Introduction to Introduction to Waterborne PathogensWaterborne Pathogens

Marylynn V. YatesMarylynn V. YatesDepartment of Environmental SciencesDepartment of Environmental Sciences

University of California, RiversideUniversity of California, Riverside

Waterborne Disease Associated with Drinking Water in the U.S., 1920-2004

0200400600800

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DeathsCases (1000s)

Outbreaks

Year

Number of Waterborne Disease Outbreaks Number of Waterborne Disease Outbreaks Associated with Drinking Water, 1971Associated with Drinking Water, 1971--20042004

CDC, 2006

Waterborne Disease Outbreaks Associated Waterborne Disease Outbreaks Associated with Drinking Water, 1986with Drinking Water, 1986--20022002

02468

101214

1986 1988 1990 1992 1994 1996 1998 2000 2002

CampylobacterNaegleria

E. coliSalmonella

Norwalk virusShigella

CryptosporidiumGiardia

AGI

Enteric PathogensEnteric PathogensExposure is via ingestion Exposure is via ingestion primary site of infection is primary site of infection is gastrointestinal tractgastrointestinal tractgastroenteritis symptomsgastroenteritis symptoms

nauseanauseavomitingvomitingdiarrheadiarrheafeverfever

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

Types of Waterborne PathogensTypes of Waterborne Pathogens

Viruses Bacteria Parasites

SalmonellaSalmonellaCauses diarrhea, fever, cramps Causes diarrhea, fever, cramps 1212--72 hours after infection72 hours after infectionillness lasts 4illness lasts 4--7 days7 dayscan also cause typhoid fevercan also cause typhoid fever40,000 cases reported annually; 40,000 cases reported annually; 1000 deaths annually1000 deaths annually0.1% population excretes 0.1% population excretes Salmonella Salmonella at a given timeat a given timemost common bacterial pathogen most common bacterial pathogen in wastewaterin wastewaterprimarily foodborne (beef, poultry, primarily foodborne (beef, poultry, milk, eggs), but also transmitted milk, eggs), but also transmitted by waterby water

Arrows indicate Salmonella cellsinvading pig epithelium

ShigellaShigellaCauses diarrhea (often bloody), Causes diarrhea (often bloody), fever, cramps 24fever, cramps 24--48 hours after 48 hours after infectioninfectionillness lasts 5 illness lasts 5 --7 days7 daysinfect only humansinfect only humans18,000 cases reported annually18,000 cases reported annuallyprimarily transmitted by direct primarily transmitted by direct contact with infected individualcontact with infected individualalso transmitted by contaminated also transmitted by contaminated food, water, recreationfood, water, recreationlow infectious dose (~10 low infectious dose (~10 organisms)organisms)

Macrophage infected with Shigella

Vibrio Vibrio choleraecholeraeReleases Releases endotoxinendotoxin that that causes mild to profuse causes mild to profuse diarrhea diarrhea ----> loss of fluids> loss of fluids---->death if untreated>death if untreatedinfects only humansinfects only humansseveral pandemics have several pandemics have occurredoccurredLatin America: 1 million cases; Latin America: 1 million cases; 10,000 deaths (199110,000 deaths (1991--1994)1994)primarily transmitted by water primarily transmitted by water and food; rarely by direct and food; rarely by direct contactcontact

PathogenicPathogenic E. coliE. coliEnterohemorrhagicEnterohemorrhagic: : E. coliE. coliO157:H7 O157:H7 -- bloody diarrhea, may bloody diarrhea, may cause acute kidney failure, deathcause acute kidney failure, deathEnterotoxigenicEnterotoxigenic: minor to severe : minor to severe diarrhea; contaminated food and diarrhea; contaminated food and water (poor sanitation)water (poor sanitation)EnteroinvasiveEnteroinvasive: dysentery: dysenteryEnteropathogenicEnteropathogenic: traveler: traveler’’s s diarrhea diarrhea -- watery diarrheawatery diarrheaEnteroaggregativeEnteroaggregative: persistent, : persistent, nonnon--bloody diarrheabloody diarrheaHealth 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

GiardiaGiardiaCauses diarrhea, abdominal Causes diarrhea, abdominal cramps, nausea for 4cramps, nausea for 4--6 weeks6 weeks11--2 week incubation period 2 week incubation period transmitted by contaminated transmitted by contaminated food/waterfood/watercan be transmitted from can be transmitted from animals to humansanimals to humansantibiotics are availableantibiotics are available

CryptosporidiumCryptosporidiumCauses diarrhea, abdominal Causes diarrhea, abdominal cramps, slight fever for 1 weekcramps, slight fever for 1 week22--10 day incubation period 10 day incubation period transmitted by contaminated transmitted by contaminated food/water, personfood/water, person--personpersoncan be transmitted from animals to can be transmitted from animals to humanshumansno antibiotics are availableno antibiotics are availablecan cause very severe illness in can cause very severe illness in individuals with weakened individuals with weakened immune systemsimmune systems

RotavirusRotavirusMost common cause of severe Most common cause of severe diarrhea worldwidediarrhea worldwidein developing countries, 1 million in developing countries, 1 million deaths/yr deaths/yr

2020--25% diarrhea deaths25% diarrhea deaths6% deaths in kids <5 yrs.6% deaths in kids <5 yrs.

Waterborne outbreaks Waterborne outbreaks documenteddocumentedvery high numbers in feces very high numbers in feces (10(101010/gram)/gram)very low infectious dose (~1)very low infectious dose (~1)

RotavirusRotavirus

CDC, 2006

NorovirusNorovirusCauses diarrhea, vomiting (1Causes diarrhea, vomiting (1--4 4 days)days)11--2 day incubation period 2 day incubation period transmitted by contaminated transmitted by contaminated food/waterfood/watermay cause up to 50% foodmay cause up to 50% food--related related gastroenteritis outbreaksgastroenteritis outbreaksmay cause 25% waterborne may cause 25% waterborne outbreaksoutbreaksHealth effects (food and water): Health effects (food and water): 23 million cases annually23 million cases annually

Recreational WaterRecreational Water--Associated Associated Outbreaks, 1978Outbreaks, 1978--20042004

CDC, 2006

Pseudomonas Pseudomonas Associated with hot tubs, Associated with hot tubs, poolspoolsTypically causes dermatitisTypically causes dermatitispreventable by maintaining preventable by maintaining adequate residual adequate residual disinfectant levelsdisinfectant levels

LegionellaLegionellaPONTIAC FEVER:PONTIAC FEVER:

Fever, muscle aches for 2Fever, muscle aches for 2--5 5 daysdaysincubation period:hoursincubation period:hours--2 2 daysdays8,000 8,000 -- 18,000 cases/year18,000 cases/yeargrow in warm (90 grow in warm (90 --105 F), 105 F), stagnant waterstagnant waterspread through aerosols spread through aerosols (cooling towers, whirlpool (cooling towers, whirlpool spas, showers)spas, showers)elderly, cigarette smokers, elderly, cigarette smokers, persons with chronic lung or persons with chronic lung or immunocompromisingimmunocompromisingdisease, and persons disease, and persons receiving receiving immunosuppressive drugs at immunosuppressive drugs at increased riskincreased risk

NaegleriaNaegleriaFound in soil and warm, Found in soil and warm, stagnant bodies of fresh stagnant bodies of fresh water, water, unchlorinatedunchlorinatedswimming pools, and in swimming pools, and in warm wastewater pools from warm wastewater pools from power plantspower plantsEntry through nose, infection Entry through nose, infection of brain and spinal cordof brain and spinal cordHeadache, fever, nausea Headache, fever, nausea and vomiting, stiff neck, and vomiting, stiff neck, confusion, loss of balance confusion, loss of balance and bodily control, seizures, and bodily control, seizures, and hallucinations. Infection and hallucinations. Infection usually results in death usually results in death within 7within 7--10 days.10 days.

Concentrations of Pathogens in Stools Concentrations of Pathogens in Stools of Infected Individualsof Infected Individuals

Organism Concentration (per g)

Giardia 5 x 106 Cryptosporidium 106 - 107 Poliovirus 103 - 106.5 Hepatitis A 108 Rotavirus 108 - 1010

from Gerba, 1995

Pathogen Removal During Sewage Pathogen Removal During Sewage TreatmentTreatment

VirusesViruses SalmonellaSalmonella GiardiaGiardia CryptosporidiumCryptosporidium

Concentration Concentration in raw sewage in raw sewage (no./liter)(no./liter)

100,000 100,000 –– 1 1 millionmillion 5,000 5,000 –– 80,00080,000 9,000 9,000 ––

200,000200,000 1 1 –– 3,9603,960

Concentration Concentration after primary after primary treatmenttreatment

1,700 1,700 ––500,000500,000 160 160 –– 3,3603,360 72,000 72,000 ––

146,000146,000 0.70.7

Concentration Concentration after secondary after secondary treatmenttreatment

80 80 –– 470,000470,000 3 3 –– 1,0751,075 6,480 6,480 ––109,500109,500

Concentration Concentration after advanced after advanced secondary secondary treatmenttreatment

0.007 0.007 –– 170170 0.000004 0.000004 –– 77 0.099 0.099 –– 29512951

Maier et al., 2000

Minimal Infective Doses for Some Minimal Infective Doses for Some PathogensPathogens

OrganismOrganism Minimal Infective DoseMinimal Infective DoseSalmonellaSalmonella sppspp.. 10,000 10,000 –– 10 million10 millionShigellaShigella sppspp.. 10 10 -- 100100E. coliE. coli 1 million 1 million –– 100 million100 millionE. coliE. coli O157:H7O157:H7 <100<100Vibrio Vibrio choleraecholerae 10001000Campylobacter Campylobacter jejunijejuni ~500~500GiardiaGiardia lamblialamblia 10 10 --100 cysts100 cystsCryptosporidiumCryptosporidium 10 oocysts10 oocystsHepatitis A virusHepatitis A virus 1 1 --10 10 pfupfu

from Bitton, 2006

Sampling for VirusesSampling for Viruses

Collection ApparatusCollection Apparatus

Collection on FiltersCollection on FiltersPositively-charged filters

+++++

COO-

NH2

Elution from FiltersElution from FiltersPositively-charged filters

+++++

COO-

NH2

+ Beef extract, pH 9.5

-----

COO-

NH2

Sample ConcentrationSample Concentration

11--liter sample concentrate:liter sample concentrate:Add acid to decrease pH to 3.5Add acid to decrease pH to 3.5Organic material (with viruses attached) Organic material (with viruses attached) precipitatesprecipitatesCentrifugeCentrifugeViruses pellet outViruses pellet outResuspendResuspend pellet in bufferpellet in buffer

Sample ProcessingSample Processing

Filter sampleFilter sample

Elute from filterElute from filter

Concentrate sampleConcentrate sample

Analyze sample:Analyze sample:cell culturecell culturePCRPCR

1000 liters1000 liters

1 liter1 liter

30 ml30 ml

up to 30 mlup to 30 ml10 10 -- 100 100 ulul

Physiological MethodsPhysiological Methods

InfectionHost Cells

Virus

ReleaseReplication

Cell CultureCell CultureConfluent MonolayerConfluent Monolayer Cytopathic EffectsCytopathic Effects

Plaque assayPlaque assay

Advantages of Cell CultureAdvantages of Cell Culture

Detects only infective particlesDetects only infective particlesAbility to quantify virusesAbility to quantify virusesHigh sensitivityHigh sensitivityEntire sample concentrate can be Entire sample concentrate can be analyzed (1000 L equivalent volume)analyzed (1000 L equivalent volume)

Limitations of Cell CultureLimitations of Cell Culture

Detects only culturable virusesDetects only culturable virusesRelatively nonRelatively non--specificspecificUp to 2Up to 2--4 weeks for analysis4 weeks for analysisCost of analysisCost of analysis

Biochemical MethodsBiochemical Methods

RIAELISA+ Y

Coat

Gene ProbesPCRRT-PCR

+NucleicAcid Oligos

Ab

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Nucleic AcidsNucleic Acids3’5’

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’

mRNAA C U G G U C A A G U

PCR: Cycles 1PCR: Cycles 1--44

Gel ElectrophoresisGel ElectrophoresisMolecular

marker Positive

control Sample

#1 Sample

#2Negative

control

311 bp

197 bp

92 bp

Advantages of PCRAdvantages of PCR

Relatively rapid resultsRelatively rapid resultsHighly sensitiveHighly sensitiveHighly specificHighly specificAbility to detect nonAbility to detect non--culturable virusesculturable virusesAbility to tailor primers for desired Ability to tailor primers for desired applicationapplication

Limitations of PCRLimitations of PCR

High potential for contaminationHigh potential for contaminationPotential for amplification of nonPotential for amplification of non--target target sequencessequencesSmall equivalent sample volume analyzed Small equivalent sample volume analyzed (0.3 (0.3 -- 3 L)3 L)Inability to distinguish between infective Inability to distinguish between infective and nonand non--infective particlesinfective particles

Virus Detection MethodsVirus Detection MethodsMethodMethod Infectivity Infectivity

test?test?Detection Detection

limitlimit TimeTime

Electron microscopyElectron microscopy nono 101055 –– 101066 < 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 << 21 d21 d

Microorganisms in Untreated Microorganisms in Untreated WastewaterWastewater

Organism 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 – 100,000 Rotaviruses 100 – 100,000 Giardia 100 – 100,000 Cryptosporidium 100 – 10,000 Ascaris 10 – 10,000

Characteristics of an Characteristics of an ““idealideal””indicator organismindicator organism

Present in waters contaminated by pathogens and Present in waters contaminated by pathogens and absent from these same waters when contamination absent from these same waters when contamination is not presentis not presentExist in concentrations that outnumber pathogenic Exist in concentrations that outnumber pathogenic organismsorganismsNonNon--pathogenic to humanspathogenic to humansEqual or greater resistance to treatment protocols Equal or greater resistance to treatment protocols and environmental factors than pathogensand environmental factors than pathogensUnable to reproduce in the environmentUnable to reproduce in the environmentDetectable by simple, rapid, and economical methodsDetectable by simple, rapid, and economical methods

Examples of indicator organismsExamples of indicator organismsTotal coliform bacteria (in use for Total coliform bacteria (in use for approximately 75 years)approximately 75 years)Fecal coliform bacteria (Fecal coliform bacteria (thermotolerantthermotolerantcoliforms)coliforms)Fecal streptococci (Fecal streptococci (EnterococciEnterococci))coliphagescoliphages

Detection of Indicator OrganismsDetection of Indicator Organisms

Total Coliform BacteriaTotal Coliform Bacteria

Fecal Coliform BacteriaFecal Coliform Bacteria

E. coliE. coli

mTEC agar modified mTEC agar

EPA, 2000

Total Coliform BacteriaTotal Coliform Bacteria

E. coliE. coli

Enterococci (Fecal Streptococci)Enterococci (Fecal Streptococci)

mEI agar, courtesy EPA

Somatic Phages (phiX174)Somatic Phages (phiX174)

Somatic phageSomatic phage

Potential Applications of an Potential Applications of an IndicatorIndicator

Being an indicator of:Being an indicator of:fecal contaminationfecal contaminationthe presence of domestic sewagethe presence of domestic sewagethe presence of pathogensthe presence of pathogensthe efficiency of a particular water or waste the efficiency of a particular water or waste treatment processtreatment processthe environmental fate of a pathogen of interestthe environmental fate of a pathogen of interestthe movement of particles suspended in water the movement of particles suspended in water during subsurface transportduring subsurface transport

Relative Sizes of Relative Sizes of MicroorganismsMicroorganisms

Bacteria: 0.1 – 10 µm

Viruses: 0.01 – 0.1 µm

Coliphage and Human Enteric Coliphage and Human Enteric Virus SimilarityVirus Similarity

GravelGravelPore Diameter > 400 umPore Diameter > 400 um

Sand Sand Pore Diameter 12Pore Diameter 12--400 um400 um

Clay Clay Pore Diameter <10 umPore Diameter <10 um

Concluding ThoughtsConcluding Thoughts

When designing a monitoring program:When designing a monitoring program:Do your homework Do your homework

Indicator vs. pathogen?Indicator vs. pathogen?All indicators are not created equalAll indicators are not created equalOne size does not fit allOne size does not fit all