MIKRO575 Outi Martikainen 071111-Eng

9/10/2015
MicrosoftPowerPointMIKRO575OutiMartikainen071111
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Escherichiacoli
FoodandenvironmentalhygieneandcontrolMIKRO575
7.11.2011
OutiMartikainen/BacteriologyUnit
17.11.2011
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Thelecturestructure
Escherichiacolibacterialspecies
Beneficialandharmfulglycols
E.coliinsulation
E.colityping
E.colicontrol
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Escherichiacolibacterialspecies
Gramnegativerods
facultativeanaerobic
Themobilealsostationarypositions
Biochemicalproperties
LactosePositive(90%ofthestrains)
Betaglukuronidaasipositiivinen(97%ofthestrains)
Indolipositiivinen(99%ofthestrains)
Positiontaxonomically
Fyyla:proteobakteerit
Class:gammaproteobakteerit
family:Enterobacteriaceae
Enterobacteriales
Type:Escherichiacoli
sect:
family:Escherichia
.LocalspeciesincludeE.Albert,E.HermanE.Ferguson,
colonizehumansandothermammalsandbirdsinthelargeintestine,partofthe
thenormalfloramicrobiota
Neonatalintestinalcolonizedwithinafewhours
childbirth
geneticallydiversespecies
.TheexchangeofgenesinSalmonellaspp,Shigellaspp.andotherE.coli
withstocks
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Escherichiacoli
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Kaperetal2004NatureReviews
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Usefulglycols
Normaalimikrobistonglycolspreventdiseasecausingmicrobes
infectingthehost'sintestineorreproducingthere
vitaminK2returninthecolon
Probiotics
E.coliNissle1917:
inthetreatmentofchronicintestinaldiseases
reducethepathogenicglycol(invasiveand
Theadhesive)adhesiontointestinalepithelialcells,and
increasestheproductionofcytokines(Huebneretal,2011)
modelorganismingenetics
KloonausisntnrecombinantDNAtechniques
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Hazardousglycols
1.IntestinalInfections
2.Urinarytractinfections
3.Meningitis/sepsis
Kommensaalikolitcancausedisease
Weakenedimmunesystem
Theintestinalepitheliumdamaged
Bacterial"wrong"place
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SuolistopatogeenisetE.coli
=Ripulikolit
Causeintestinalinfectionsinhumans
vomiting,andpossiblecomplicationssuchashaemolytic
uremicsyndrome(HUS)
diarrhea,bloodydiarrhea,and
Ripulikolitcanbedividedintoatleastfiveclasses=damgroups:
EnteropathogenicE.coli(EPEC)
EnterotoxigenicE.coli(ETEC)
EnterohemorraaginenE.coli(EHEC)
EnteroinvasiveE.coli(EIEC)
EnteroaggregatiivinenE.coli(EAEC)
Ripulikolitoccurinanimals,especiallyruminants,intestines
suchasthenormalfloraE.coli.Theyhavealsobeenfoundtobe
soilandwatersystems.
Humanscangetinfectedfoodofanimalorigin
throughdrinkingorfaecesofanimalsorhumaninfected
water.
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Ripulikolienvirulenssimekanismit
Differentgroupsofdifferentvirulencepathogens
Somefeaturesmayoccurinmoredamgroup
thevirulencegenesarelocatedonmobileelements(integrated
transposons,bacteriophagesandPathogenicity)ofthebacterium
chromosomeandplasmids
subjectofthevirulencegenesofintestinalepithelialcells
Intestinalepithelialcolonization
Penetrationintocells
Returnontoxinsandexcreteepithelialcells
Nonpathogenicglycolmaychangebyhavingapathogen
kolikannoiltaorothervirulencegenesfromotherspecies.
Variousdamgroupscanbenefitfromeachothervirulencegenes
new
hypervirulenttejapositions?
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EHECbacteria
AKA
=VTECVerotoxigenicE.coli
toxicforVerocells(monkey
kidneycells)
STEC=shigatoksiiniaproducingE.coli
virulencefactors
Shigatoksiinitstx1andStx2(stx
=Intimiiniadhesionprotein(eae)
andstx
)
2
Enterohemolysiini(EHEChly/EHX)
STECautoagglutinoivaadhesin,Make
(Gets)
Diarrhea/bloodydiarrhea
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sequelaehaemolyticuraemic
syndrome(HUS)
EHECO157serogroupstrains
theclinicalpicture
mostwantedserogroup
serious
Kaperetal2004NatureReviews
Highriskgroups:childrenunder5yearschildren,theelderly
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Patogroupsvirulenssimekanismit
Kaperetal
2004Nature
Reviews
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EHECinfectionroutes
Cattle,sheep(animal
usuallyasymptomatic)
Pets
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Poorlycookedorraw
mincedbeef
Unpasteurised
(milk)products
Freshfruits,salads,sprouts
Waterlakes,ponds
Humantransmittedtoanother
contagion
Hospitalacquiredinfections(food
salad,kotileivokset)
LaboratoryInfections
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Theinfectiousdoses
EHEC10100cells
EPEC10
cell
ETEC10
cell
EIEC1010
10EAEC
6 cell
8
10
10
cell
EHEC,EPEC,ETECandtheAPEC(avianpathogenicE.
coli)inanimalpathogens
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InfectionTreatment
Diarrhoeasselflimiting
EHECantibioticsisnotrecommended
Lysis
Releaseofshigatoksiinin
Symptomsgetworse
AsymptomaticCarrier
EAECdamgroups,aswellassufferingfromdiarrheathat
healthychildreninAfrica(Bonkoungouetal2011)
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E.coliisolation
Food
Environmentalsample
Human(feces,urine,blood)
Concentrates
Selektiivimaljat
SMAC(sorbitolMacConkey)
CTSMAC(cefiximetelluriitti
Meatsamplespeptonisuola
solution.
SMAC)
CLED(cysteine
lactoseelectrolyte
deficientmedium)
Greatweakness
Thepathogenicglycolisnodifferentinappearanceorbiochemical
thecharacteristicsofthenormalflora
Searchforvirulencegenes(PCR,DNADNAhybridization)
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Selektiivimaljat
SMAC
SMAC:
sorbitol
Sorbitol
Fermentation
fermentative
EHECO157serogroup
stem
positionsoften
sorbitolinegatiivisia,but
Alsosorbitolipositiivisia
strainsareoftenisolatedfromthe
CLED
lactose
Fermentation
SMAC:
Strainwhichisnot
Enterohemolysiininreturn
ferments
pointingbowl(TBVP)
sorbitol
showsonlytheenteric
hemolyticstrains
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Screeningofcolonies
mixedculturebyPCR
Individualcolonies
Colony"pools"
manyofcolonytesting
inonereaction
Itcanbeadauntingtaskifthepathogenconcentration
samplelow
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Colonyhybridization
bowlcoloniesleaveaDNAimprintmembrane
stamped
probepairswithpathogeniccolonywithimprint
+Colorbind
Positivecontrol
antibody
pathogenpositioningtheplate
Negativecontrol
Healthreplicatedhybridisaatiomembraanille.
Positivecontrol
Positivecontrol
Afterhybridization,themembranes.
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Immunomagneticenrichment(IMS)
serogroupsbasedenrichmentmethod
serogroupsO26,O103,O111,O145,O157
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Thebeadsonthesurfaceofserogroupantibody
bacterialsurfacestructure
thebacteriacanbesearched
sticks
recoveredwiththebeadsandtheotherendto
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Ifyoucannotisolate...
MonialukkeinenPCRmethod,themixedcultureripulikolien
todetect
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E.colityping
Serotyping
serogroup=Ostructure(O=somaticantigen
LPS)
serotype=OstructureH(H=aflagellarantigen)
EPECO26,O55,O86,O111,O125,O126,n.127,O128
EHECO26,O45,O91,O103,O111,O113,O145,O157
(O157:H7,O157:H)
EHECO157phagetypes,
serotyping,moredetaileddistinctions
Otherfenotyypitysmenetelmt
genotyping
Virulencegenes
Pulsed
geneticfingerprint
Sequencing(antigen/totalgenomic)
Suchasthevirulencegenesstxsubtypingofspecificprimersor
ThePCRRFLPmethod
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Serotyping
Otyping
Htyping
Lasiagglutinaatio
RealtimePCR
7mostcommonhumanpathogensE.coliOgroup
(O26,O55,O91,O157,O103,O111,andO145)and
flagellarantigenH7
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Phagetypes,
EHECO157strains
60%
50%
40%
30%
20%
10%
0%
OccurringinFinlandEHECO157
PT2
PT4
PT49
PT8
Other
phagetypes
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Otherfenotyypitysmenetelmt
antimicrobialsusceptibility
Biochemicalidentifioimistestit
Lactoseandsorbitolfermentation
glucuronidasereturn(PGUA)
Enterohemolysiininreturnindication
Shigatoksiininreturnindication
Verosolukoe
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Lateksiagglutinaatio
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Pulsed(PFGE)
Comparisonofthesameserotype
(OHstructure)strains
between
bacterialchromosomal
DNAcleavage
restrictionenzyme
EHEC
XbaI
Electrophoresis,wherein
electriccurrentchangesdirection
atcertainintervals
Epidemiologicalstudies
eg.foodpoisoning
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PFGE
geneticstrains
fingerprint
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EHECdiagnosticsTabain
Where
TABAofposted
PCR
PCRpositivecolony
FaecalPrimaryculture/
stx
,Stx
1
ehlymay
Pureculture
,Eae,
isolatedand
identifiedby
API20E
O:Hserotypes
Finalanswers
Phagetypes(O157)
+EHEC(O157)
Stx1/Stx2return
VTEC+(nonO157)
Ehly
EHEC
Antibiogrammi
EPEC+
Sorbitolfermentation
PFGE
(XbaI)
Stx
subtypes
2
Eaesubtypes
Betaglucuronidase
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Ripulikolidiagnostiikka
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FindFinlandandmanyothercountries
usuallyonlytheEHECpathogenO157serogroupGroup
dangerousserogroup(HUS)
ManystudiesbreakdownofEHECO157and
VTECnonO157,althoughtherearealsootherserotypes
hasbeenfoundtobehighlyvirulent(e.g.O26,
O103,O111,O145)
Otherripulikoliryhmiusuallyarenotsearchedwhen
explainingthecauseofintestinalinfections
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Food
bacterialinfectionsin20002010
Cases
Details:THL/TTRegister
4500
4000
3500
3000
2500
2000
1500
1000
500
0
20002001200220032004200520062007200820092010
Campylobacter
Salmonella
Yersinia
Shigella
EHEC
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EHECcasesannually
CertifiedEHECcasesin19962010
70
60
61
9
1
50
NonO157(n=135)
38
13
30
51
29
21
20
29
10
0
sor+O157:H7/H
(n=25)
43
40
7
0
2
6
9
9
1
11
7
9
9
1
8
9
9
1
18
151515
12July7
9
January1 0
0
July7
6
6
9
9
9
1
0
0
0
2
1
0
0
2
2
0
0
2
10
10
6
0
45
3
0
0
2
4
0
0
2
13
21
5
0
0
2
14 12
March6
0
2
9 5
6
0
3
6 7
0 0
0 0
2 2
3
813
8
0
0
2
9
0
0
2
23
20
15
11
0
5
9
3
0
1
1
1
0
0
2
2
sorO157:H7/H
(n=168)
Total(n=351)
Photo:LordoftheLienemann/THL
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EHECepidemicinFinland
Donegal,in1997
SerogroupO157
N.15infection
Bathingwater
SouthernFinland,19982002
SerogroupO157
Minorepidemicclusters
Mincedmeat,cattle
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GermanEHECO104:H4outbreak
"Thekillercucumbers"
TheoutbreakstrainofEHEC+EAECfeatures
Stx
,AGGR
2
Intimiininegative,negativeenterohemolysiini
Multiresistant
uncommonserotypeO104:H4
Affected2684,39dead(by20.7.2011)
HUSsyndromesufferers1/3
EFSA(EuropeanFoodSafetyAuthority)andthe
ECDC(EuropeanCentreforDisease)riskassessment
Germanyand
Frenchepidemicsprobablecommonfactor
Egyptianoriginfenugreekseedsprouts
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RipulikolitinBurkinaFaso
TheAcademyofFinlandproject
20082011:Food&
waterborne
enteropatogeenisten
bacteriaEpidemiology
BurkinaFaso
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microbiologicalresearch
sustainable
publichealth
Insupportofthestrategy
Nopreviousknowledge
enteric
epidemiology
DetectionofRipulikolien
foodandpeople
stools,EHECstrains
Isolationandtyping
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Food"hygiene"inOuagadougou
reason
ripulikolienhighincidenceraa'oissameats
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Comparisonoftheprevalenceofvirulencegenes
ripulikolipositiivisissameatsamples,and
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children'sstoolsamples
16PCRprimersinamixedculture
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BurkinaFasoisolatedEHECstrains
EristysmenetelmincolonyhybridizationandPCRscreening
21positions:20meat(beefandvealproducts,mutton)and1
achildsufferingfromdiarrheastool
Alltheheelsofstx
,Sideofthestx
1
stx
morevirulent.
2
Allstrainsintimiininegatiivisia(eaegene),but
seveninfavorofintimiiniapotentialsubstituteSTEC
autoagglutinoivaadhesin(shallgene)(adhesiontotheintestinal
epithelialcells)
Verocellexperimenttestedstrainproducedshigatoksiinia
SerotypesO8:H,O38:H26,O82:H8,O179:Hand
OX183:H18hasbeenpreviouslyisolatedfrompatientsofdiarrhea
serotypeO2:H2ispreviouslyisolatedfromapatientHUS
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Thestudyconclusions
Ripulikolejaoccurredin43%oftheopenairmarkets
forsale,aswellasinrawmeats
59%ofdiarrheapatients
youngchildreninBurkinaFaso
Babyfoodisoftenpreparedby
underthesameconditionswiththecrude
processedmeat
Contaminatedmeat
mayhaveyoungchildrenripulikoli
infectionsintheback
EPECandETECdamgroups
oftenperformedaswellasfaecalthat
meatsamples
potential
infectiousaccess
meatandsicklychild
isolatedEHECstrainsare
apossiblehuman
pathogenic
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E.colicontroltoimprovethe
Foodcontrol
Studiesrelatedtofoodqualityare
primarilytheresponsibilityoftheoperator
Monitoringofselfsupervision
E.Usingcoliasanindicator(products,productionfacilities)
indicatesfaecalcontamination
Thereductionofcontaminationoffarmanimals
Aftertheslaughterandprocessingofcarcasseswashingwithdiluted
acid+coldchain
Waterqualitymonitoring
Thermotolerantcoliforms,E.coli
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Householdwater
Bathingwater
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Mikrobikriteeriasetuksen(2073/2005)
studiesinaccordancewith
E.coliLookfor:
Meats:mincedmeat,meatpreparations,mechanicallyseparated
meat(cattleandsheep:EHECadditionalresearch)
PISCES:shelledandcookedcrustaceansandmolluscs,
livebivalvemolluscs
MILK:destinedforconsumptioninpacked
rawmilk,rawmilkpasteurizationorweaker
heattreatedmilkmade
fromcreamandbutter,
pasteurisationorastrongerheattreatedmilk
unripenedsoftcheesesobtainedfromwhey,or
agedcheeses
VEGETABLESANDFRUIT:Precutfruitand
vegetables,whenintendedforconsumption(eg.
salads,grated,frozenvegetables),fruitand
vegetablejuices(unpasteurised,suchdrinkable)
additionalstudytolookforEHECbacteria
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EHECsurveillance
InfectiousDiseasesAct
(989/2006)andDecree(1376/2006)
PeopleEHECinfectionsareclassifiedasdangerousforthegeneral
Communicablediseases
Foodcontrol
MeatInspection
.Forexample,basedontheELISAmethodasarapidtest(antigen
antibodycomplexformation)ofEHECO157:a
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THL
Humanstrainstyping
Epidemicsstatistics
EVIRAin
Foodstuffsandtypingofstrainsisolatedfromanimals
EscherichiacoliO157,O145,O111,O103andO26
serogroupsdemonstrationofcultivationmethodand
byrealtimePCRmethod
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MinistryofAgricultureandForestryMinistry'sregulationofbovineEHEC
studiesinaslaughterhouseandvenue
(24/EEO/2006)
" Naccordancewith20ofslaughterhousesandsmallslaughterhousesFoodAct
housecontrolplanshallbeincludedinthesamplingEHEC
bacteriafightingmeasurestotheslaughterhouseorasmall
receivedfromthefaecesofcattleinaccordancewithAnnex1.Samples
mustbesenttotheadmissibilityoftheFoodAct39subsection2.
thelaboratoryunder.ThesamplesmustbeexaminedE.coliO157
stocksincase.Researchlaboratory,samplesshallbesubmitted
haveisolatedE.coliO157strainsofbacteriaFoodsafety
Agency.FinnishFoodSafetyAuthorityshallensurethatthe
Thebacterialstrainsarealsoprovided."
" FinnishFoodSafetyAuthoritymustbedrawnupeachyearslaughterhouses
onasamplingplanfortheEHECsurveillance.
Theplanindicatedthesamplesbasedontheslaughtervolumes
Numberofteurastamokohtaisesti.Attheslaughterhousemustbesampled
inaccordancewiththesamplingplan.Thesamplesmustbe
sporadicandbedistributedasevenlyaspossiblethroughouttheyear
period.Smallplaceofatleasttwosamplesmustbetaken
peryear.Ifyoursmallabattoirstoslaughterlessthan100headofcattleayear,
samplesdonotneedtotake."
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Authorities:foodpoisoningThestudy,
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THLTART,Taba
TheNationalFoodAgency
Infectiousdiseases
Foodpoisoning
register
Hospitaldistricts
Veterinaryandfood
Researchequipment
register
Theprovincialgovernment
HealthCentre
Healthprotection
Authority
LocalAuthority
FoodLaboratory
Foodpoisoninginvestigationgroup
Niemietal.2004
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Summary
EHEC(O157serogroup)themostimportantcausesoffoodpoisoning
glycol
Casesannuallyfewotherfoodbornepathogen
incomparison,butcauseseveresymptoms
Ripulikolikantojentransformationasathreat
Seriousinfections
Preventionofinfectionsthroughappropriatemonitoring
Tracingoutbreaksrequirescooperationbetweenauthorities
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THANKYOU!
outi.martikainen@thl.fi
17.11.2011
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