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Clostridia

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Clostridia

LargeGrampositiveanaerobicrods

Formofsporesresistanttooxygen,drought,
freezingandcookingtemperatures

Thesoilandintheintestinesofwarmblooded

Causethefood,theenvironmentandthepointsof
throughdrugpoisoningandinfectious
thetoxininfections

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A.Clostridiumbotulinum

1.Theclassicbotulism
2.Theinfantbotulism
3.Thewoundbotulism

B.Clostridiumperfringens

1.Theclassicfoodpoisoning

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2.enteritisnecroticans

C.clostridiumtetani

1.Tetanus

D.Clostridiumdifficile

1.Clostridiumdifficileassossioitunutdiarrhea(CDAD)

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A.Clostridiumbotulinum

1.Classicalbotulism

Foodpoisoning,theriskoffoodproducts,especiallymeat,fish,vacuumpacked
Productsandpreservedhomeproducts

Clostridiumbotulinumsecretesproteiinieksotoksiinia,whichisthestrongest
knownastoxic(lethaldose:0.1ng/kg)

Neurotoxinsaresynthesizedasaninactivepolypeptide,i.e.,
protoksiineina(size150kDa),whichareactivatedwhentheproteasecleaves
polypeptidechain

C.botulinumareclassifiedintosevenserotypesAGduringgrowth
onthebasisoftheantigeniccharacteristicsoftheirproductionoftoxinstypesA,
B,EandFcausebotulismpeople

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Afewotherklostridilajit(C.butyricum,C.Barati)toproduce
thesametoxinsasC.Botulinum
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Functionalformofbotulinumtoxincanbind
nervemusclejointsandpreventstheneurotransmitter
releaseofvesiclescontainingacetylcholine.Since
themusclemaynolongerbeanykindofmessagestothenervecells,muscle
paralyzed.

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thetoxinisabsorbedinthestomachandsmallintestine,fromwhereit
spreadsthroughoutthebody,canalsobeabsorbedbyinhalation

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Symptomsbegin1236hoursafteringestionoffood

Thefirstbotulismsymptomsincludefatigue,weakness,
vomiting,thenvoiceanddifficultyinswallowingandvisualdisturbances

Respiratorydepressioncanultimatelyleadtodeath

Mortalityofthediseaseisabout65%,indevelopedcountriesis10%..

Riskgroup:dangerousforeveryone

Evenoneperson'sillnessbotulismepidemicisinterpreted
Duetothenatureofthe

aseriousdisease

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2009

InearlySeptember,itwasfoundinFranceinthreeatthesame
thefamilyofapersonwhoisClostridiumbotulinum
botulismcausedbybacteria,thesuspectedsource
PurchasedfromFinlandtohotsmokedwhitefish.Vacuumpackedfish
waspurchasedinEasternFinlandinretailtradeinAugust
Attheend,transportedbyplanetoFrance
andbyaprivateindividualeatentwoweeks

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lateronthelastdayofuse.Journey,thefish
hadbeenstoredinacoolbox
unknowntemperaturefor14hoursandthen
Afterthehomerefrigerator.Beforeeatingfishisnot
washeated.

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2006

ClostridiumbotulinumcausedinJuly2006forone
epidemic,whichcontractedbytwopersonsofthesame
family.Thevehicleswere
likelytobehotsmokedwhitefish,whichwaspurchased
packedinalocalsupermarket.Fishfinal
datewasweeksawaysymisajankohdasta.Fish
thecountryoforiginwasCanada,itwasdeliveredtoFinland
frozen.Defrostingandsmoking,aswellasthefinishedproduct
packagingtookplaceinadomesticestablishment.

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Olderepidemics

1981twoGermantouristsgotsickaftereating
silykemaksapasteijaa

1999onepersonillwithwhitefishroe

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Botulismtreatment:

ABEtrivalentantitoxin(givenbeforesymptoms
thestart)
Gastrointestinaltractiscleared
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Missedthegutbacteriaaredestroyedpenicillin
Artificialrespiration
Forecasttheworsethesoonerthesymptomsappear
Recoverymaytakemonths
Waitingfornewnervemusclejunctionsformation

Smalldosesofpurifiedbotulinumtoxincanbe
useofCNSlihastondysregulation,
inthetreatmentofdiseases,thecosmeticuseofbiological
warfare

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2.Theinfantbotulism

C.botulinumproducestoxininthebaby'sgastrointestinaltract(n.80
spores/g)

Riskfoodshoney,vegetablesandfruits

Lessthanhalfyearsmorelikelytogetinfantbotulism
immatureintestinalflora,andahigherpHinthedigestivetract
allowbacterialsporegrowthandyieldofthetoxininthegut

Symptomsofconstipation,muscleslackening,weepingsluggish

Thesymptomsaresurprisinglymild,andmortalityonlyafewpercent,
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causingsuddeninfantdeathsyndrome

Poweroftreatmentisusuallyrescued

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3.Thewoundbotulism

C.botulinumspore'swound,wheretheygerminate

Thediseaseresemblestetanus

Theuseofinjectingdrugs

CasesdetectedintheAmericas,Europe,rare

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Clostridiumbotulinum
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taskanalysisinmice

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Clostridiumbotulinum

PCR/PFGE/ELISA
M.LindstrmFinland,studiedthemethod:
kytettnprimeriparia4,whichare
specificneurotoxinsbotulinumtoxinA,B,Eor

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F.Fourserotypesimultaneousdetection
possible.

ELISAreadyforadaywhenthetoxininthesample

Aviablecellgrowthandtoxin
generatingidentificationofgenesbyPCR

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B.Clostridiumperfringens

1.Theclassicfoodpoisoning

C.perfringensTypeAenterotoxinsecretedlast
years,themostcommonlyreportedcauseoffoodpoisoning
InFinland

Theriskoffoodmeatandfish,incompletelycooked,
slowlychilled,attoohighatemperature,storedand
incompletelycookedfoods

Causingdiseasedoseof10^6to10^8cells

Theincubationperiodof824h
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C.perfringenscellsincreaseinfood,someofthecellsdieofthestomach
acidity,butsomecellsremainaliveinthesmallintestine,where
theybegintoproliferate

Whenthebacterialcellitii,consistspoisoningcausingCPE
enterotoxin,ifthepositioniscpegeneencodingit(only5%
strains)

CPEtoxindamagethesurfaceoftheepithelialcellsofthesmallintestine
bymakingholesinthecellmembranestosmallmoleculessuchasanions,
cationsandorganicacidsleakout

Symptomsofabdominalpain,nauseaandintensediarrheathatlastfor1to2
days,theelderlyC.perfingenscancausepersistentdiarrhea

Enterotoxincanbedemonstratedinfaeces

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2.Enteritisnecroticans(pigbel)

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ClostridiumperfingensTypeCsecretedby
enterotoxincancausesevereenteritis
necroticansin(necrotizinginflammationofthebowel)

Thebacteriasecreteatoxinfromthefood,which
leadtoparalysis,haematomasandregional
necrosis,especiallyinthesmallintestine

Symptoms:vomiting,diarrhea,abdominalpainHard,
hemorrhage

Rare

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Clostridiumperfringens

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Clostridiumperfringensplating

TSCAgar(TryptoseSulfite
CycloserineAgar)

toproduceblackcolonies

cycloserinetopreventotherbacteria
growth

Inadditiontokanamycinandpolymyxin
inhibitionofbacterialflora

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TSC

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ClostridiumperfringensPCRandmicroarray

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Microarraytechnologywithantibodies
IdentificationofC.perfingenstoxins

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C.Clostridiumtetani

1.Tetanus,orlockjaw

C.tetaniproducestwopoison,andtetanoslysiinia
tetanospasmiinia,ofwhichthelatterissaidto
tetanustoxin

Tetanusneurotoxin,andthesecondstrongestpoison
Afterbotulinumtoxin

Thebacteriacancomeintothewound,whereitbeginstoincrease
underanaerobicconditions

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Infectionislocal,butthetoxinspreads
viathebloodstreamandlymphcirculation,aswellasdirectlytothe
thenervefibersalongthespinalcordandbrainstem

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toxinaffectsthecentralnervoussystem
hermosynapseihinandmotor
endplates,blockstheinhibitory
neurotransmitters(e.g.glycine)
release,whereinthemuscle
ispreventedandtherelaxationofthe
musclespasms
Theincubationperiodofdaystomonths
Atthebeginningofmusclespasmsofthewound
close,spreadstothemusclesofthejawand
allpartsofthevoluntarymuscles
Tetanusleads
ifuntreated,isalmostalways
death,manicuredmortality
1030%
Preventedthroughvaccination

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Clostridiumtetani
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C.tetanipenetration

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Clostridiumtetanidetermination

GeneticallyIgGELISAIgGclassaredetermined
antibodies
MouseTest

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Clostridiumtetani,theMousetest

Thelefthindlegofthemouseinjected
Tetanustoxin

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Clostridiumdifficile(40006000cases/yr)

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InsulationCDS(C.difficile
SELECTIVE)agarcontainingaDcycloseriniaand
Cefoksitinia

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Clostridiumdifficile

Clostridiumdifficileassociateddiarrhea
(CDAD)
DiagnostictestsforCDAD:in
thedeterminationofthemarket.They
oftoxinB(CytoToxassay)
ortoxinsAandB(immunoassay)
thegeneticanalysis.

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QuickTest:ClostridiumdifficileToxinAdetection
ofC.difficiletoxinAinfaeceswith
Rapydtest

1015min.

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Sources:
Austin,JWandDoddsKL1997.Clostridiumbotulinum.In:FoodMicrobiology.ASMPress.Pages
288304.
Hielm,S.clostridia,thequiet.In:M.SalkinojaSalonen(ed.).Microbiologicalcriteria.Microbiology
publications49/2000.
MicrobiologyDepartmentofAppliedChemistryandMicrobiology,UniversityofHelsinki.Pages652
655.
SagittariusSomer,H.andRistola,M.2003.Clostridiumspecies.In:P.Huovinen(ed.).
Microbiologyand
infectiousdiseases.Book1.DuodecimMedicalPublications.Pages228236.
Madigan,MT2003.BrockBiologyofMicroorganism.PearsonEducational.
Guidemicrobiologicalfoodanddrinkingwaterhazards.EvidentialEELAOpas1/2003.Pages3744.
McClaneBAin1997.Clostridiumperfringens.In:FoodMicrobiology.ASMPress.Pages305326.
SalkinojaSalonen,M.clostridialtoxins.In:M.SalkinojaSalonen(ed.).Microbiologicalcriteria.
Microbiologypublications49/2000.
DivisionofMicrobiology,DepartmentofAppliedChemistryandMicrobiology,Helsinki
University.Pages548549.

Pictures:
http://www.agen.ufl.edu/chyn/age2062/lect/lect_25/FG19_012.GIF
http://wwwuser.gwdg.de/hbruegg/ct/tetanus2.gif
http://www.agen.ufl.edu/chyn/age2062/lect/lect_25/FG19_011.GIF

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Fountains

Lindstrm,MiiaKeto,RiikkaMarkkula,AnnukkaNevas,MariHielm,
SebastianKorkeala,Hannu.MultiplexPCRAssayforDetectionand
IdentificationofClostridiumbotulinumtoxinTypesA,B,E,and...&Applied
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EnvironmentalMicrobiology,Dec2001,Vol.67Issue12,p5694,6p,4
charts,1bw(AN6063605)
http://www.cfsan.fda.gov/ebam/bam17.html
www.google.fi

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