Microbial Pathogens

Living organisms that cause disease
– Can be
Viruses
 Bacteria
 Protozoa
 Helminths

– But not all are pathogens
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Viruses

Intracellular parasites
– very small (20-100 nm), very simple
– not composed of cells
– need host cells to replicate
– infection usually person-to-person, not
through water
– hepatitis, gastroenteritis....
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Aside - Units

nm
– Nano = 1/1,000,000,000
– ~ 3 to 6 atoms end to end constitute a
nanometer
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Detection of Viruses



Not recommended for routine analyses
Should be done only by competent and
specially trained water virologists
Three Steps
– Collect representative sample
– Concentrate viruses in sample
– Identify and quantify

Beyond our capability
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Problems, Virus Methods
Very small (20 to 100 nm)
 Generally present at low concentration,
but variable in amount and type
 Unstable as biological entities
 Other compounds interfere
 Current methods are limited

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Bacteria

microscopic, single-celled organisms
– 500-5000 nm
– procaryotic (DNA not enclosed in membrane)
– most are not pathogens


perform valuable functions in environment,
our bodies, & wastewater treatment
Proliferate in:
– feces: 1 - 1000 X 106 / gram
– wastewater: ~ 10,000 / ml

Pathogenic bacteria cause typhoid, cholera....
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Vibrio cholerae (Microbe causing cholera)
From www.bact.wisc.edu/microtextbook/TOC.html
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Detection Methods - Specific Species


Not recommended for routine analyses
Three Steps
– Collect representative sample
– Concentrate bacteria in sample / Grow bacteria
colonies
– Identify and quantify


Stains, size, shape, growth patters, what they grow on...
Beyond our capability
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Protozoa

Microscopic, single-celled "animals", more
complex and larger than bacteria
–
–
–
–
10000-15000 nm
eucaryotic (DNA in nucleus within cell)
Most not pathogenic
Form Cysts / Oocysts


Resistant forms which allows Protozoa to survive under
adverse conditions
Pathogenic protozoans cause diarrhea
(Cryptosporidium), dysentery, gastrointestinal
infection (Giardia lamblia)...
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Giardia lamblia
111 waterborne outbreaks between
1965 and 1990, >26,000 cases
 Causes diarrhea

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Cryptosporidium parvum
Of increasing concern
 Causes cholera-like diarrhea

– can be life-threatening to immunodeficient
persons
1993, Milwaukee
- 400,000 sick
- 50 dead
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Entamoeba histolytica
Causes amebic dysentery
 Averages 28 deaths / year
 Has not been a frequent cause of
waterborne outbreaks in recent times

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Protozoa Detection
Not recommended for routine analyses
 Crypto and Giardia

– concentrate, purify and distribute organisms in
monolayer on membrane filter
– label with fluorescent antibody reagents
– identify cysts and oocysts by specific criteria
(immunoflorescence, size, shape, internal
morphological characteristics)

Beyond our capability
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Helminths (worms)





Humans can ingest worm eggs in
contaminated water
Worm can grow inside body, causing disease
Some (e.g., Hookworms) can infect by
penetrating skin
Worms can cause joint arthritis, damage
lymph nodes, damage tissue and organs
Not of Concern in US
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Intestinal nematodes - from www.life.sci.qut.edu.au/LIFESCI/darben/paramast.htm
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Drinking Water Standards & Pathogens

Maximum Contaminant Level Goal
– zero pathogens

Maximum Contaminant Level
– We will accept a limited number of positive
samples (indicator organism)


to account for inadvertent contamination
re-check water
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Indicator organisms

Too difficult to identify all pathogens, so
we use indicator organisms
– May not be pathogens themselves

Find indicator organisms?
– sample might be pathogen contaminated

Don't find indicator organisms?
– very unlikely sample is contaminated
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Common Indicator Organisms
Total Coliform
 Fecal Coliform

Bacteria

E. Coli

Common denominator is fecal coliform
– found in intestines
– evidence of fecal contamination
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General Types of Tests

Prescriptive tests
– Positive result good indication of presence
of indicator organism, but not definitive

Confirmatory
– Positive result indicates definite presence
of indicator organism
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Specific Tests
Membrane Filtration
 Presence/Absence
 Fermentation tube

– (confirmatory)
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Membrane Filtration

Filter known volume through sterile filter
– with proper dilution, deposit isolated bacteria

Place filter in petri dish w/ sterile agar
– promotes organism of interest,
inhibits others

Incubate (time / temperature)
– isolated bacteria grow into easily
identified colonies

Count colonies
– Concentration = Colonies /
Volume of sample
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Presence/Absence
 Add
100 mL sample to broth
 Incubate
(time / temperature)
– yellow color indicates presence of coliforms
 Determines
only
presence or absence
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Fermentation Tubes
 (1)
Presence/Absence
– Inoculate tube containing special
broth
– Incubate (time / temperature)

gas production in tubes indicates
presence
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Fermentation Tubes
 (2)
Concentration
– Inoculate series of tubes with various
amounts of sample

# of bacteria introduced proportional to
sample amount
– Incubate

Observe which tubes generate gas
– Statistically relate to most likely
concentration
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WWW Resources

EPA Pathogen Document
– www.epa.gov/enviro/html/icr/gloss_path.html

Germ Tutorial
– www.mwra.state.ma.us/germs/intro.htm

Pathogenic Bacteria Photo Gallery
– www.geocities.com/CapeCanaveral/3504/

Cryptosporidium Newsletter
– www.fspubl.com/index.html

Online Microbiology Textbook
– www.bact.wisc.edu/microtextbook/TOC.html
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