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Microbial PathogensMicrobial Pathogens

Living organisms that cause Living organisms that cause diseasedisease– Can be Can be

VirusesViruses BacteriaBacteria ProtozoaProtozoa HelminthsHelminths

– But not all are pathogensBut not all are pathogens

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VirusesViruses

Intracellular parasites Intracellular parasites – very small (20-100 nm), very simplevery small (20-100 nm), very simple– not composed of cellsnot composed of cells– need host cells to replicateneed host cells to replicate– infection usually person-to-person, infection usually person-to-person,

not through waternot through water– hepatitis, gastroenteritis....hepatitis, gastroenteritis....

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Aside - UnitsAside - Units

nmnm– Nano = 1/1,000,000,000Nano = 1/1,000,000,000– ~ 3 to 6 atoms end to end constitute ~ 3 to 6 atoms end to end constitute

a nanometera nanometer

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Detection of VirusesDetection of Viruses

Not recommended for routine analysesNot recommended for routine analyses Should be done only by competent and Should be done only by competent and

specially trained water virologistsspecially trained water virologists Three StepsThree Steps

– Collect representative sampleCollect representative sample– Concentrate viruses in sampleConcentrate viruses in sample– Identify and quantify Identify and quantify

Beyond our capabilityBeyond our capability

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Problems, Virus MethodsProblems, Virus Methods

Very small (20 to 100 nm)Very small (20 to 100 nm) Generally present at low Generally present at low

concentration, but variable in concentration, but variable in amount and typeamount and type

Unstable as biological entitiesUnstable as biological entities Other compounds interfereOther compounds interfere Current methods are limitedCurrent methods are limited

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BacteriaBacteria microscopic, single-celled organisms microscopic, single-celled organisms

– 500-5000 nm500-5000 nm– procaryotic (DNA not enclosed in membrane)procaryotic (DNA not enclosed in membrane)– most are not pathogensmost are not pathogens

perform valuable functions in environment, perform valuable functions in environment, our bodies, & wastewater treatmentour bodies, & wastewater treatment

Proliferate in:Proliferate in:– feces: 1 - 1000 X 10feces: 1 - 1000 X 106 6 / gram/ gram– wastewater: ~ 10,000 / mlwastewater: ~ 10,000 / ml

Pathogenic bacteria cause typhoid, Pathogenic bacteria cause typhoid, cholera....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 Detection Methods - Specific SpeciesSpecies

Not recommended for routine analysesNot recommended for routine analyses Three StepsThree Steps

– Collect representative sampleCollect representative sample– Concentrate bacteria in sample / Grow Concentrate bacteria in sample / Grow

bacteria coloniesbacteria colonies– Identify and quantifyIdentify and quantify

Stains, size, shape, growth patters, what they Stains, size, shape, growth patters, what they grow on...grow on...

Beyond our capabilityBeyond our capability

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ProtozoaProtozoa Microscopic, single-celled "animals", Microscopic, single-celled "animals",

more complex and larger than bacteriamore complex and larger than bacteria– 10000-15000 nm10000-15000 nm– eucaryotic (DNA in nucleus within cell)eucaryotic (DNA in nucleus within cell)– Most not pathogenicMost not pathogenic– Form Form Cysts / OocystsCysts / Oocysts

Resistant forms which allows Protozoa to survive Resistant forms which allows Protozoa to survive under adverse conditionsunder adverse conditions

Pathogenic protozoans cause diarrhea Pathogenic protozoans cause diarrhea ((CryptosporidiumCryptosporidium), dysentery, ), dysentery, gastrointestinal infection (gastrointestinal infection (Giardia Giardia lamblialamblia)...)...

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Giardia lambliaGiardia lamblia

111 waterborne outbreaks 111 waterborne outbreaks between 1965 and 1990, >26,000 between 1965 and 1990, >26,000 casescases

Causes diarrheaCauses diarrhea

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Cryptosporidium parvumCryptosporidium parvum Of increasing concernOf increasing concern Causes cholera-like diarrheaCauses cholera-like diarrhea

– can be life-threatening to can be life-threatening to immunodeficient personsimmunodeficient persons

1993, Milwaukee- 400,000 sick- 50 dead

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Entamoeba histolyticaEntamoeba histolytica Causes amebic dysenteryCauses amebic dysentery Averages 28 deaths / yearAverages 28 deaths / year Has not been a frequent cause of Has not been a frequent cause of waterbornewaterborne outbreaks in recent outbreaks in recent timestimes

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Protozoa DetectionProtozoa Detection

Not recommended for routine analysesNot recommended for routine analyses Crypto and GiardiaCrypto and Giardia

– concentrate, purify and distribute organisms concentrate, purify and distribute organisms in monolayer on membrane filterin monolayer on membrane filter

– label with fluorescent antibody reagentslabel with fluorescent antibody reagents– identify cysts and oocysts by specific identify cysts and oocysts by specific

criteria (immunoflorescence, size, shape, criteria (immunoflorescence, size, shape, internal morphological characteristics)internal morphological characteristics)

Beyond our capabilityBeyond our capability

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Helminths (worms)Helminths (worms)

Humans can ingest worm eggs in Humans can ingest worm eggs in contaminated watercontaminated water

Worm can grow inside body, causing Worm can grow inside body, causing diseasedisease

Some (e.g., Hookworms) can infect by Some (e.g., Hookworms) can infect by penetrating skinpenetrating skin

Worms can cause joint arthritis, damage Worms can cause joint arthritis, damage lymph nodes, damage tissue and organslymph nodes, damage tissue and organs

Not of Concern in USNot of Concern in US

16Intestinal nematodes - from www.life.sci.qut.edu.au/LIFESCI/darben/paramast.htm

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Drinking Water Standards & Drinking Water Standards & PathogensPathogens

Maximum Contaminant Level GoalMaximum Contaminant Level Goal– zero pathogens zero pathogens

Maximum Contaminant Level Maximum Contaminant Level – We will accept a limited number of We will accept a limited number of

positive samples (indicator organism) positive samples (indicator organism) to account for inadvertent contaminationto account for inadvertent contamination re-check waterre-check water

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Indicator organismsIndicator organisms

Too difficult to identify all pathogens, Too difficult to identify all pathogens, so we use indicator organisms so we use indicator organisms – May not be pathogens themselvesMay not be pathogens themselves

Find indicator organisms? Find indicator organisms? – sample might be pathogen contaminatedsample might be pathogen contaminated

Don't find indicator organisms? Don't find indicator organisms? – very unlikely sample is contaminatedvery unlikely sample is contaminated

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Common Indicator OrganismsCommon Indicator Organisms

Total ColiformTotal Coliform Fecal ColiformFecal Coliform E. ColiE. Coli

Common denominator is Common denominator is fecal fecal coliformcoliform– found in intestinesfound in intestines– evidence of fecal contaminationevidence of fecal contamination

Bacteria

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General Types of TestsGeneral Types of Tests

PrescriptivePrescriptive tests tests– Positive result good indication of Positive result good indication of

presence of indicator organism, but presence of indicator organism, but not definitivenot definitive

ConfirmatoryConfirmatory– Positive result indicates definite Positive result indicates definite

presence of indicator organismpresence of indicator organism

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Specific TestsSpecific Tests

Membrane FiltrationMembrane Filtration Presence/AbsencePresence/Absence Fermentation tubeFermentation tube

– (confirmatory)(confirmatory)

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Membrane FiltrationMembrane Filtration Filter known volume through sterile filterFilter known volume through sterile filter

– with proper dilution, deposit isolated bacteriawith proper dilution, deposit isolated bacteria Place filter in petri dish w/ sterile agarPlace filter in petri dish w/ sterile agar

– promotes organism of interest, promotes organism of interest, inhibits othersinhibits others

Incubate (time / temperature)Incubate (time / temperature) – isolated bacteria grow into easily isolated bacteria grow into easily

identified coloniesidentified colonies Count coloniesCount colonies

– Concentration = Colonies / Concentration = Colonies / Volume of sampleVolume of sample

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Presence/AbsencePresence/Absence

Add 100 mL sample to brothAdd 100 mL sample to broth

Incubate (time / temperature)Incubate (time / temperature)– yellow color indicates presence of yellow color indicates presence of

coliformscoliforms

Determines presence or Determines presence or absenceabsence only only

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Fermentation TubesFermentation Tubes

(1) Presence/Absence(1) Presence/Absence– Inoculate tube containing special Inoculate tube containing special

brothbroth– Incubate (time / temperature)Incubate (time / temperature)

gas production in tubes indicates gas production in tubes indicates presencepresence

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Fermentation TubesFermentation Tubes (2) Concentration(2) Concentration

– Inoculate series of tubes with Inoculate series of tubes with various amounts of samplevarious amounts of sample # of bacteria introduced # of bacteria introduced

proportional to sample amount proportional to sample amount – IncubateIncubate

Observe which tubes generate gasObserve which tubes generate gas– Statistically relate to most likely Statistically relate to most likely

concentrationconcentration

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WWW ResourcesWWW Resources EPA Pathogen DocumentEPA Pathogen Document

– www.epa.gov/enviro/html/icr/gloss_path.htmlwww.epa.gov/enviro/html/icr/gloss_path.html Germ TutorialGerm Tutorial

– www.mwra.state.ma.us/germs/intro.htmwww.mwra.state.ma.us/germs/intro.htm Pathogenic Bacteria Photo GalleryPathogenic Bacteria Photo Gallery

– www.geocities.com/CapeCanaveral/3504/www.geocities.com/CapeCanaveral/3504/ Cryptosporidium NewsletterCryptosporidium Newsletter

– www.fspubl.com/index.htmlwww.fspubl.com/index.html Online Microbiology TextbookOnline Microbiology Textbook

– www.bact.wisc.edu/microtextbook/TOC.htmlwww.bact.wisc.edu/microtextbook/TOC.html