/wiki/Cholera

1915

A Model for Cholera Epidemic

SunlightPrecipitationTemperatureNutrients

Zooplankton & Animal

larvae

Increase of V. cholerae population

Infectious dose is now readily available

Phytoplankton: micro & macro-algae

Disease Outbreak

Phage – another player?

Infectious Dose of Bacteria that Can Cause Cholera

(Cash et al, 1974)

Inoculum Symptom10,000 with antacids Mild

diarrhea1,000,000 with food Severe

diarrhea>1,000,000 with water Severe

diarrhea

Vibrio spp 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

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

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

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

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

One Member of the GI Microflora

Enterococcus faecalis 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

Enterococcus faecalis infecting lung tissue. Source: Wikipedia

ColiformsColiform 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 coliformsIn 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

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

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

Enterococcus faecalis. Sources: CDC Public Health Image Library (PHIL), Photo by Janice Haney Carrhttp://phil.cdc.gov/Phil/details.asp

How might pathogens reach our water?

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

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.

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

Sterile Technique Forceps removed

from ethanol, flamed

THEN handed to students

Place Filter 1 Peel cover off filter

(best done by instructor or partner)

Grab edge with sterilized forceps

Place Filter 2 Place paper filter

grid side up on top of metal screen

Paper must completely cover screen to get proper filtration

Reassemble Filtration Apparatus

Place filter funnel on top of paper filter

Clamp glassware in place

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

Wash Filter Funnel With clean syringe,

wash the sides of the funnel to get any splashes

Remove Filter Unclamp filter

funnel Flame forceps Grab edge of filter

and break vacuum seal

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

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

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

What issues affect the Chesapeake Bay?

Chesapeake Bay Foundation

http://www.youtube.com/watch?feature=player_embedded&v=o7kB7-UN7m4

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