introduction to waterborne pathogens

8/12/2019 Introduction to waterborne pathogens

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Introduction toIntroduction toWaterborne PathogensWaterborne Pathogens

Marylynn V. YatesMarylynn V. Yates

Department of Environmental SciencesDepartment of Environmental SciencesUniversity of California, RiversideUniversity of California, Riverside


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Waterborne Disease Associated with

Drinking Water in the U.S., 1920-2004

0

200

400600

800

20-30

31-40

41-50

51-60

61-70

71-80

81-90

91-00

01-

DeathsCases (1000s)

Outbreaks

Year


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Number of Waterborne Disease OutbreaksNumber of Waterborne Disease Outbreaks

Associated with Drinking Water, 1971Associated with Drinking Water, 1971--20042004

CDC, 2006


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Waterborne Disease Outbreaks AssociatedWaterborne Disease Outbreaks Associated

with Drinking Water, 1986with Drinking Water, 1986--20022002

0

2

4

6

8

10

1214

1986 1988 1990 1992 1994 1996 1998 2000 2002

Campylobacter

Naegleria

E. coli

Salmonella

Norwalk virus

Shigella

Cryptosporidium

Giardia

AGI


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Enteric PathogensEnteric Pathogens

Exposure is via ingestionExposure is via ingestion

primary site of infection isprimary site of infection is

gastrointestinal tractgastrointestinal tract gastroenteritis symptomsgastroenteritis symptoms

nauseanausea

vomitingvomiting

diarrheadiarrhea feverfever

may spread to other sites (blood,may spread to other sites (blood,

liver, nervous system)liver, nervous system)

shed in fecal materialshed in fecal material fecalfecal--oraloral route of transmissionroute of transmission


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Types of Waterborne PathogensTypes of Waterborne Pathogens

Viruses Bacteria Parasites


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SalmonellaSalmonella

Causes diarrhea, fever, crampsCauses diarrhea, fever, cramps

1212--72 hours after infection72 hours after infection

illness lasts 4illness lasts 4--7 days7 days can also cause typhoid fevercan also cause typhoid fever

40,000 cases reported annually;40,000 cases reported annually;

1000 deaths annually1000 deaths annually

0.1% population excretes0.1% population excretesSalmonellaSalmonella at a given timeat a given time

most common bacterial pathogenmost common bacterial pathogen

in wastewaterin wastewater

primarily foodborne (beef, poultry,primarily foodborne (beef, poultry,milk, eggs), but also transmittedmilk, eggs), but also transmitted

by waterby waterArrows indicate Salmonella cells

invading pig epithelium


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ShigellaShigella

Causes diarrhea (often bloody),Causes diarrhea (often bloody),

fever, cramps 24fever, cramps 24--48 hours after48 hours after

infectioninfection illness lasts 5illness lasts 5 --7 days7 days

infect only humansinfect only humans

18,000 cases reported annually18,000 cases reported annually

primarily transmitted by directprimarily transmitted by directcontact with infected individualcontact with infected individual

also transmitted by contaminatedalso transmitted by contaminated

food, water, recreationfood, water, recreation

low infectious dose (~10low infectious dose (~10organisms)organisms)

Macrophage infected with

Shigella


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VibrioVibrio choleraecholerae

ReleasesReleases endotoxinendotoxin thatthat

causes mild to profusecauses mild to profuse

diarrheadiarrhea ----> loss of fluids> loss of fluids---->death if untreated>death if untreated

infects only humansinfects only humans

several pandemics haveseveral pandemics have

occurredoccurred

Latin America: 1 million cases;Latin America: 1 million cases;

10,000 deaths (199110,000 deaths (1991--1994)1994)

primarily transmitted by waterprimarily transmitted by waterand food; rarely by directand food; rarely by direct

contactcontact


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PathogenicPathogenic E. coliE. coli

EnterohemorrhagicEnterohemorrhagic:: E. coliE. coli

O157:H7O157:H7 -- bloody diarrhea, maybloody diarrhea, may

cause acute kidney failure, deathcause acute kidney failure, death EnterotoxigenicEnterotoxigenic: minor to severe: minor to severe

diarrhea; contaminated food anddiarrhea; contaminated food and

water (poor sanitation)water (poor sanitation)

EnteroinvasiveEnteroinvasive: dysentery: dysentery EnteropathogenicEnteropathogenic: traveler: travelerss

diarrheadiarrhea -- watery diarrheawatery diarrhea

EnteroaggregativeEnteroaggregative: persistent,: persistent,

nonnon--bloody diarrheabloody diarrhea Health effects (food and water)Health effects (food and water)

~ 73,000~ 73,000 E. coliE. coli cases each yearcases each year

~ 61 deaths~ 61 deaths


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GiardiaGiardia

Causes diarrhea, abdominalCauses diarrhea, abdominal

cramps, nausea for 4cramps, nausea for 4--6 weeks6 weeks

11--2 week incubation period2 week incubation period

transmitted by contaminatedtransmitted by contaminated

food/waterfood/water

can be transmitted fromcan be transmitted fromanimals to humansanimals to humans

antibiotics are availableantibiotics are available


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CryptosporidiumCryptosporidium

Causes diarrhea, abdominalCauses diarrhea, abdominal

cramps, slight fever for 1 weekcramps, slight fever for 1 week

22--10 day incubation period10 day incubation period transmitted by contaminatedtransmitted by contaminated

food/water, personfood/water, person--personperson

can be transmitted from animals tocan be transmitted from animals to

humanshumans no antibiotics are availableno antibiotics are available

can cause very severe illness incan cause very severe illness in

individuals with weakenedindividuals with weakened

immune systemsimmune systems


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RotavirusRotavirus

Most common cause of severeMost common cause of severe

diarrhea worldwidediarrhea worldwide

in developing countries, 1 millionin developing countries, 1 milliondeaths/yrdeaths/yr

2020--25% diarrhea deaths25% diarrhea deaths

6% deaths in kids


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RotavirusRotavirus

CDC, 2006


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NorovirusNorovirus

Causes diarrhea, vomiting (1Causes diarrhea, vomiting (1--44

days)days)

11--2 day incubation period2 day incubation period transmitted by contaminatedtransmitted by contaminated

food/waterfood/water

may cause up to 50% foodmay cause up to 50% food--relatedrelated

gastroenteritis outbreaksgastroenteritis outbreaks may cause 25% waterbornemay cause 25% waterborne

outbreaksoutbreaks

Health effects (food and water):Health effects (food and water):

23 million cases annually23 million cases annually


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Recreational WaterRecreational Water--AssociatedAssociated

Outbreaks, 1978Outbreaks, 1978--20042004

CDC, 2006


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PseudomonasPseudomonas

Associated with hot tubs,Associated with hot tubs,

poolspools

Typically causes dermatitisTypically causes dermatitis

preventable by maintainingpreventable by maintaining

adequate residualadequate residual

disinfectant levelsdisinfectant levels


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LegionellaLegionellaPONTIAC FEVER:PONTIAC FEVER: Fever, muscle aches for 2Fever, muscle aches for 2--55

daysdays

incubation period:hoursincubation period:hours--22daysdays

8,0008,000 -- 18,000 cases/year18,000 cases/year

grow in warm (90grow in warm (90 --105 F),105 F),

stagnant waterstagnant water spread through aerosolsspread through aerosols

(cooling towers, whirlpool(cooling towers, whirlpoolspas, showers)spas, showers)

elderly, cigarette smokers,elderly, cigarette smokers,

persons with chronic lung orpersons with chronic lung orimmunocompromisingimmunocompromisingdisease, and personsdisease, and personsreceivingreceivingimmunosuppressive drugs atimmunosuppressive drugs atincreased riskincreased risk


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NaegleriaNaegleria

Found in soil and warm,Found in soil and warm,stagnant bodies of freshstagnant bodies of fresh

water,water, unchlorinatedunchlorinatedswimming pools, and inswimming pools, and inwarm wastewater pools fromwarm wastewater pools frompower plantspower plants

Entry through nose, infectionEntry through nose, infectionof brain and spinal cordof brain and spinal cord

Headache, fever, nauseaHeadache, fever, nauseaand vomiting, stiff neck,and vomiting, stiff neck,confusion, loss of balanceconfusion, loss of balance

and bodily control, seizures,and bodily control, seizures,and hallucinations. Infectionand hallucinations. Infectionusually results in deathusually results in deathwithin 7within 7--10 days.10 days.


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Concentrations of Pathogens in StoolsConcentrations of Pathogens in Stools

of Infected Individualsof Infected Individuals

Organism Concentration (per g)

Giardia 5 x 106

Cryptosporidium 106- 107

Poliovirus 103- 10

6.5

Hepatitis A 108

Rotavirus 108- 1010

from Gerba, 1995


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Minimal Infective Doses for SomeMinimal Infective Doses for Some

PathogensPathogensOrganismOrganism Minimal Infective DoseMinimal Infective Dose

SalmonellaSalmonella sppspp.. 10,00010,00010 million10 million

ShigellaShigella sppspp.. 1010 -- 100100

E. coliE. coli 1 million1 million100 million100 million

E. coliE. coli O157:H7O157:H7


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Sampling for VirusesSampling for Viruses


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Collection ApparatusCollection Apparatus


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Collection on FiltersCollection on Filters

Positively-charged fil ters

+

+

+

++

COO-

NH2


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Elution from FiltersElution from Filters

Positively-charged fil ters

+

+

+

++

COO-

NH2

+ Beef extract,

pH 9.5

-

-

-

-

-

COO-

NH2


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Sample ConcentrationSample Concentration

11--liter sample concentrate:liter sample concentrate:

Add acid to decrease pH to 3.5Add acid to decrease pH to 3.5 Organic material (with viruses attached)Organic material (with viruses attached)

precipitatesprecipitates CentrifugeCentrifuge

Viruses pellet outViruses pellet out ResuspendResuspend pellet in bufferpellet in buffer


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Sample ProcessingSample Processing

Filter sampleFilter sample

Elute from filterElute from filter

Concentrate sampleConcentrate sample

Analyze sample:Analyze sample:

cell culturecell culture

PCRPCR

1000 liters1000 liters

1 liter1 liter

30 ml30 ml

up to 30 mlup to 30 ml

1010 -- 100100 ulul


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Physiological MethodsPhysiological Methods

Infection

Host Cells

Virus

ReleaseReplication


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Cell CultureCell CultureConfluent MonolayerConfluent Monolayer Cytopathic EffectsCytopathic Effects


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Plaque assayPlaque assay


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Advantages of Cell CultureAdvantages of Cell Culture

Detects only infective particlesDetects only infective particles

Ability to quantify virusesAbility to quantify viruses High sensitivityHigh sensitivity

Entire sample concentrate can beEntire sample concentrate can beanalyzed (1000 L equivalent volume)analyzed (1000 L equivalent volume)


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Limitations of Cell CultureLimitations of Cell Culture

Detects only culturable virusesDetects only culturable viruses

Relatively nonRelatively non--specificspecific Up to 2Up to 2--4 weeks for analysis4 weeks for analysis

Cost of analysisCost of analysis


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Biochemical MethodsBiochemical Methods

RIA

ELISA+

Y

Coat

Gene Probes

PCRRT-PCR

+Nucleic

Acid Oligos

Ab

Polymerase Chain ReactionPolymerase Chain Reaction


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Polymerase Chain ReactionPolymerase Chain Reaction

(PCR)(PCR)


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Nucleic AcidsNucleic Acids

35

A C T G G T C A A G TT G A C C A G T T C A

3 5

Double-stranded DNA

5 3

mRNA

A C U G G U C A A G U


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PCR: Cycles 1PCR: Cycles 1--44


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f CR


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Advantages of PCRAdvantages of PCR

Relatively rapid resultsRelatively rapid results

Highly sensitiveHighly sensitive Highly specificHighly specific

Ability to detect nonAbility to detect non--culturable virusesculturable virusesAbility to tailor primers for desiredAbility to tailor primers for desired

applicationapplication

Li it ti f PCR


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Limitations of PCRLimitations of PCR

High potential for contaminationHigh potential for contamination

Potential for amplification of nonPotential for amplification of non--targettargetsequencessequences

Small equivalent sample volume analyzedSmall equivalent sample volume analyzed(0.3(0.3 -- 3 L)3 L)

Inability to distinguish between infectiveInability to distinguish between infective

and nonand non--infective particlesinfective particles

Vi D t ti M th dVi D t ti M th d


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Virus Detection MethodsVirus Detection Methods

MethodMethodInfectivityInfectivity

test?test?DetectionDetection

limitlimitTimeTime

Electron microscopyElectron microscopy nono 101055101066 < 24 h< 24 h

ELISAELISA

viral antigensviral antigens nono 101055 < 2 h< 2 h

antiviral antibodiesantiviral antibodies yesyes 101055 < 2 h< 2 h

RealReal--time (RT) PCRtime (RT) PCR nono 101000 -- 101011 < 8 h< 8 h

Plaque assayPlaque assay yesyes 101000 -- 101011


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Microorganisms in UntreatedMicroorganisms in Untreated

WastewaterWastewaterOrganism No. per liter

Total coliform bacteria 100 million 1 billion

Fecal coliform bacteria 10 million 100 millionSalmonella 1000 100,000

Shigella 10 10,000

Enteroviruses 10,000 100,000Rotaviruses 100 100,000

Giardia 100 100,000

Cryptosporidium 100 10,000Ascaris 10 10,000

Characteristics of anCharacteristics of an idealideal


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indicator organismindicator organism Present in waters contaminated by pathogens andPresent in waters contaminated by pathogens and

absent from these same waters when contaminationabsent from these same waters when contamination

is not presentis not present

Exist in concentrations that outnumber pathogenicExist in concentrations that outnumber pathogenic

organismsorganisms

NonNon--pathogenic to humanspathogenic to humans

Equal or greater resistance to treatment protocolsEqual or greater resistance to treatment protocols

and environmental factors than pathogensand environmental factors than pathogens

Unable to reproduce in the environmentUnable to reproduce in the environment

Detectable by simple, rapid, and economical methodsDetectable by simple, rapid, and economical methods


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Examples of indicator organismsExamples of indicator organisms

Total coliform bacteria (in use forTotal coliform bacteria (in use for

approximately 75 years)approximately 75 years)

Fecal coliform bacteria (Fecal coliform bacteria (thermotolerantthermotolerant

coliforms)coliforms)

Fecal streptococci (Fecal streptococci (EnterococciEnterococci)) coliphagescoliphages

D t ti f I di t O iD t ti f I di t O i


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Detection of Indicator OrganismsDetection of Indicator Organisms

T t l C lif B t iTotal Coliform Bacteria


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Total Coliform BacteriaTotal Coliform Bacteria

Fecal Coliform BacteriaFecal Coliform Bacteria


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Fecal Coliform BacteriaFecal Coliform Bacteria

E coliE coli


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E. coliE. coli

mTEC agar modified mTEC agar

EPA, 2000



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E coliE coli


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E. coliE. coli


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Enterococci (Fecal Streptococci)Enterococci (Fecal Streptococci)

mEI agar, courtesy EPA


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Somatic Phages (phiX174)Somatic Phages (phiX174)

S i h


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Somatic phageSomatic phage

Potential Applications of anPotential Applications of an


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IndicatorIndicatorBeing an indicator of:Being an indicator of:

fecal contaminationfecal contamination the presence of domestic sewagethe presence of domestic sewage

the presence of pathogensthe presence of pathogens

the efficiency of a particular water or wastethe efficiency of a particular water or waste

treatment processtreatment process

the environmental fate of a pathogen of interestthe environmental fate of a pathogen of interest the movement of particles suspended in waterthe movement of particles suspended in water

during subsurface transportduring subsurface transport

Relative Sizes ofRelative Sizes of


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MicroorganismsMicroorganismsBacteria: 0.1 10 m

Viruses: 0.01 0.1 m

Coliphage and Human EntericColiphage and Human Enteric

Vi Si il it


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Virus SimilarityVirus Similarity

GravelGravel


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GravelGravel

Pore Diameter > 400 umPore Diameter > 400 um


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ClayClay


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C ayy

Pore Diameter


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Concluding ThoughtsConcluding Thoughts

When designing a monitoring program:When designing a monitoring program:

Do your homeworkDo your homework Indicator vs. pathogen?Indicator vs. pathogen?

All indicators are not created equalAll indicators are not created equal

One size does not fit allOne size does not fit all

introduction to waterborne pathogens

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