1. Yeggie Dearborn, PhD Cel Analytical Inc. October 29, 2008
Classical Techniques and New Targets in Water Quality Monitoring for Pathogens
Yeggie Dearborn, PhD
Cel Analytical Inc.
October 29, 2008

2. Waterborne Pathogens Pathogens are organisms that cause disease.
Waterborne pathogens are most commonly organisms such as bacteria, viruses or parasites found in feces of humans and animals.
Waterborne pathogens may be found in drinking water, groundwater, rivers, lakes, streams, and oceans.

3. Sources of Drinking Water
Drinking water comes from surface water and ground water.
Large-scale water supply systems tend to rely on surface water resources,
smaller water systems tend to use ground water, (slightly more than half of the population receives its drinking water from ground water sources).
Surface water includes rivers, lakes, and reservoirs.
Ground water is pumped from wells that are drilled into aquifers.
Aquifers are geologic formations that contain water. The quantity of water in an aquifer and the water produced by a well depend on the nature of the rock, sand, or soil in the aquifer where the well withdraws water. Drinking water wells may be shallow (50 feet or less) or deep (more than 1,000 feet).
Water utility or Public works department can tell you the source of your public drinking water supply- is Hetch-Hetchy

4. Exposure To Waterborne Pathogens
There are several ways a person might be exposed to a waterborne pathogen:
• Drinking contaminated water
• Direct physical contact with the pathogen such as swimming in contaminated water at a beach
• Inhalation or breathing in a pathogen contained in water vapor

5. How to make water safe?
Water suppliers use a variety of treatment processes to remove contaminants from drinking water.
The most commonly used processes include: filtration, flocculation and sedimentation, and disinfection.
Some treatment also include ion exchange and adsorption.
A typical water treatment plant would have only the combination of processes needed to treat the contaminants in the source water used by the facility.

6. USA Water Regulations
Equipment
Vendors
Consulting
Engineers
Local
Utilities
The States
With Primacy
USA Federal
The EPA
Clean Water Act (1977) is a 1977 amendment to the Federal Water Pollution Control Act of 1972, which set the basic structure for regulating discharges of pollutants to waters of the United States.
Safe Drinking Water Act (1974)
Under SDWA, EPA sets standards for drinking water quality and oversees the states, localities, and water suppliers who implement those standards. The standards for drinking water which fall into two categories -- Primary Standards and Secondary Standards. Primary Standards are based on health considerations, and are designed to protect people from three classes of toxic pollutants: pathogens, radioactive elements and toxic chemicals.
Certified Laboratories
Laboratory Certification:
Another area in which some states have important responsibilities that complement federal regulation is the certification of testing laboratories. As with any food establishment, the states perform unannounced plant inspections, and some states perform annual inspections.
Only certified Lab can perform EPA or Public Health Approved Test Methods

7. Eradicated Waterborne Diseases
Advances in Drinking Water filtration and disinfection coupled with stringent water quality monitoring have largely eliminated many deadly waterborne diseases of the past century.
1. Cholera (essentially gone since1960), V. cholerae (Bacteria)
2. Typhoid fever (few cases since1971), Salmonella typhi (Bacteria)
3. Hepatitis A (only 4% of cases since 1971), Virus

8. Waterborne Diseases by Pathogens “Emerging Concerns”
Newly identified waterborne pathogens of emerging concern : • Norwalk-like virus • Rotavirus • Yersinia- bacteria • Campylobacter - bacteria • E. coli 0157:H7 E. coli-bacteria • Cyclospora/Cryptosporidum/Giardia protozoa (cyst) • Aeromonas - bacteria

9. "Indicator" Organisms Classical Approach
Water pollution caused by fecal contamination is a serious problem due to the potential for contracting diseases from pathogens.
The presence of pathogens is determined with indirect evidence by testing for an "indicator" organism possessing the following criteria:
Come from the same sources as pathogenic organisms.
Relatively easy to identify,
Usually present in larger numbers than more dangerous pathogens,
Respond to the environment, wastewater treatment, and water treatment similarly to many pathogens

10. Indicator Organisms: Bacteria
Total coliforms include large group of bacteria that are found in the soil, in water that has been influenced by surface water, and in human or animal waste.
Most basic indicator of the sanitary condition of a water supply.
Escherichia coli (E. coli) is generally not found growing and reproducing in the environment (only animal and human fecal matter). Consequently, E. coli is considered to be the species of coliform bacteria that is the best indicator of fecal pollution and the possible presence of pathogens.
Test Methods include:
-membrance filteration techniques on selective media
-Multiple tube fermentation-culturing techniques on selective media
Regulatory Limits in drinking water= 0 CFU/ml
Treatment Technology: Chlorination/UV

11. Indicator Organisms: Bacteria
Enterococci /Streptococci
In 2004, Enterococcus sp. took the place of fecal coliform as the new federal standard for water quality at public beaches.
It is believed to provide a higher correlation than fecal coliform with many of the human pathogens often found in city sewage.
Test method: membrane filtration techniques on selective media
Regulatory limits: Geometrical Mean 35 CFU/100 ml (minimum if 5 samples over 30 days)

12. Indicator Organism Bacteria
Aeromonas morphologically resembles Coliform Bacteria (E.coli & Salmonella). Fourteen species of Aeromonas have been described, most of which have been associated with human diseases. The most important pathogens are A. hydrophila, , and A. veronii biovar sobria.
Aeromonas occur naturally in freshwaters. Such waters are frequently used as raw sources for treated drinking water
Aeromonas may be found in non-chlorinated or low-flow parts of chlorinated water distribution systems. And in some cases associated with particles in raw water that evades treatment and enter distribution system.
Once in the system the bacteria may find environments conducive to their survival, especially when temperature and nutrient levels are elevated and when chlorine residuals are low (e.g. in the remote parts of the distribution system and in biofilm material on the inside of pipes).
“Agent of traveler’s diarrhea”
Test: Membrane filtration techniques using selective media
Treatment: Chlorination/UV
Unregulated contaminant under EPA Primary Drinking Water Contaminant

13. Indicator Organism Heterotrophic Bacteria
HPC tests are simple culture-based methods designed to recover a wide variety of organisms (selected members of the culturable population).
Unlike E. coli and total coliforms, it is not feasible or necessary to reduce the level of heterotrophic organisms to zero in drinking water.
The 100 bacteria/ml -standard in many European countries
the USA and Canada adopted a 500 bacteria/ml guideline.
Although the level of heterotrophic bacteria in drinking water is not related to contamination by pathogens, it is still present in most national laws on drinking water quality as an indicator of the
overall quality of the water (van der Kooij, 1993; Anon, 1999).
Simplate Method
(UV detection)
Pour Plate Method

14. Limitation of Classic Methods for Indicators of Water Quality Pathogens
Most of the indicator applications described in previous Slides rely on biological measurements of bacteria.
The classical laboratory techniques at best take 24-48hrs for processing. Therefore no real-time data
Underestimate the true concentrations of viable and potentially infectious organisms
Pathogens not amenable to culture methods are not detected

15. Pathogens Missed by Bacterial Indicators: “Parasites”
Giardiasis caused by the flagellate protozoan Giardia lamblia - The cyst stage is the "life stage" that will be found in the environment and is responsible for the spread of the organism. Giardia cysts are elliptically shaped and range in size from 6 to 10 microns. the structure of the cyst makes the organism very resistant to environmental factors and disinfection.
After viable oocysts are ingested, there is an incubation period of 2 to 12 days. Typically the average incubation period is 7 days. The primary symptoms include: diarrhea, abdominal cramps, headaches, nausea, vomiting, and low-grade fever, which can lead to weight loss and dehydration.
There are some people that have Giardia in their system, but do not get ill.  These individuals are asymmptomatic and could act as carriers or host and spread the disease or infect the other members of the household or family.  This why all members of the household should be tested.
Recent changes in the EPA Drinking Water regulations and the development of the SOURCE Water Protection strategies have required water suppliers using springs or groundwater sources to evaluate drinking water sources for the presence of trophozoite).

16. Pathogens Missed by Bacterial Indicators: “Parasites”
Cryptosporidium is a protozoan organism which causes the parasitic infection, cryptosporidiosis. It exists in either the free-swimming (trophozoite) form or the oocyst (dormant) form.
Cryptosporidium parvum is now recognized as a human pathogen which can cause severe diarrheal illness.
Ingestion of drinking water contaminated with viable Cryptosporidium oocysts, the environmentally resistant form of the organism, is the major mode of transmission.
Cryptosporidium is introduced into the water supply via animal excreta containing oocysts. Important reservoirs of the oocysts include cattle, sheep and pigs. Cryptosporidium is also found in wild animal populations.
Development of Cryptosporidium occurs more rapidly than many textbooks imply, and each generation can develop and mature in as little as hours. Due to the rapidity of the life cycle, plus the autoinfective cycles, huge numbers of organisms can colonize the intestinal tract in several days.  

17. Pathogens Missed by Bacterial Indicators: “Viruses”
Norovirus is the new official name for a group of viruses described as “Norwalk-like viruses” (NLV) RNA-Virus . Noroviruses cause the “stomach flu,” or gastroenteritis, and have been associated with recent outbreaks on cruise ships and in communities, camps, schools, institutions, and families. Accounts for 90% of epidemic non-bacterial outbreaks of gastroenteritis around the world.
Infection is usually spread from person to person.  Eating contaminated food is the most common and efficient means of transmission, but noroviruses can also be transmitted through water and by direct person-to-person contact.
Noroviruses are found in every part of the United States and throughout the world.  Noroviruses may be found in water sources such as private wells that have been contaminated with the feces from infected humans.  Waste can enter the water through various ways, such as sewage overflows or sewage systems that are not working properly.

18. Emerging Technologies for Indicator Organisms
“The advent of increasingly sophisticated and powerful molecular biology techniques provide new opportunities and alternative approaches to improve upon present indicators and pathogens by both culture and non-culture methods.”
Quote and Images from:
Indicators for Waterborne Pathogens
Author:Committee on Indicators for Waterborne Pathogens,
National Research CouncilPublisher:The National Academies Press
Date:2004Copyright © 2004, National Academy of Sciences

19. Nucleic Acid
DNA/RNA: microbial identification, viability, and infectivity or culturability.
-Broad identification of a family or genus of microorganism,
specific identification of species, strain, or subtype.(Genome projects)
-messenger RNA (mRNA) of cellular microbes or mRNA production by viruses in infected cells, can be used as measure of viability or infectivity.
Methods of Nucleic acid amplifications such as qPCR, RT-PCR alone or in combination hybridization can even provides two levels of discrimination.
Fluorescent In situ Hybridization Assay (Fish) are emerging in Environmental monitoring.

20. Nucleic Acid Application
Examples of applications currently in use:
Norovirus (norovirus RNA) in Shellfish (clams and oysters) by RT-PCR (reverse transcription polymerase chain reaction) methods are being employed by governmental laboratories such as the FDA in the USA. This tells us a lot about shell fish growing water as well!
Nucleic acid detection methods such as PCR is coupled with microbial culture methods. Initial amplification of the microbes through culturing is then followed by methods to detect their nucleic acids. This has been done successfully for enteric viruses to overcome problems of detecting inactive virus and is referred to as integrated cell culture-PCR
More recently, real time detection of Enterococci in Recreational water following beach closures
In the works: Detection of organisms not amenable to culture methods

21. Immunological Methods
Surface proteins on bacteria, protozoa, and viruses can be unique to the microbe and detected by immunological methods of analysis.
An antibody (polyclonal or monoclonal) is raised against
- the microorganism
- the purified (surface) protein
the enzyme linked immunosorbent assay (ELISA) in which a capture antibody bound to a surface is used to bind and concentrate the analyte. A second antibody, labeled with an enzyme, is then bound to a second recognition site on the analyte. Finally, a chromogenic or fluorogenic substrate is added that is converted to an observable product, which can be detected.
Immunomagnetic separation This method can be employed as both a processing (recovery, concentration, and purification) method and a detection method (or at least part of a detection method). In some applications, reaction of the target microbe antigen with the antibody on the bead is a sufficient basis for detection using an electronic sensor.
Flow cytometry: microbial cells are labeled with a fluorescently-labeled antibody. Multiple antibodies are employed, with each antibody specific to a particular microorganism. The labeled mixture is then passed through the flow cytometer and the numbers of each microorganism based on the occurrence of each label is determined.

22. Examples of Antibody-based detection methods currently in use
Detection of Giardia in Drinking Water
The procedure used for Giardia is known as the immunofluorescent antibody method. The procedure requires that a minimum of 100 to 1000 gallons of water is filtered through a 1 micron filter. The filter is then cleaned and the material collected and centrifuged. The centrifuged pellet is then floated using a Percoll - Sucrose density gradient technique. The floated material is then cleaned and centrifuge and the pellet is then stained with a fluorescent antibody. 
Slides of the pellet are prepared and the slides are examined for the cysts. If a cyst is suspected, the slide is then examined using to confirm the proper shape, size.
Source: EPA Website

23. Examples of Antibody-based detection methods currently in use
Testing procedures are available for detecting Cryptosporidium oocysts in both raw and treated drinking water.
The testing procedure involves filtering a large volume of water through a 1 micron, yarn-wound, polypropylene filter. The filter is then treated to remove any oocysts which may be present and the sample is concentrated.
A monoclonal antibody to Cryptosporidium is added to the sample to bind to oocyst wall antigens. The reaction can be visualized by the addition of fluorescein isothiocyanate (FITC) - conjugated anti-immunoglobulin and scanning with an epifluorescence microscope
Source: EPA Website

24. Water Quality Monitoring Challenges
Waterborne pathogens continue to evolve & New Disease outbreaks continue to emerge, even the eradicated ones like Cholera!
There is an increasing need for better detection technologies in testing processes (source water protection, treated water etc…)
Water suppliers must be diligent to protect their customers-However, their biggest problem is the aging water Infrastructure (pipe erosions, combined sewer overflows into source water or recreational water etc..)
Proper sampling and knowledge of NEW and existing testing techniques for Water quality are vital in this diligence
There is need for new “technology savvy” analysts and field sampling technicians in this field

25. for more information Visit http://www. epa. gov/safewater/ http://www
for more information Visit remember biolgical contaminants are only one part of the story- chemical contaminants are the other part!