Microbial Pathogens

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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,
  gt26,000 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.a
u/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
• E. Coli
• Common denominator is fecal coliform
• found in intestines
• evidence of fecal contamination

Bacteria
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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 presence or absence only

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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