TESTING THE WATER

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TESTING THE WATER Molecular Tools for Microbial
Assays
Joan B. Rose Homer Nowlin Endowed Chair for
Water Research
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Microbial Risks
PLAGUES
CONTAGION
OUTBREAKS
EPIDEMICS
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• The first recorded account of a pestilence or
  plague as it was often referred to, was in
  approximately 3180 BC in Egypts First Dynasty.
• epidemic fevers which was written in a papyrus
  ca. 1500 B.C. discovered in a tomb in Thebes,
  Egypt.
• Plagues were described and in particular
  associated with the decimation of the Greek Army
  near the end of the Trojan War (ca. 1190 B.C.)
  and with massive epidemics in Roman history in
  790, 710 and 640 B.C

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Fecal contamination of water remains the largest
threat to the biological safety of water today.
Cholera, an ancient disease still ravages the
developing world. Yet new biological risks have
begun to emerge in the developed world as well.
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SAFE WATERPathogen DiscoveryAdvances in
MicrobiologyAdvances in MedicineAdvances in
Engineering

• 1500-1700s advances first in microbiology lead
  the way for discoveries in medicine which
  solidified the idea of bacteria and led to the
  germ theory, pathogen discovery and the
  understanding of disease transmission.

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• Medicine Science Engineering
• 3180 B.C. First recorded epidemic in Egypt
• 2000 B.C. Boiling and sunlight radiation for
  drinking water
• 430 B.C. Plague of Athens
• 580 Wealth responsible for a dysentery
  epidemics
• 1403 Quarantine established
• 1546 "seminaria" cause infection and epidemics.
• 1590 Microscope invented
• 1676 Microscopic observation of bacteria
• 1773 First description of bacteria
• 1783 Ozone identified
• 1800 Chlorine kills germs
• 1801 UV discovered
• 1849 Waterborne transmission of cholera
• 1854 John Snow removed handle from water pump
• 1856 Fecal-oral transmission of typhoid fever
• 1859 Chlorination of sewage
• 1876 Germ theory by Robert Koch
• 1881 Culture plate technique

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Types of Waterborne Pathogens
Viruses Bacteria Parasites
THE DISEASES diarrhea, respiratory illness,
liver damage, kidney failure, heart disease,
cancer, nervous system disorders birth defects,
death.
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Emerging Microbial Risks in Wastewater

• Enteric pathogens are shed in large
  concentrations by infected population.
• Includes bacteria, parasites and viruses.
• Less than 1 have been cultivated and studied.

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WATERBORNE PATHOGENS THE CHANGING FOREGROUND
VIRAL DIARRHEAS
OTHER BACTERIAL INFECTIONS
CHOLERA
TYPHOID
DYSENTARY
PROTOZOAL DIARRHEAS
HEPATITIS A
HEPATITIS E
Slide Courtesy of DR. SYED SATTAR
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THE CHANGING FOREGROUND (contd..)
CHOLERA
TYPHOID
VIRAL DIARRHEAS
ENVIRON. MYCOBACTERIA
TOXOPLASMA
DYSENTERY
CRYPTOSPORIDIUM
HEPATITIS A
Coxsackie viruses and Noroviruses
Slide Courtesy of DR. SYED SATTAR
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Monitoring Tools, Approaches and Applications

• Watershed assessment association with
  environmental variables.
• Source Tracking and Direct Pathogen monitoring.
• Molecular monitoring approaches, PCR, microarrays
  and NASBA.
• WHAT CONTAMINANTS?
• WHAT SOURCES?
• WHAT RISK?
• WHO IS RESPONSIBLE?
• HOW DO WE FIX THE PROBLEM?

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Advanced Technologies for Water Quality and
Health. Direct Pathogen monitoring Source
Tracking Environmental Transport Health Risk
Genosensor
The REAL-TIME PCR
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Pathogen monitoring
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Why Worry About Cryptosporidium?

• Globally one of the most important pathogens in
  Water
• Crosses species barriers
• Zoonotic
• Animal and human fecal waste
• A number of transmission routes
• Resistant to routine water disinfectants
• Drinking water
• Waste water
• Cryptosporidiosis is not treatable
• High mortality in immunocompromised

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Life Cycle of Cryptosporidium
Host ingests oocyst
Animal reservoir
Obligate intracellular parasite infects humans
and animals
Crypto hominus restrictive to human to human
transfer
Crypto parvum
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Strategy for detection of waterborne
Cryptosporidium
Environmental water sample
Method 1623
50 IMS
25 IMS
25 IMS
IFA
PCR
CC
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RESULTS in WASTEWATER
Sample IFA Oocysts/100 L PCR
Result Species/Genotype 1
4 8 - - 2
107 209 C. parvum
genotype 2 3 5 61
- - 4 5 10
- - 5 83 162
C. parvum genotype 2 6
151 319 C. parvum
genotypes 1 and 2 7 26
55 - - 8 3
6 - - 9 2
8 - - 10 40
120 C. parvum genotypes 1 and
2 11 26 53
C. parvum genotypes 1 and 2 12 26
104 C. parvum genotype
2
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Crypto FARMS off site impacts on Water Quality

• Cryptosporidium was found in 11 surface water
  sites near Concentrated Animal Feeding Operations
  farms which may have been the source of the
  oocysts. The site with the highest detected
  level of Cryptosporidium was at the tile that
  drains into Rice Lake Drain with levels as high
  as 5990 oocysts per 10L. Giardia was detected at
  8 of the surface water sites. Viable and
  infectious oocysts were also detected. High
  levels of E.coli bacteria were reported as well.
• C. Andersonii C. parvum were most frequently
  identified genetically, but C. hominus was also
  found.

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• EMERGING PATHOGENS ASSOCIATED WITH CANCER AND
  SEVERE HEALTH CONSEQUENCES
• The WHO has classified H. pylori as a Class I
  carcinogen because of the association of H.
  pylori and gastric malignancies.
• About 50 of the U.S. populations are thought to
  be symptomatic or asymptomatic carriers, even
  though the source of human infection is not well
  understood.
• German group ,Rolle-Kampczyk et al. ( 2004)
  found a significant correlation between well
  water contaminated with H. pylori detection by
  PCR and colonization status in humans.
• Water supplies contaminated with fecal material
  may be a potential source of H. pylori
  transmission (Hulten et al., 1996).

Helicobacter pylori in Michigan Sewage
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Adenoviruses respiratory and enteric disease
Coxsackie viruses caus mycarditis. Cancer
causing viruses Polyomaviruses in Wastewater.
VIRUSES ARE BIO NANO PARTICLES
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MICROARRAY TECHNOLOGY FOR VIRUSES AND
PATHOGENDISCOVERYWhat is a microarray?

• Chip platform with synthesized genetic sequences
• Hybridization detection
• Multiple pathogens

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• Unique 3-D nano-reactors
• Size 2 cm x 1.8 cm
• Current capacity 10,800
• Future capacity 30,000

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Microfluidic Microarray Chip showing the 3D
chambers In which probes capture target sequences
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Virus Discovery from SEWAGE using Microarrays
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Microbial Source Tracking

• Tools are now available to determine the
  molecular fingerprint of the fecal pollution.
• Health risks
• Remediation
• Prioritization
• Responsibility

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Microbial Source Tracking (MST)

• Indicator bacteria doesnt provide source of
  pollution
• Track sources of fecal contamination in water
• MST can be library dependent
• or library independent

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Culture vs nonculture

• Culture dependent methods target viable organisms
  only
• Non- culture methods target both viable and non
  viable cells

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Host specific

• Host specific method is library independent
• For Library-dependent, DNA libraries are built
  using isolates from animals human sources in
  the area
• Using these libraries to match to the unknown
  environmental sample
• Host specific method requires no library, the
  marker is specific to the host

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Esp marker

• Host specific genetic marker
• Differentiate Human/non human pollution
• Present in Indicator bacteria (Enterococci)
• Traditional MF method and PCR used to detect the
  gene
• A culture dependent library independent method

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Human Pollution Marker Validation
Source esp marker
Sewage 55/55
Septic tank 8/10
Poultry 0/6
Swine 0/9
Cattle 0/32
Canada Goose 0/12
Seagull 0/28
Pelican 0/7
Wild Birds 0/8
Scott et al., 2005. Env. Sci. Technol., 39
283-287
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Year long assessment of Untreated Sewage
Membranes assayed Cfu/plate (range) Esp ( membranes positive)
11 0-30 2 18
17 31-50 5 29
26 51-70 13 50
18 71-90 12 66
11 91-110 4 36
14 gt110 13 93
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Effluent samples
Membranes assayed Cfu range Results Esp ()
5 0-17 -- 0
6 10-16 -- 0
9 1-21 -- 0
4 68-100 100
4 54-139 -- --
6 gt100 67
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Enterococci Source Tracking

• Preliminary ResultsCan separate out human fecal
  bacteria with 99.9 certainty.

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THE WORLDS WATERA GLOBAL CHALLENGE

• The United Nations warns that 80 percent of
  diseases in developing countries result from
  unsafe water.
• More people die from unsafe water annually than
  from all forms of violence, including war.
• According to the WHO, the water consumed by more
  than one billion people one out of five on
  Earth is unsafe, potentially leading to disease
  and death.

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Cholera and Typhoid

• These diseases
• continue to ravage populations and communities.
• The current disease burden of typhoid is
  estimated at 17 million per year and 600,000
  deaths (WHO http//www.who.int/vaccine_research/d
  iseases/typhoid/en/)
• while cholera is reportable and the numbers range
  from 100,000 to 250,000 cases per year with about
  2000-3000 deaths (WHO, http//www.who.int/wer/2005
  /wer8031.pdf).

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Use of Enterococci ESP sewage marker to track
source and impact
More than 500 sewage treatment systems in the
Gulf Coast region battered by Hurricane Katrina
have been rendered inoperable, damaged or at
reduced service, including 25 large and 35
intermediate-sized facilities.
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Impact Assessment
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Wells and reservoirs
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THE U.S. WATER CHALLENGEs

• Climate Variability floods and droughts.
  Prediction of Global Warming, sea level, changes
  in precipitation.
• Infrastructure EPA Gap report suggests 52
  billion dollars needed in the next decade.
• New Technology Detection Technology New
  Contaminants at Lower levels. Treatment
  Technology, Membranes and UV disinfection.
• Sensitive Populations Immunocompromised and
  Elderly, 30 of our communities.
• Home Land Security Water Vulnerability.

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Great Lakes

• Over 30 million people
• 10,210 miles of shore line
• 43 Areas of Concern
• 26 in the U.S.
• 13 AOCs in Michigan

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Water Quality Data
Hazard identification



Exposure estimation
E.coli Enteroocci Coliphage levels
Sewage and


Source Tracking
Fecal Loading
Prevention Treatment Strategies
Environmental

Parasite tesing
Survival

Transport Runoff
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Virus testing

Surface Water/ Ground Water Concentrations
Risk Estimation

Dose-response
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Water Ethics Data Access Communication Educati
on Training Networks Safety goals Sensitive
populations Shared Responsibility
Hydrogeological Setting/Land use Patterns
C L I M A T e I M P A C T s
M O N I T O R I N G
HAZ ID Transport Fate Models Exposure RISK
Prevention Early Warning Response Recovery
Watershed to the Tap HACCP WSP Decision-Support
Systems
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The Molecular Future for Water Quality

• Molecular methods provide the best approach to
  meet the future assurance of safe water for the
  future.
• A microbial waterborne genomics program for
  pathogen discovery and assessment should be
  supported
• Virulence-Factor Activity Relationships (VFAR),
  Pathogen loading, identification (species,
  genotypes), toxin production, source tracking
  will then move forward
• PCR techniques and Gene Chip Micro Arrays will
  help to address water concerns

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Risk

• Risk assessment and management

Microbial detection
Environmental
Water Safety

• Target organisms
• Genetic variation
• Detection technologies

• Surface water, groundwater,
• Transport from Source to the Beach Modeling
  Fate

Treatment Needs

• Determine how much treatment and what targets.

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Thank You QUESTIONS?