Académique Documents
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May2004
BacteriologicalAnalyticalManual
Chapter9
Vibrio
Authors:CharlesA.KaysnerandAngeloDePaola,Jr.
RevisionHistory:Chapter9substantiallyrewrittenandrevisedMay2004
INTRODUCTION
MembersofthegenusVibrioaredefinedasGramnegative,asporogenousrodsthatarestraightorhavea
single,rigidcurve.Theyaremotilemosthaveasinglepolarflagellum,whengrowninliquidmedium.Most
produceoxidaseandcatalase,andfermentglucosewithoutproducinggas(7).Threespecies,V.cholerae,
V.parahaemolyticus,andV.vulnificus,arewelldocumentedhumanpathogens(54,78,79,90,101).V.
mimicus(24,103,111),isarecognizedpathogen(103)withsimilarcharacteristicstoV.cholerae,exceptan
abilitytofermentsucrose.Otherspecieswithinthegenus,suchasV.alginolyticus(51),V.fluvialis(71),V.
furnissii(15),V.metschnikovii(39,70),andV.hollisae(40)areoccasionalhumanpathogens(1,39,96).
Vibriospeciesaccountforasignificantproportionofhumaninfectionsfromtheconsumptionofrawor
undercookedshellfish(96).AFloridastudyofillnessesfromrawshellfishconsumptionreportedthefollowing
speciesindescendingorderoffrequencyV.parahaemolyticus,nonO1/O139V.cholerae,V.vulnificus,V.
hollisae,V.fluvialis,O1V.cholerae(64,72).
AnumberofsubstantialchangeshavebeenmadeinthisversionoftheVibriochapterincludinggreater
emphasisonmolecularmethodssuchasDNAcolonyhybridizationandPCRforidentificationand
characterizationofpathogenicVibriospp.Withtheadditionoftheseoptions,lessemphasishasbeenplaced
onsomeoftheoldermethodsandinsomecasesomesectionsrequiringdangerousordifficulttoobtain
reagents(i.e.O/129reagent)havebeeneliminatedbutmaybementionedinthetextortables.Therehave
beenconsiderableadvancesinmoleculardetectiontechniquessuchasrealtimePCRandasthese
methodsarevalidated,theywillbeincorporatedintothewebversionofthischapter.
V.cholerae
V.cholerae(6),thetypespeciesofthegenusVibrio,isthecausativeagentofcholeraoutbreaksand
epidemics(34,54,126).Variousbiochemicalpropertiesandantigenictypescharacterizeit.Itcanbe
differentiatedfromotherVibriospecies,exceptV.mimicus,becauseitsobligaterequirementforsodiumion
(Na+)(6)canbesatisfiedbythetraceamountspresentinmostmediaconstituents.Choleraenterotoxin
(CT)istheprimaryvirulencefactorofthediseasecholera.AgeneticpathogenicityislanddesignatedVPI
(vibriopathogenicityisland),whichcontainsmostgenesnecessarytocausecholerawasdemonstratedto
regulatetheCTgene(55).MostV.choleraestrainsrecoveredfromepidemiccholeracasescontaina
commonsomaticantigenandincludeserogroupO1(54).Over150knownsomaticantigenictypeshave
beenidentified.StrainsthatareagglutinableinInabaorOgawaserotypesofO1antiserumarewell
documentedhumanpathogens.Untilrecently,onlytheO1serogroupwasassociatedwithcholera
epidemics.However,in1993,alargeoutbreakofcholeraoccurredinIndia/Bangladeshfromanew,until
thenunknownserogroup,O139(3).Numerouscaseswererecordedinwhichpatientshadthetypical
symptomsofclassicalcholera,choleragravis,previouslyonlyseenwiththeO1serogroup.ExceptfortheO
antigenandthepresenceofapolysaccharidecapsule,thisserogroupisnearlyidenticaltotheseventh
pandemicstrainofV.cholerae(10).TheO139strainhasbecomeendemicintheBengalregionandisthe
causeofwhatmaybeknownastheEighthCholeraPandemic(34,117).
V.choleraestrainsthatareidenticalto,orcloselyresemble,clinicalstrainsinbiochemicalcharacteristics,but
failtoagglutinateineitherantiO1orO139seraarenowreferredtoasV.choleraenonO1/O139
(34,53,54).Theseserologicallydiversestrainsareabundantinestuarineenvironments.Evidenceindicates
thatnonO1/O139strainsaresporadicallyinvolvedincholeralikediarrhealdisease(22,73,83,96,105),but
rarelyinoutbreaks.Indeed,thepermeabilityfactorproducedbyanonO1/O139strainduringan
investigationofacholeraoutbreakwasfoundtobebiologicallyandimmunologicallyindistinguishablefrom
CT.SomenonO1/O139strainsalsoareinvasive,produceaheatstabletoxin,andhavecausedseptic
infectionsinindividualswithpredisposingmedicalconditions(83,85,93,99).MoststrainsdonotproduceCT,
thekeydifferencebetweentheseandepidemicV.choleraeO1/O139.
V.mimicus
V.mimicus(24,102)hasbeenassociatedwithdiarrheafollowingconsumptionofraworundercooked
seafood(96).IsolatedfromsamplesduringasearchforV.cholerae,V.mimicuscanbedifferentiatedfrom
thatcloselyrelatedpathogenbysucrosenonfermentation.Theorganismwillappearasgreencolonieson
thiosulfatecitratebilesaltssucrose(TCBS)agarandwillgrowinmostcommonmediawithoutaddedNaCl.
Virulenceispoorlycharacterized,butsomestrainshavebeenfoundtopossessthecholeratoxingene
(111),producedemonstrableCTinatissuecultureassay,andthectxgenecanbedetectedbyPCR
amplification.
V.parahaemolyticus
V.parahaemolyticus(36,81),theleadingcauseofbacterialdiarrheaassociatedwithseafoodconsumptionin
Florida(64)andprobablytheUSandoccasionallycausessepticemia(96).Itisahalophilicestuarine
organismfoundincoastalwatersofvirtuallyalltemperateregions(27,52,101).Intemperateregions,a
seasonaloccurrenceinshellfishandinhumaninfectionshasbeenreported,themajorityinthewarmer
monthsoftheyear.InsubtropicalregionssuchasFlorida,illnesscanoccuryearround.Allstrainssharea
commonHantigen,but,todate,12O(symatic)typesandover70K(capsular)antigenshavebeen
described,thoughmanyotherstrainsareuntypable(81,101).MostclinicalisolatesofV.parahaemolyticus
aredifferentiablefromenvironmentalstrainsbytheirabilitytoproduceathermostabledirecthemolysin
(TDH),termedtheKanagawaphenomenon(82,120).Thetdhgenehasbeenclonedandsequenced
(86,87).DNAprobesnowareavailabletotestforthisvirulencemarkerinV.parahaemolyticusisolates
(42,77,87).Athermostablerelatedhemolysin(TRH),whichshares60%homologywithTDH,hasalsobeen
associatedwithstrainscausinggastroenteritis(45,46).Presently,thereisnoinvitrotesttodetectTRH
production.ManyclinicalstrainsofV.parahaemolyticus,producebothTDHandTRH(8,106).Taniguchiet
al.(123)describedathermolabilehemolysin,TLH,foundinallV.parahaemolyticusstrains,butnotinother
species.PCRproceduresandgeneprobeshavebeendevelopedtodetectthetlh,tdhandtrhgenesinV.
parahaemolyticus(8,37,49,76,77).
V.vulnificus
V.vulnificus(33),theleadingcauseofdeathintheUSrelatedtoseafoodconsumptionandnearlyalways
associatedwithrawGulfCoastoysters(90,104),resemblesV.parahaemolyticusonTCBSagar,butcanbe
differentiatedbyseveralbiochemicalreactions,includinggalactosidaseactivity(31).Epidemiologicaland
clinicalinvestigationshaveshownthatV.vulnificuscausessepticemiaanddeathfollowingingestionof
seafoodorafterwoundinfectionsoriginatingfromthemarineenvironment(43,118,129).Recentgeneprobe
assays(29,134),PCRprocedures(41),fattyacidprofiles(68)andenzymeimmunoassays(31,122)have
beendevelopedtodetectandidentifythispathogen.
Otherspecies
Thefollowingspecieshavealsobeenisolatedfromhumanstoolsand/orfrompatientswithgastroenteritis,
withtheconsumptionofshellfishasthepredominantsourceofinfection(96).V.metschnikoviidiffersfromall
otherVibriospeciesinlackingcytochromeoxidase(7).Somestrains(biotypeII)ofV.fluvialissp.nov.(now
designatedV.furnissii)producegasduringDglucosefermentation(15).V.hollisaeisahalophilicspecies
thatgrowspoorly,ifatall,onTCBSagarwhichexhibitsadelayedmotilitypattern(>48hr)uncharacteristicof
theothervibrios(7).AvariantofthetdhgenevirulencemarkerofpathogenicV.parahaemolyticusstrains
wasdetectedinsomeV.hollisaestrains(40).
Differentiationofspecies
Table1presentsthedifferentialcharacteristicsofthespeciesmostoftenassociatedwithhumanillness
relatedtoseafoodconsumption.Tablescanalsobefoundinseveralpublications,includingBaumannand
Schubert(7),Elliotetal.(31),McLaughlin(78)andWestetal.(131).
Table1.BiochemicalcharacteristicsofhumanpathogenicVibrionaceaecommonlyencounteredinseafood*
V.alginolyticus
V.cholerae
V.fluvialis
V.furnissii
V.hollisae
V.metschnikovii
TCBSagar
NG
mCPCagar
NG
NG
NG
NG
NG
CCagar
NG
NG
NG
NG
NG
AGS
KA
Ka
KK
KK
Ka
KK
Oxidase
0%NaCl
3%NaCl
Arginine
dihydrolase
Ornithine
decarboxylase
Lysine
decarboxylase
Growth
in
(w/v):
6%NaCl
8%NaCl
10%NaCl
nd
Lactose
Arabinose
nd
nd
Gelatinase
Urease
Growthat42C
Sucrose
D
Cellobiose
Acid
from:
D
Mannose
D
Mannitol
ONPG
Voges
Proskauer
10g
O/129
Sensi
150g
tivity
O/129
to:
*AdaptedfromElliotetal.(31)
**Aeromonashydrophila,Plesiomonasshigelloides
Abbreviations:TCBS,thiosulfatecitratebilesaltssucrosemCPC,modifiedcellobiosepolymyxinBcolistinAGS,arg
Y=yellowNG=noorpoorgrowthS=susceptiblend=notdone
G=greenV=variableamongstrainsR=resistantP=purple,V=variable
KK=Slantalkaline/ButtalkalineKA=Slantalkaline/Buttacidic,Ka=Slantalkaline/Buttslightlyacidic
DistributionandSourcesofContamination
V.cholerae
V.choleraeO1isexcretedingreatnumbersinthefecesofcholerapatientsandconvalescents(34,54).The
diseaseistransmittedprimarilybythefecaloralroute,indirectlythroughcontaminatedwatersupplies
(30,78,80,116,126,130).Directpersontopersonspreadisnotcommon.Foodsuppliesmaybe
contaminatedbytheuseofhumanfecesasfertilizerorbyfresheningvegetablesformarketwith
contaminatedwater(30,57,58,80,94).CholeraoutbreaksinseveralcountriesandtheUSarethoughtto
haveresultedfromtheconsumptionofraw,undercooked,contaminated,orrecontaminatedseafood.
ToxigenicV.choleraeO1israrelyisolatedfromUSenvironmentsandfoodsandnoisolationsofserogroup
O139havebeenreportedinthiscountry.Incontrast,nonO1/O139strainsarecommonlyisolatedfrom
estuarinewaterandshellfish(5,126).EvidencesuggeststhatV.choleraeO1isacomponentofthe
autochthonousfloraofbrackishwater,estuaries,andsaltmarshesofcoastalareasofthetemperatezone,
posinganongoinghazardtopublichealth(11,126).VariousO1strainshavebecomeendemicinmany
regionsintheworld,includingAustraliaandtheGulfCoastregionoftheUS(19,127).
V.parahaemolyticus
Thisorganismisfrequentlyisolatedfromcoastalwatersandseafoodintemperatezonesthroughoutthe
world.ItisthemostfrequentcauseoffoodbornediseaseinJapan(89),wheremanyresidentseatrawfish.
AnumberofcommonsourcegastroenteritisoutbreaksattributedtoV.parahaemolyticushaveoccurredin
theUS(57),associatedwithoysterconsumption(88,96).SomefoodsimplicatedintheUSarecrab,shrimp,
andlobster,whichunlikefishinJapan,typicallywerecookedbeforeeating.Mishandlingpractices,suchas
improperrefrigeration,insufficientcooking,crosscontamination,orrecontaminationaresuspectedinthese
outbreaks.Recently,consumptionofrawoysterswasassociatedwithlargeoutbreaksofV.
parahaemolyticusgastroenteritisontheWestCoastin1997(17),andinTexasandNewYorkin1998(18).
ClinicalstrainsfromtheWestCoastwereureasepositiveandpossessedbothtdhandtrhgenes.TheTexas
andNewYorkoutbreakswerecausedbyaureasenegativeO3:K6serotype,possessingonlytdh.This
strainappearstohavebecomepandemicandisthemostprevalentstraininAsia(10,23,74,135).
V.vulnificus
Theinvasivespecies,V.vulnificus,thecausativeagentofsepticemicshock(63,90,118),isacommon
organismincoastalwatersofsomeareasoftheUSandothercountries(60,90,122,124).Itisreportedto
cause20to40U.S.caseseachyearofprimarysepticemiawitha50%mortalityrateamongindividualswith
liverdiseaseandelevatedserumironlevels(104).Areviewofcaseshasdeterminedanassociation
betweensepticemiaandconsumptionofrawoysters,nearlyallfromGulfCoastwaters.Thisspecieshas
alsobeenresponsibleforwoundinfectionsinindividualswhoareassociatedwithmarineenvironments(90).
ThishalophilicspecieswillgrowonorinmanylaboratoryformulationsofmediathatcontainNaCla0.5%
minimumconcentrationisrecommended.Althoughvirulenceisassociatedwithacapsule,noreliablemarker
hasbeenidentifiedmosttestscannotdistinguishclinicalfromenvironmentalstrains(79,108,132).
Otherhalophilicvibrios
LikeV.parahaemolyticus,V.cholerae,andV.vulnificusV.alginolyticus,V.fluvialis,V.furnissii,V.
metschnikovii,andV.hollisaearerecoveredfrombrackishcoastalwaters,sediment,andsealifetakenfrom
thetemperateestuarineenvironment(7).Thesespeciesarenormalcomponentsofthatenvironment,
appearonaseasonalbasis,andhavebeenassociatedwithhumanillness(12,96).
MethodsofIsolation
Vibriospecies,likemanyotherGramnegativebacteria,growinthepresenceofrelativelyhighlevelsofbile
salts.Theyarefacultativelyanaerobicandgrowbestunderalkalineconditions.Isolationfromfoodsis
facilitatedbytheuseofmediaformulatedwithanalkalinepH.Alkalinepeptonewater(APW)isused
commonlyforisolatingseveralspeciesofconcern.
ThestricthalophilicnatureofV.parahaemolyticusprobablyaccountsforthefactthatillnessescausedby
thisorganismwerenotdocumentedintheUSuntilworkersbeganexaminingfoodandfecesonappropriate
mediacontainingaddedsalt.MediausedfortestingthebiochemicalreactionsofV.parahaemolyticusshould
contain2%or3%NaCl.V.vulnificusrequiresNaClforgrowth.Aminimumof0.5%NaCl,theconcentrationof
mostpreparedmedia,isadequate.Diluentusedfortransferofcellsuspensionsordilutionpreparationmust
containNaClforexample,phosphatebufferedsaline,PBS(31).
TCBSagar(31)isamediumcommonlyusedforisolatingV.cholerae,V.parahaemolyticus,andother
speciesfromseafood.Thismediumsupportsgoodgrowthofmostspecieswhileinhibitingmostnonvibrios
(65).RecentformulationsforselectiveagarsfortheisolationofV.vulnificushavealsoprovedeffective.
Amongthesemedia,modifiedcellobiosepolymyxincolistin(mCPC)(31)andCC(44)agarswereformulated
todifferentiateV.vulnificusfromothervibrios.V.choleraestrains,excepttheClassicalbiotype,willgrowon
mCPCagar,whilemostV.parahaemolyticusstrainsandotherspecieswillnot.Tofacilitatetheidentification
ofsuspectisolates,therapiddiagnostickitAPI20Ecanbeusedinlieuofthemanybiochemicalmedia
neededforidentification(31,92)Additionally,DNAprobesorPCRcanbeusedforidentificationofV.
vulnificusandV.parahaemolyticus(29,41)
GENERALCONSIDERATIONS
StorageofSample
Thesampleshouldbecooledimmediatelyaftercollection(about7Cto10C),thenanalyzedassoonas
possible.Directcontactwithiceshouldbeavoidedtomaximizesurvivalandrecoveryofvibrios.Vibrioscan
beinjuredbyrapidcooling,butgrowrapidlyinseafoodatambienttemperatures(20,21).Despitethe
recognizedfragilityofthevibriostoextremesofheatandcold,theirsurvivalisenhancedundermild
refrigeration(13,14,16,38,50,95).Whenfrozenstorageofthesampleisrequired,atemperatureof80Cis
recommended,iffeasible(14).
ShellfishsamplesshouldbehandledaccordingtorecommendedproceduresdescribedbytheAmerican
PublicHealthAssociation(4).Tentotwelveanimalsarepooled,asepticallyshuckedtoasterileblenderjar,
andblendedathighspeedfor90sec.ThiscompositeisusedtopreparedilutionsusingaNaClcontaining
solution,suchasPBS.
Tofacilitatethestorageandfurtheranalysisofnumerousisolatesfromasample,thefollowingprocedureis
recommended.Thismethodallowsforthegeneprobeanalysisofmanyisolatesobtainedfromasample,in
contrasttotheminimalnumberthatcanbefeasiblyhandledusingtraditionalbiochemicaltests.Asterile96
wellmicrotiterplateisfilledwith100l/wellofAPW.Numerouscoloniesofpresumptivevibriosarepicked
fromaselectiveagarplateusingasteriletoothpickorwoodtransfersticktoindividualwells.Theinoculation
patternisrecordedandtheplateisincubated35horovernightat352C.A48prongreplicatorisusedto
replicate/transferisolatesinthewellstoanagarplateforgeneprobeanalysis.Afterreplication,100lTSB
1%NaCl24%glycerol(TSG)isasepticallydispensedtoeachwell.Theplateiswrappedinadoublelayerof
foilorplasticandplacedinanultralowfreezer,72to80C,forstorageofcultures.Whenneeded,the
plateispartiallythawedandtheculturesfromthewell(s)transferred,orreplicatedtoanewmicrotiterplate
ortubedmedium.PurityoftheculturecanbedeterminedbystreakingtoanagarmediumsuchasT 1N3.
GeneticBasedTechniques
Thesenewertechnologieshavetheadvantageofmorerapiddetectionandidentificationandareincluded
forthoselaboratorieswiththeproperequipment.PCRbasedidentificationoffersaonedayanalysis
(5,8,9,35,41,66,67,107,109,119,125),whilegeneprobeprocedures,includingthosepresentedinthis
chapter,areonetotwodayanalyses(29,37,42,61,69,76,77,87,97,100,133,134,136,137).Thetraditional
qualitativeprocedureandthemostprobablenumber(MPN)techniquerequirefourtosevendaysto
complete(31).Alkalinephosphatase(AP)labeledprobestoidentifythepresenceofV.parahaemolyticus
andstrainsharboringthetdhgene,anddetectingV.vulnificusinasampleareavailablecommercially.One
lotofcommerciallyAPlabeledprobeisenoughtoprocessapproximately200filters.Inexpensivepaper
filters(Whatman541)canbeusedforcolonylifts.
Digoxigenin(dig)labeledampliconprobes(97)arealsopresentedforthethreespeciesofconcern.
Advantagesofthediglabeledprobeprocedureare:(a)canbepreparedinhouse(b)inexpensiveto
prepare(c)morereportergroupsperprobemolecule(d)twicethenumberofcopiesoftheprobeprepared
asthereversecomplimentisalsolabeled,(e)probesolutioncanbeusedseveraltimes,(f)thehybridization
andwashtemperatureisthesameforalldigprobes,(g)thenylonmembranecanbestrippedofaprobe
andhybridizedwithanadditionalprobe(s),and(h)usinganylonmembraneallowsforthetransferbetween
agarsurfaces,i.e.,fromanonselectiveagarforresuscitatingcellspriortomovingtoaselectiveand
differentialagar.HybridizationtimesaregreaterthanwithAPlabeledprobesandnylonmembranesare
moreexpensivethanpaperfilters
RecommendedControls
Morethanoneplatingmediumshouldbeusedforvibriosbecausestrainsmayvaryintheirgrowth
characteristics.T 1N3agarworkswellforallvibriosrelevanttohumanhealth.Positiveandnegativecontrol
strainsshouldbeusedforallphenotypicandgenotypicassaystoensureappropriateinterpretationofthe
reactions.
Media,Reagents,SuppliesandEquipment
1. 1.Alkalinepeptonewater(APW)(M10)
2.AKImedium(M7)
3.Arginineglucoseslants(AGS)(M16)
4.Bloodagar(5%sheepredbloodcells)(M20)
5.Casaminoacidsyeastextract(CAYE)broth(M34)
6.modifiedCellobiosepolymyxincolistin(mCPC)agar(M98)
7.Cellobiosecolistin(CC)agar(M189)
8.Motilitytestmedium1%NaCl(M103)
9.Oxidasereagent(1%N,N,N,N'tetramethylpphenylenediamine.2HClindH2O)(R54)
10.PeptoneTweensaltdiluent(PTS)(90)
11.Phosphatebufferedsaline(PBS)(R59)
12.PolymyxinBdisks,50U(Difcoorequivalent)(R64)
13.Salinesoln0.85%indH2O(R63)
14.2%NaClsoln(R71)
15.Sodiumdesoxycholate0.5%insteriledH2O(R91)
16.Thiosulfatecitratebilesaltssucrose(TCBS)agar[M147]
17.T 1N1andT 1N3agars(1%tryptoneandeither1%or3%NaCl)(M163)
18.T 1N0,T 1N3,T 1N6,T 1N8,T 1N10broths(M161)
19.Trypticsoyagarmagnesiumsulfate3%NaCl(TSAMS)(32)Trypticase(ortryptic)soybroth(TSB),
agar(TSA)(M152)(withaddedNaCl,2%)
20.TSB1%NaCl24%glycerol
21.Ureabroth(M171)(orChristensen'sureaagar(M40)withaddedNaCl(2%)(R71)
22.V.choleraepolyvalentO1andO139antiserum
23.VETRPLATD920Aenterotoxindetectionkit(Oxoid,Inc.)
24.Vibrioparahaemolyticussucroseagar(VPSA)(M191)
25.Vibriovulnificusagar(VVA)(M190)
26.API20Ediagnosticstripsandreagents(BioMerieux)
2.ProbeReagents,EquipmentandMaterialsRequired
1.Shakingwaterbath(s)capableofupto65C.(tempsneeded,42,54,55and65C)
2.Shakerplatformatroomtemperature
3.Microwave
4.LongwaveUVlightboxorUVCrosslinker(254nmwavelength)
5.Heattolerantbags(andsealer)orplastictubswithlids(300500mlcapacity)
6.96wellmicrotiterplateswithlids
7.8or12channelmicropipetter
8.48prongreplicator
9.Whatman541filters,85mm(specialorderforthisdia,1541085fromWhatman)
10.Whatman#3orequivalentabsorbentfilterorpad
11.Nylonmembranes(MagnaGraphTransfermembrane)(positivecharge),82mm(Osmonics,Inc,
Westboro,MA,griddedNJOHG08250,plainNJOHY08250)
12.Fiberglassmeshscreens,householdwindowscreenavailableathardwarestores(59)
13.Sterilehockeysticks
14.Steriletoothpicksorwoodapplicatorsticks
15.Glasspetridishes,100mm
16.Lysissoln(0.5MNaOH,1.5MNaCl)(MaasI)(R94)addtoreagentslist
17.Neutralizingsoln(1.0MTrisHCl,pH7.0in2.0MNaCl)fornylonmembranes(MaasII)(R95)
18.2Mammoniumacetatebuffer(forAPlabeledprobeand541filters)(R1)modifyreagent1toinclude
this.
19.1SSC,5SSC,20SSC(standardsalinecitrate)(R77)edittoadd1,5,20
20.1SSC1%SDS(sodiumdodecylsulfate)and3SSC1%SDS(R93)addnewreagent
21.10%Sarkosylsoln(Nlauroylsarcosine,sodiumsalt)(R96)addnewreagent
22.10%SDSsoln(sodiumdodecylsulfate)(R92)addnew
23.1MTris,pH7.5(TrizmabaseSigmaCat.No.T1503)
24.1MTris,pH9.5(TrizmabaseSigmaCat.No.T1503)
25.3MNaCl
26.1MMgCl2
27.ProteinaseKstocksolution(20mg/ml)
28.Hybridizationsolution(BSA,SDS,PVPin5SSC)(forAPlabeledprobe)
29.NBT/BCIPcolorreagent[nitrobluetetrazoliumchloride/5bromo4chloro3indolylphosphate],
toluidinesalt,RocheDiagnosticsCat.No.1697471(forcolorimetricdetection)
30.Digbuffers1,2,3and4(97)
31.10mMTrisHCl,1mMEDTA,pH8.0
32.Blockingreagent(RocheDiagnosticsCat.No.1096176)
33.WashsolnAandB(97)
34.AntiDigAP[Antidigoxigeninalkalinephosphatase,Fabfragments](RocheDiagnoticsCat.No.
1093274)
35.dig11dUTP(RocheDiagnosticsCat.No.1093088)
36.CSPDRocheDiagnosticsCat.No.1755633(forchemiluminescentdetection)
PRECAUTIONS
AgoodselectiveenrichmentbrothhasnotbeendevelopedforV.cholerae.However,duetoitsrapid
generationtime,shortincubationperiodsareeffectiveforisolation.APWprovidessuitableenrichmentfor
incubationperiodsof6to8h,butothercompetingmicrofloramayovergrowV.choleraeduringlonger
enrichmentperiodsforcertaintypesofsamples.Overnightperiods(16to18h),althoughnotdesirable,
havebeenusedtofacilitatesampleanalysisduringworkhours.Iftheproductwassubjectedtoaprocessing
step,i.e.heating,freezing,drying,orlowdensitiesareexpected,incubationovernightisrecommendedto
thoroughlyresuscitateinjuredcells.TheincubationofrawoystersamplesinAPWat42Cfor6to8hhas
proveneffectiveforisolationofV.choleraeandisrecommended(26).However,DePaolaandHwang(28)
foundthatenrichmentincubationfor18to21h,insteadof6to8h,gaveahigherrecoveryofO1V.
choleraewhenlowinoculawereused.Becauseoftheseconsiderations,itisrecommendedtostreakAPW
enrichmentsbothafter6to8handafterovernightincubation.Itisalsorecommendedforrawoysterstouse
a1:100ratioofoystertoAPW(28).
PROCEDURES
V.cholerae:
1.Enrichmentandplating
1.Weigh25gofsampleintoataredjar(capacityapproximately500ml).Productssuchasseafoodor
vegetablesmaybeblendedorcutintosmallpieceswithsterilescissors.
2.Add225mlAPWtojar.Thoroughlymixthesampleorblend2minathighspeed.
3.IncubateAPWat352Cfor6to8h.Reincubatethejarovernightifthesamplehadbeenprocessed
insomeway.Foranalysisofrawoysters,includeasecondtaredflaskwith25gofproductplus2475
mlAPW.Thisflaskshouldbeincubated18to21hat420.2Cinawaterbath(28,31).An
enumerationtechniquebymostprobablenumber(MPN)mayalsobeperformedifdesired.
4.PreparedriedplatesofTCBSagar.ModifiedCPCorCCagarsmayalsobeincluded.
5.Transfera3mmloopfulfromthesurfacepellicleofAPWculturetothesurfaceofadriedTCBSplate
(andmCPCorCC),andstreakinamannerthatwillyieldisolatedcolonies.
6.IncubateTCBSovernight(18to24h)at352C.IncubatemCPCandCCovernightat3940Cifa
3940Cincubatorisnotavailablethen3537Cisusuallyadequateasselectivityisdetermined
primarilybyantibioticsinformulationthanbyhightemperature.
7.TypicalcoloniesofV.choleraeonTCBSagararelarge(2to3mm),smooth,yellowandslightly
flattenedwithopaquecentersandtranslucentperipheries.
8.TypicalcoloniesofV.choleraeonmCPCorCCagararesmall,smooth,opaque,andgreentopurple
incolor,withapurplebackgroundonextendedincubation.
9.Forbiochemicalidentification,coloniesfromcrowdedplatesmustbestreakedtoanonselectiveagar
(T 1N1,T 1N3,orTSA2%NaClagar)forpurity.Incubateovernightat352Candproceedwith
identificationusingasingleisolatedcolony.
10.SubculturethreeormoretypicalcoloniesfromeachplatingmediumtoT 1N1agarslantsormotilitytest
mediumstabs.Incubateslantsorstabsovernightat352C.
2.ScreeningandConfirmation
1.Arginineglucoseslant(AGS).InoculateeachsuspectT 1N1culturetoAGSbystreakingtheslantand
stabbingthebutt.IncubateAGSwithloosecapovernightat352C.V.choleraeandV.mimicus
cultureswillhaveanalkaline(purple)slantandanacid(yellow)butt,asarginineisnothydrolyzed.No
gasorH2Sisproduced.
2.Salttolerance.FromT 1N1culture,lightlyinoculateonetubeeachofT 1N0andT 1N3broths.Incubate
tubesovernightat352C.V.choleraeandV.mimicuscultureswillgrowwithoutNaCl.
3.Stringtest.Thestringtest(110)isausefulpresumptivetestforsuspectedV.choleraeasallstrainsare
positive.EmulsifyalargecolonyfromaT 1N1agarcultureinasmalldropof0.5%sodium
desoxycholateinsteriledH2O.Within60secthecellslyse(lossofturbidity)andDNAstringswhena
loopfulislifted(upto2to3cm)fromtheslide.
4.Oxidasereaction.TransfertheovernightT 1N1growthusingaplatinumwire(nichromewireshouldnot
beused)orwoodapplicatorsticktoafilterpapersaturatedwithoxidasereagent(1%N,N,N,N'
tetramethylpphenylenediamine.2HCl).Adarkpurplecolordevelopingwithin10secindicatesa
positivetestgrowth.Alternatively,addadropofreagenttothegrowthonaT 1N1slantoragarplate.V.
choleraeandV.mimicusareoxidasepositive.
5.Serologicagglutinationtest.SerotypingofsuspectV.choleraeculturespassingthestringtestusing
somaticorOantigensgivesimportantepidemiologicalevidence.Twomajorserotypesofserogroup
O1,OgawaandInaba,andserogroupO139arerecognizedashumanpathogens.Thetwoserotypes
ofO1areseeninboththeclassicalV.choleraeandtheElTorbiotypes.TheO139serogroup
resemblesonlytheElTorbiotype.
1.Foreachculture,markoffthreesections(withwaxpencil)about12cmontheinsideofaglass
petridishorona23inchglassslideandaddonedropof0.85%salinesolutiontothelowerpart
ofeachmarkedsection.Withasteriletransferlooporneedle,emulsifytheT 1N1cultureinthe
salinesolutionforonesection,andrepeatfortheothersection.Checkforagglutination.
2.AddadropofpolyvalentV.choleraeO1antiserumtoonesectionofemulsifiedcultureandmixwith
asterilelooporneedle.AddadropofantiO139toaseparatesection.(Thirdsection)
3.Tiltthemixturebackandforthforoneminandobserveagainstadarkbackground.Apositive
reactionisindicatedbyarapid,strongagglutinationinaclearbackground.
4.Ifpositive,testseparatelywithOgawaandInabaantisera.TheHikojimaserotypereactswithboth
antisera.
5.AntibodiestotheInabaandOgawa,andgroupO1antigenarecommerciallyavailable(i.e.
ColumbiaDiagnosticsInc.,Springfield,VA).Similarly,O139antiserumiscommerciallyavailable.
ResultsofnonagglutinableculturesshouldbereportedasnonO1/O139V.cholerae.
3.Biochemicaltests
Table2presentstheminimalnumberofcharactersneededtoidentifyV.choleraestrains.TheabilityofV.
choleraetogrowin1%tryptonewithoutaddedNaCldifferentiatesitfromothersucrosepositivevibrios.
TheAPI20Ediagnosticstriphasbeenusedsuccessfullyforidentificationandconfirmationofisolates
(92).Themicrotiterplatesystemforstorageofsuspectisolatescanbeusedhere.
4.DifferentiationofElTorandClassicalbiotypes.
AlthoughtheClassicalbiotypeisrarelyencountered,thefollowingareoptionalteststodifferentiatethem
fromtheElTorbiotype:
1.Betahemolysis.ThemostcommonmeansofdifferentiatingthebiotypesofO1V.cholerae,and
perhapstheeasiest,istodeterminehemolyticabilityonsheepbloodagar.ElTorstrainsare
hemolytic,whileclassicalstrainsdonotproduceahemolysin.Inoculateabloodagarplatewithtest
culturesbyspottingtothesurfaceandincubate1824hat352C.Betahemolysincanbe
determinedbyaclearzonearoundthegrowthoftheculture.
2.PolymyxinBsensitivity.StreakasuspectculturetoadryT 1N1agarplateandplacea50unitdiscof
polymyxinBonthesurface.Inverttheplate,incubateovernightat352C,andrecordtheresult.
Classicalstrainsaresensitive(>12mmzone)ElTorstrainsareresistant.Ifthesuspectculturegrows
onmCPCagar,whichcontainspolymyxinB,itisconsideredtobeoftheElTorbiotype.
5.Determinationofenterotoxigenicity
MoststrainsofV.choleraeisolatedfromfoodsortheenvironmentdonotproducecholeratoxin,(CT)and
arenotconsideredvirulent.IsolatesidentifiedasV.choleraeorV.mimicusshouldbetestedforthe
productionofCTorthectxgene(111).
1.Y1mouseadrenalcellassay(98).CThasbeenshowntostimulatetheenzymeadenylatecyclase
withtheproductionofcyclicadenosinemonophosphatethatultimatelyinfluencesseveralcellular
processes.IntheY1cellassay,CTpromotestheconversionofelongatedfibroblastlikecellsinto
roundrefractilecells.
Themaintenanceandpassageofcellcultures,preparationofmicrotiterassayplatesandconductand
interpretationofassayarecarriedoutasinChapter4Escherichiacoliofthismanual.
1.InoculatetestculturesfromT 1N1slantstotubesofCAYEbrothandincubateovernightat302C.
2.Inoculatea10mlportionofCAYEbrothina50mlErlenmeyerflaskfromeachstationaryculture
incubatefor18hrwithshaking.Centrifugeeachtestculturefilterthesupernatantthrougha0.22
mfilter.Refrigeratedfiltratesmaybestoredforupto1week.
3.Addaliquotsof25lfromeachfiltrate,bothunheatedandheatedto80Cfor30min,towellsofthe
microtiterassayplate.Inadditiontofiltratesfromknowntoxigenicandnontoxigeniccultures,add
0.025mlaliquotsfrompreparationscontaining1.0and0.1ngCT/ml.Suppressionofcellrounding
bytreatmentoftestfiltrateswithantiCTserumisanadvisablecontrolfornonspecificreactions.
2.ImmunoassayforCT.Acommerciallyavailableimmunoassayhasbeendevelopedtodetectthe
presenceofCTinculturalfiltratesofV.choleraeandV.mimicus(VETRPLA,Oxoid,Inc.,Ogdensburg,
NY).
1.InoculatetestculturesintoAKImediumandincubateat352C18hwithshakingat100rpm.
Centrifuge5to7mlofcultureat8,000gfor10min.Filtersterilizethesupernatantthrougha0.2
mfilterorusedasis.Testthesupernatantorfiltratefollowingthemanufacturer'sprotocolusing
conical96wellmicrotiterplates.Incubatetheplateovernight,undisturbedatroomtemperature.
3.Othertoxins
Thesignificanceofothertoxinsinhumanpathogenicityispoorlyunderstoodandtheseassaysarenot
recommendedforroutineanalysis.Maddenetal.(73)alsodemonstratedclinicalisolatesthatwere
pathogenicforinfantrabbits.AheatlabilecytolysinproducedbyV.choleraenonO1/O139wasfound
byMcCardelletal.(75)tobecytotoxictoY1mouseadrenalandChinesehamsterovarycells,tobe
rapidlyfataluponintravenousinjectionintoadultmice,andtocausefluidaccumulationinrabbitileal
loops(112).Culturesmaybetestedforheatstableenterotoxin(ST)(75,121)orcytotoxin(83,100)if
desired.
6.Genotypicdetectionofthecholeratoxingenebypolymerasechainreaction(67)
TheCTgenemaybepresentinstrainsofV.choleraeandV.mimicus,butnotexpressedunder
experimentalconditions.ThusagenotypicassaysuchasPCRamplificationofthectxgeneis
recommended.Thisprocedureoffersamorerapidresultandislesscomplicatedthanphenotypicassays.
1.CholeratoxinPCRprimers,10pmol/lstocksolutions.
1.Forward5'tgaaataaagcagtcaggtg3'
2.Reverse5'ggtattctgcacacaaatcag3'
ThePCRproductisa777bpfragment.
2.APWenrichment.Fromsamplepreparationaboveofthe621hincubation,prepareacrudelysatefor
PCRbyboiling1mlofAPWenrichmentmixtureina1.5mlmicrocentrifugetubefor10min.Lysatecan
beusedimmediatelyforPCRorstoredat20Cuntiluse.ForsuspectV.choleraeandV.mimicus
isolatesandcontrolcultures,inoculate1mlvolofAPW,incubate18hat352Candproceedwith
boilingstep.
3.TominimizecrosscontaminationofPCRreagents,itisrecommendedthataPCRmastermixbe
preparedandaliquotsstoredfrozen(20C)untiluse.Mastermixescontainallnecessaryreagents
exceptTaqpolymeraseandthelysate(template)tobeamplified.Thefinalreactioncontains:10mM
TrisHCl,pH8.31.5mMMgCl2200MeachofdATP,dTTP,dCTP,dGTP2to5%(v/v)APWlysate
(template)0.5Mofeachprimerand2.5UTaqpolymeraseper100lreaction.Volumesof25to
100lmaybeused.AddTaqpolymerasetothemastermixandaddtemplateupondistributionto0.6
mlmicrocentifugetubereactionvessels.Somethermocyclersmayrequireamineraloiloverlay(5070
l).Thefollowingthermocyclerconditionsshouldbeused:
Thermocyclerconditions:
time
temperature
(min)
(C)
Initialdenaturation
94
Denaturation
94
Primerannealing
55
Primerextension
72
Finalextend
72
No.cycles:Nomorethan35
4.AgarosegelanalysisofPCRproducts.Mix10lPCRproductwith2l6loadinggelandloadsample
wellsof1.5to1.8%agarosegelcontaining1g/mlethidiumbromidesubmergedin1TBE.Usea
constantvoltageof5to10V/cm.IlluminategelwithaUVtransluminatorandvisualizebandsrelative
tomolecularweightmarkermigration.Theprimerslistedgivea777bpfragmentofctxAB.Polaroid
photographscanbetakenofthegelfordocumentation.Positiveandnegativeculturecontrolsand
reagentcontrolshouldbeincludedwitheachPCRrun.
ProbeshavebeendevelopedtoalsodetectthepresenceofctxAB(133)Adiglabeledprobecanalso
bepreparedofthePCRamplificationproductfordetectionofthectxABgeneusingcolony
hybridization.Thepreparationoftheprobe,thehybridizationconditionsforcolonyblotsofsuspect
isolatesandwashprotocolfollowthoseoutlinedinthetechnicalliteratureofRocheDiagnostics,
Indianapolis,IN(97).
5.Finalreport.
ThefinalreportforV.choleraeshouldincludebiochemicalandserologicalidentificationoftheisolate
andenterotoxicityresults.Theminimalnumberofcharacterstoidentifythespeciesarepresentedin
Table2.
Table2.MinimalNumberofCharactersneededtoIdentify
V.choleraeandV.parahaemolyticusStrains
PositiveReaction
Percentage
Gramnegative,asporogenousrod
100
oxidase
100
String
100
Llysinedecarboxylase
100
Largininedihydrolase
Lornithinedecarboxylase
98.9
growthin1%tryptonebrotha
99.1b/0c
aNosodiumchlorideadded.
bV.cholerae(andV.mimicus)
cV.parahaemolyticus
FromHughandSakazaki(48)
OtherVibrios
V.parahaemolyticus
ThreeanalyticalschemesforenumeratingV.parahaemolyticusarepresented.ThefirstistheMPN
procedurecommonlyusedbymanylaboratories.Inaddition,thisprocedureisnearlyidenticalfor
enumerationofV.vulnificus.Thesecondisamembranefiltrationprocedureusinghydrophobicgrid
membranefilter(HGMF)(32).ThethirdisadirectplatingmethodusingDNAprobesforidentificationofthe
totalV.parahaemolyticuspopulation(76)andpathogenic(TDHcontaining)strains(77).Inaddition,aTRH
geneprobeprocedureandaPCRconfirmationanalysis(8)arealsoincluded.
1.Seafoodsamples:Enrichment,isolation,andenumeration.
1.Weigh50gofseafoodsampleintoablender.Obtainsurfacetissues,gills,andgutoffish.Shellfish
samplesincludemeatandliquor.Normally12animalsarepooled,blendedathighspeedfor90sec
and50gofhomogenateusedforanalysis.Forcrustaceanssuchasshrimp,usetheentireanimalif
possibleifitistoolarge,selectthecentralportionincludinggillandgut.Note:sameforV.vulnificus
2.Add450mlPBSdilutionwaterandblendfor1minat8,000RPM.Thisconstitutesthe1:10dilution.
Prepare1:100.1:1000,1:10,000dilutionsorhigher,ifnecessary,inPBS.
1.Formolluscanshellfish,pool12animals.Blend90secwithanequalvolofPBS(1:2diln)(4).
Preparea1:10dilutionbytransferring20.0g(weighingisrecommendedbecauseairbubblesinthe
1:2dilutionpreventaccuratevolumetrictransfer)ofthe1:2to80mlofPBS.Additional10fold
dilutionscanbepreparedvolumetrically(i.e.1mlof1:10to9.0mlofPBSfora1:100dilution.
2.Forproductthathasbeenprocessed,i.e.heated,dried,frozen,inoculate310mlportionsofthe
1:10dilutioninto3tubescontaining10mlof2APW.Thisrepresentsthe1gportion.Similarly,
inoculate31mlportionsofthe1:10,1:100,1:1000,and1:10,000dilutionsinto10mlofsingle
strengthAPW.IfhighnumbersofV.parahaemolyticusareexpected,theexaminationmaystartat
the1:10dilutionofproduct.
3.IncubateAPWovernightat352C.
4.Streaka3mmloopfulfromthetop1cmofAPWtubescontainingthethreehighestdilutionsofsample
showinggrowthontoTCBS(andmCPCorCCagarsforV.vulnificusisolation)
5.IncubateTCBSplatesat352C(andmCPCorCCplatespreferablyat3940Cor3537Cif39
40Cisnotavailable)overnight.
V.parahaemolyticusappearasround,opaque,greenorbluishcolonies,2to3mmindiameteron
TCBSagar.Interfering,competitiveV.alginolyticuscoloniesare,large,opaque,andyellow.Most
strainsofV.parahaemolyticuswillnotgrowonmCPCorCCagar.Ifgrowthoccurs,colonieswillbe
greenpurpleincolorduetolackofcellobiosefermentation.
Purifyisolatesasdescribedpreviouslyandinoculateamicrotiterplateforfreezerstorage.
2.ScreeningandConfirmation
1.Biochemicalidentificationofisolates.Unlessotherwisespecified,allmediainthissectionareprepared
tocontain2%or3%NaCl.TheAPI20Ediagnosticstripcanbealternativelyusedhere(92).Preparea
cellsuspensionofthesuspectculturesin2%NaClfortheAPI20E.
1.ScreensuspectculturesofV.parahaemolyticus(andV.vulnificus),usingAGS,andT 1N0andT 1N3
brothsasdescribedpreviously.Incubatetubesat352Cfor1824h.
2.TransfertwoormoresuspiciouscoloniesfromTCBSagarwithaneedletoarginineglucoseslant
(AGS).Streaktheslant,stabthebutt,andincubatewiththecaplooseovernightat352C.BothV.
parahaemolyticusandV.vulnificusproduceanalkaline(purple)slantandanacid(yellow)butt
(argininedihydrolasenegative),butnogasorH2SinAGS.
3.ForTSBandTSAslants(supplementedwith2%NaCl),inoculatebothmediaandincubate
overnightat352C.TheseculturesprovideinoculaforothertestsaswellasmaterialfortheGram
stainandformicroscopicexamination.BothV.parahaemolyticusandV.vulnificusareoxidase
positive,Gramnegative,pleomorphicorganismsexhibitingcurvedorstraightrodswithpolar
flagella.
4.Inoculateatubeofmotilitytestmediumbystabbingthecolumnofthemediumtoadepthof
approximately5cm.Incubateovernightat352C.Acircularoutgrowthfromthelineofstab
constitutesapositivetest.V.parahaemolyticusandV.vulnificusaremotile.
5.V.parahaemolyticusandV.vulnificuswillonlygrowinT 1N3butnotinT 1N0.Onlythesaltrequiring
culturesneedtobetestedfurther.
Onlymotile,GramnegativerodsthatproduceanacidbuttandanalkalineslantonAGS,donot
formH2Sorgas,andaresaltrequiringrequirefurtherexamination.
6.TheidentifyingcharacteristicsofV.parahaemolyticusandV.vulnificusarepresentedinTable1
Biochemically,V.parahaemolyticusandV.vulnificusarephenotypicallysimilar,butcanbe
differentiatedbydifferencesoftheONPG,salttolerance,cellobioseandlactosereactions(Table1).
Byusingselectedbiochemicaltraits,V.parahaemolyticusandV.vulnificuscanbedistinguished
frommostinterferingmarinevibriosandothermarinemicroorganisms.
AllV.parahaemolyticusisolatesshouldbetestedforthepresenceofurease,byeitherusingurea
brothsupplementedwith2%NaCloronChristensen'sureaagarsupplementedwithNaCl,2%final
concorusingtheAPI20E.ClinicalstrainsfromtheUSWestCoastandfromAsiancountrieshave
beenpredominantlyureasepositive.Ureaseproductioniscorrelatedwiththepresenceofthetdh
and/ortrhgenes(2,49,62,88,91,114,115).Theureasereactionisavaluablescreeningtestfor
potentiallypathogenicstrains(62).
Inoculateureabroth3%NaClwithaheavyinoculumofcultureorspottheculturetosurfaceof
Christensen'sureaNaClagarplateorslant.Incubate352C1824h.
Productionofureaseisdeterminedbyapink(alkaline)colortothemedium.
Negativeculturesshouldbeincubatedanadditional24hfortherare,slowureaseproducing
strains.
WhenthecoloniesarefinallyidentifiedbiochemicallyasV.parahaemolyticusrefertotheoriginal
positivedilutionsintheenrichmentbrothandapplythe3tubeMPNtables(Appendix2)forfinal
enumerationoftheorganism.
7.Alternatively,isolatescanbeidentifiedasV.parahaemolyticusorV.vulnificusbyDNAprobe
hybridizationorPCRasdescribedinthefollowingsections.
2.Hydrophobicgridmembranefiltrationenumerationprocedure(HGMF)(25,32)
Theapparatus,filters,andspecificinstructionsmaybeobtainedfromQALaboratories,SanDiego,CA.
1.Preparea1:10dilutionofseafoodsamplewithpeptonetweensaltdiluent(PTS),andblend60sec
athighspeed.Filter1.0mlorothervolumeofhomogenatethroughHGMFusingsterilediluentasa
carrier.Withforceps,asepticallytransfertheHGMFfromthefiltrationapparatustothesurfaceofa
drytrypticsoyagarmagnesiumsulfateNaClplate(TSAMS)M152.Incubate4hat352C.Ten
folddilutionsmaybepreparedifhighlevelsofV.parahaemolyticusareexpected.
2.Withforceps,asepticallytransfertheHGMFfromtheTSAMStothesurfaceofadryV.
parahaemolyticussucroseagar(VPSA)plateM191.Invertplateandincubateat42Cfor18to20
h.
3.OnVPSA,V.parahaemolyticuscolonieswillbegreentobluefillingatleastonehalfofthegrid
square.Thisisapresumptiveenumeration.Atleastfiverepresentativecoloniesmustbeidentified.
Othergrowthwillnormallybeyellowduetosucrosefermentation.Confirmedsquaresmustbe
multipliedfortotaltypicalcoloniesandtheMPN/gofseafoodcalculated.Forexampleif3of5
presumptivecoloniesarebiochemicallyconfirmedasV.parahaemolyticus,thenthetotalnumberof
presumptivecoloniesshouldbemultipliedby0.6toestimatetheV.parahaemolyticusdensity.V.
vulnificuscolonieswillalsobeblue/greenincolor.DNAprobescandifferentiatethespecies
(29,61,76,134).
3.Serologictyping(47,81)
V.parahaemolyticuspossessesthreeantigeniccomponents:H,O,andK.TheHantigeniscommonto
allstrainsofV.parahaemolyticusandisoflittlevalueinserotyping.TheK,orcapsularantigen,maybe
removedfromthebacterialbodybyheatingtheisolatefor1or2hrat100C.Thisprocessexposes
theO,orsomatic,antigen,whichisthermostable.SincetheKantigenmaskstheOantigen,itis
necessarytoremovetheformerbyheatingbeforeperformingtheOagglutinationtests.
Thereare12Ogroupandover70knownKantigens(47).FiveoftheKantigenshavebeenfoundto
occurwitheitheroftwoOgroupantigenstherefore,thereare76recognizedserotypes(Table3).
SerologictestsbythemselvesarenotusedtoidentifyV.parahaemolyticusbecauseofcrossreactions
withmanyothermarineorganisms.However,duringinvestigationsoffoodborneoutbreaks,serologic
testsbecomeavaluableepidemiologictool.
Table3.AntigenicschemeofV.parahaemolyticus(1986)a
Ogroup
Ktype
1,25,26,32,38,41,56,58,64,69
3,28
4,5,6,7,27,30,31,33,37,43,45,48,54,57,58,59,65
4,8,9,10,11,12,13,34,42,49,53,55,63,67
5,15,17,30,47,60,61,68
6,18,46
7,19
8,20,21,22,39,70
9,23,44
10
l9,24,52,66,71
11
36,40,50,51,61
12
52
Total12
65
aTheantigenicschemewasfirstestablishedbySakazakietal.(101)andlaterextendedbythe
CommissionoftheSerotypingofV.parahaemolyticus(Japan)Kantigens,2,14,16,29,35,and62
wereexcludedbytheCommission(47).
Ktypes4,5,6,7,8,9,and19occurwithmorethanoneOgroup.
V.parahaemolyticusdiagnosticantiserumkitsareproducedcommerciallyinJapanandavailablefrom
NichimenCo.,1185AvenueoftheAmericas.31stFloor,NewYork,NY10036(212)7191000or
AccurateChemicalandScienceCorp,SanDiego,CA8002559378.Becausetheantiserumis
expensive,itisnotrecommendedformostlaboratories.CDChasserotypingcapability.
4.Determiningpathogenicity
Katoetal.(56)showedthatV.parahaemolyticusisolatesfromthestoolsofpatientswithenteric
infectionsarehemolyticonaspecialhighsalthumanbloodagar,whereasV.parahaemolyticus
isolatesfromseafoodandmarinewaterusuallyarenot.Wagatsuma(128)latermodifiedthisspecial
agartoavoidconfusionwiththeregularnormalhemolyticactivityofV.parahaemolyticuson
conventional5%sheepbloodagar.ThespecialagarwasnamedWagatsumaagarandthespecial
hemolyticresponsetheKanagawaphenomenon.Freshlydrawnhuman,dogorsheepbloodisusedin
preparationoftheagar.
ThecorrelationhasbeenwellestablishedthatV.parahaemolyticusstrainsthatcauseillnessin
humansarealmostalwaysKanagawapositiveandisolatesrecoveredfromseafoodarealmostalways
Kanagawanegative(81,82,101,102,106).Inaddition,extensiveinvestigationinanimalmodels
suggeststhattheKanagawahemolysinistheprimaryvirulencefactorinV.parahaemolyticus(82,120).
TheKanagawatest,orhybridizationwiththetdhgeneprobeprovidesreliableinformationonthe
presenceofpathogenicstrainsisolatedfromfoods.Duetothedifficultyofobtainingfreshbloodand
thestrongcorrelationbetweenKanagawaphenomenonandpresenceofthetdhgene,itis
recommendedtouseDNAprobemethodsdescribedinthischaptertodeterminepotentialvirulenceof
V.parahaemolyticusisolatesinsteadoftheKanagawaphenomenon.
1.GenotypicdetectionofhemolysingenesofV.parahaemolyticus.
AlkalinephosphataseanddigoxigeninlabeledDNAprobescanbeusedfortheidentificationofV.
parahaemolyticus.Athermolabilehemolysingene,tlh,hasbeenfoundinallstrainsofV.
parahaemolyticus,butnotinotherspecies(123)andDNAprobeshavebeenusedforidentification.
TwoDNAprobeproceduresthathavebeenshowntobeequivalentarepresented.DNAprobes
havealsobeenconstructedtodetectthethermostabledirecthemolysin,tdh(87)andthermostable
relatedhemolysin,trh(46),genesthatareassociatedwithpathogenicstrains.
Analkalinephosphataselabeled(AP)tlhprobe(76)iscommerciallypreparedforusewith
Whatman541colonylifts.ThehybridizationanddetectionprocedurefortheAPtlhandAPtdh
probes(77)arepresentedbelow,usingahybridizationandwashtemperatureof54C.Digoxigenin
labeledprobesfortlhandtrhwereconstructedofPCRamplificationproductsusingtheprimersets
reportedbyBejetal.(8).Thetdhprobewasconstructedusingaprimersetbasedonthe
oligonucleotideprobesofNishibuchietal.(87),usingtdh1astheforwardprimerandthereverse
complimentoftdh4(tdh4c)asthereverseprimer.Theprobesarelabeledwithdigoxigeninduring
amplificationaccordingtotheproceduredescribedbyRocheDiagnostics(97).Theseampliconsare
ofthefollowingsizes450bptlh,424bptdhand500bptrh.
1.Alkalinephosphataselabeledoligonucleotideprobes(APtlhandAPtdh)(76,77)
Storeprobesintherefrigeratorforonetotwoyearsdonotfreeze.
Probesequencesare:
Speciesspecific
thermolabilehemolysin(tlh)
APlabeled
5'Xaaagcggattatgcagaagcactg3'
(whereXistheAPlabel)
Thermostabledirecthemolysin(tdh),
APlabeled
theKanagawahemolysin,
5'Xggttctattccaagtaaaatgtatttg3'
ProbescanbepurchasedfromDNATechnologyApS,ScienceParkAarhus,GustavWledsd
Vej10,DK8000,AarthusC,Denmark.Phone4586203388,Fax4586202121,email
oligo@dnatech.aau.dk.
2.SamplepreparationanddilutionsarethesameaswiththeMPNprocedure.Inaddition,the
preparationofsampleandthehybridizationconditionsarethesameforthesimultaneous
enumerationofV.vulnificus,exceptplatingtoVVAM190anda55Chybridizationtemperature
(29).Justbeforeuse,thoroughlydryT 1N3M161(andVVA)agarplatesinvertedwithlidscracked
openfor1hat35C.Thisisanonselectiveagar.
1.Pool1012oystermeatsandhomogenizeinequalpart,byweight,withPBSfor90secathigh
speed(1:1dilution)(4).
2.Weigh0.20gofthisoyster:PBS(1:2)homogenatedirectlyfromblender(represents0.1gor
1dilutionofoystertissue)ontoT 1N3plateusingbalancewith0.01gsensitivitytotareplate.
Pipet100lof1,2and3dilutionsontoT 1N3plateswiththedilutions.Forshellfish
harvestedfromDecemberthroughMarch,plating1and2dilutionsisadequate,whileduring
MaythroughOctober,summermonths,1,2,and3dilutionsareadequateforplating.The
detectionlevelis10CFU/g.
3.Forseafoodotherthanoysters,theinitialdilutionof1:10shouldbeused,becauseofthe
productdebrisfromhomogenizing.InoculatethesurfaceofT 1N3with100lofthis1:10
dilutionofsample.Thusthedetectionlevelwillbe100CFU/g.
4.UsesterilehockeystickstospreadinoculumevenlyontoT 1N3agarplates.Dryplatesand
uniformdistributionofinoculumareessentialforadequatecolonyisolation.
5.Incubateplates1824hat352C.Allplatesshouldbeusedforcolonyliftsandhybridization
unlessthereisconfluentgrowth.
3.Filterpreparation
1.OverlayT 1N3(andVVA)plateswithlabeled(samplenumber,dilution)#541Whatmanfilters
(85mm)for1to30min.Forplatestobeusedfortdhdetection,markfilterandplatefor
alignmentandsubsequentcolonyrecovery.Storeplateinrefrigerator.Transferfilterswith
colonysideuptoplasticorglassPetridishlidcontaining1mloflysissolution.Microwavein
glasspetridishes(fullpower)for1520sec/filterdependingonwattageofmicrowaverotate
disheswithfiltersandrepeatmicrowaving.Filtersshouldbehotandalmostcompletelydrybut
notbrown.Caution:Microwavetimeshouldbemonitoredcloselywhenanewordifferent
microwaveovenisusedasfilterscanburnwithflamesifoverdone.Microwavemaximumof6
filtersatonetime.
2.Neutralizefilters5min.onshakeratroomtemperatureinaroundvesselwithammonium
acetate(4ml/filter).
3.Brieflyrinse#541Whatmanfilter2timesin1SSCbuffer(10ml/filter).Upto10filterscanbe
combinedinonecontainer.(Filterscanbedriedandstoredatthispoint.)
4.ProteinaseKtreatment
Incubateupto30filtersinproteinaseKsolution(10ml/filter)for30minat42Cwithshaking
(50rpm)inplasticcontainertodestroynaturallyoccurringalkalinephosphataseanddigest
bacterialprotein.
5.Rinsefilter3timesin1SSC(10ml/filter)for10minatroomtemperaturewithshaking,50
rpm.(Filterscanbedriedandstoredatthispoint.)
4.Hybridization
1.Inaplasticbag,presoakfilterinhybridizationbufferfor30minat54C(55CforV.vulnificus)
withshaking,50rpm.Usemaximumof5filters/bagwith1015mlofbuffer.
2.Pouroffbufferfrombagandadd10mlfreshprewarmedbuffer/filter.Addprobe(finalconcis
0.5pmol/ml)tobagwithfiltersandincubate1hourat54C(55CforV.vulnificus)with
shaking.Thetemperatureiscriticalforhybridizationandwashingsteps.
3.Rinsefilter2timesfor10mineachin1SSC1%SDS(fortlh)or3SSC1%SDS(fortdh)
(10ml/filter)at54C(55CforV.vulnificus)withshaking.Inplasticcontainer,rinsefilter5
timesfor5mineachin1SSCatRTwithshaking,100rpm.
5.Colordevelopment
1.Inpetridish,addplace20mlofNBT/BCIPsolution.Addfilters(5orless)todishandincubate
withshakingatRT,covertoomitlight.Checkdevelopmentofpositivecontrolevery30min
reactionisusuallycompleteby12h.
2.Rinseintapwater(10ml/filter)3timesfor10mineachtostopdevelopment.Donotexpose
filterstolightastheywillcontinuetodevelop.Countpurpleorbrownspots,comparedtoa
seriesofcontrolsonseparatefilters.Storeindarkorunderacetateholders.
3.Recovertdh+coloniesbyaligningdevelopedfilterswithcorrespondingplate.Swabareaof
agarsurfacecorrespondingtopositivesignalwithasterileloopandstreakaTCBSagarplate.
Test5to10colonieswithtlhandtdhprobestoconfirmpathogenicV.parahaemolyticus
6.FilterpreparationforV.parahaemolyticusenumerationbydigoxigeninlabeledprobes(97)
1.TheprimersequencesforampliconpreparationofdigoxigeninlabeledprobesandthePCR
conditionsforconstructionaredetailedbelow:
gene
proteinencoded
location
LTLH
tlh
tdh
trh
thermolabilehemolysin(8)
thermostabledirecthemolysin
(Kanagawahemolysin)(87)
sequence
5'aaagcggattatgcagaagcactg
3'
RTLH
5'gctactttctagcattttctctgc3'
tdh1
5'ccatctgtcccttttcctgcc3'
tdh4c
5'ccactaccactctcatatgc3'
VPTRHL
5'ttggcttcgatattttcagtatct3'
VPTRH
5'cataacaaacatatgcccatttccg
3'
thermostablerelatedhemolysin
(8)
2.Thefollowingthermocyclerconditionsshouldbeused:
tlhandtrh
tdh
PCRconditions
Temperature
Time
Temperature
Time
Hold
94C
3min
94C
10min
Denature
94C
1min
94C
1min
Anneal
60C
1min
58C
1min
Extend
72C
2min
72C
1min
Hold
72C
3min
72C
10min
Hold
8C
indefinite
8C
indefinite
25cycles
25cycles
3.TriplicateprelabeledmembranesshouldbeinoculatedifhybridizationswiththethreeV.
parahaemolyticusdiglabeledprobesaredesired.DensitiesofV.parahaemolyticuscanbe
determinedwiththetlhprobeandthedensitiesofthepathogenicstrainscanbedetermined
usingtdhandtrhhybridizations.Theresultsarereportedastherespectivenumber/g.
Althoughthemembranesarenotsterile,carefulhandlingwillnotinterferewithanalysis.
4.Dilutionsofasamplearepreparedaspreviouslydescribed.Onehundredlvolumes(0.2gof
1:2oysterhomogenate)aredirectlyplatedtolabelednylonmembranesonthesurfaceofwell
driedT 1N3agarplates.Aseparatemembraneisusedforeachofthethreeprobes.Normally,
the1,2,and3dilutionsareadequate.Theminimumdetectionlevelis100/g(10/gfor
oyster).Asterilehockeystickisusedtogentlyspreadtheinoculumoverthemembrane
surface.
5.IncubatetheT 1N3platesfor3hat352C.Withforceps,transferthemembranetothe
surfaceofaTCBSagarplateandincubateovernightat352C.
6.Afterincubation,estimatethenumberofgreencoloniesandproceedwiththehybridization
steps.Ifnogreencoloniesarepresent,nohybridizationisnecessary.Themicrotiterplate
systemofretentionofsuspectisolatescanbeusedatthispointbypickinggreencoloniesto
platewellswithsteriletoothpicks.
7.Thecoloniesonmembranesarelysedbyplacingthemcolonysideuponanabsorbentpad
containing4mllysissoln.(MaasI)for30minatroomtemperature.Aslightheatingby
microwavingfor20secmayalsobeused.
8.Themembranesarethentransferredwithforcepstoanabsorbentpadcontaining4ml
neutralizingsoln.(MaasII)for30minatroomtemperature.
9.Drymembranesbrieflyonapaperorclothtowel,thencrosslinktheDNAtothemembranefor
3minunderUVlightsource,254nm,orinaUVcrosslinker.
7.Day1.Hybridizationwithdiglabeledprobes&colorimetricorchemiluminescentdetection.(97)
1.Warmshakerwaterbathto65C.
2.Placemembrane(s)inheattolerant,sealablebagorplasticcontainerwithlid.Membranescan
bestackedbacktobackwithafiberglassmeshscreenspacerbetweeneachpair(59).
3.Coverthestackwithprehybridizationsoln.Incubatesubmergedat65Cfor1h.
4.Removemembrane(s)frombag,containerandgentlywipeeachwithalabtissuewettedwith
prehybridizationsoln.Thisstepremovesexcesscolonymaterial.(Thisstepisoptional
dependingoncolonysize).
5.Covermembrane(s)withprehybridizationsolnandincubateat65Csubmergedfor2h.
Longerprehybridizationtimesareacceptable.
6.Boildoublestrandeddigprobefor10min.
7.Pouroffprehybridizationsolnandaddprobewhilestillhot.Hybridizesubmergedat65C
overnight.
8.Day2.Washanddetectionsteps
1.Afterhybridization,saveprobesolninaheatresistantplastictubeinfreezer.Probecanbe
storeduptooneyear.
2.Removemembrane(s)toplastictrayandwashtwice(inastack)bycoveringinWashsolnA
for5minatroomtemperatureonshaker,50100rpm.
3.Washmembrane(s)twicebycoveringwithprewarmedWashsolnBinabagorcontainerat
65Cfor15min.
4.PrepareGeniusDigbuffer2byadding0.25gpowderedblockingreagentto50mlofGenius
DigBuffer1,agitatevigorously,andmicrowaveorputin65Cbathtodissolveblocking
reagent.Agitateevery10minuntildissolved,thencooltoroomtemperature.
9.Colorimetricdetection(option1)orseeoption2below.
1.PreparecolorsolutionbyaddingoneNBT/BCIPtabletor0.2lNBT/BCIPstocksolution
dissolvedin10mlofDigbuffer3.
2.Pouroutantibodysoln.CovermembranewithDigbuffer1inplastictraywithshakingfor15
minatroomtemperature,50rpm.(Useafreshlywasheddishorbag,onethathasnotbeen
incontactwithantiDig)
3.PouroffDigbuffer1andcoveragaininDigbuffer1.Incubate15minatroomtemperature
withshaking.
4.PouroffBuffer1andcoverintraywithDigbuffer3for3minatroomtemperature.
5.Addapproximately10mlNBT/BCIPcolorsubstratesolutionper24membranes.Membranes
canbeplacedbacktoback(colonysidesexposed).Incubateinbagordishindarkatroom
temperature.Donotshakecontainerwhilecolorisdeveloping.Thecolorprecipitatestartsto
formwithinafewminutesandisusuallycompleteafter12h,butevidentin34h.
6.Oncedesiredspotsaredetected,comparingwithknowncontrolspots,washmembranewith
50mlDigbuffer1for5mintostopreaction.Countthepurpletobrownspotsandcalculate
thenumber/gofsample.
7.Themembranecanbestored,damp,inabagafterabriefrinseinDigbuffer4toretaincolor.
8.Probestripping
Nylonmembranescanbestrippedandreprobedifdesired.Thespecificsareoutlinedinthe
RocheDiagnosticsmanual(97).
10.Forchemiluminescentdetection(option2).
1.WarmCSPDreagenttoroomtemperature.
2.Removemembrane(s)frombag/container,coverindishwithDigbuffer1for1minatroom
temperature.
3.Inacontainer/bag,covermembrane(s)withDigbuffer2andincubateatroomtemperature
onshakertrayfor1h,50rpm.Longerblockingtimesareacceptable.
4.DrainoffDigbuffer2.
Covermembrane(s)withDigBuffer2withaddedantidigalkalinephosphataseat1:5000
(add5lantidigforeach25mlDigbuffer2).
5.Incubatefor30minatroomtemperatureonshakertray,50rpm.
6.Wipeoffacetatedocumentholderwith95%EtOH.Placemembraneonacetate,add0.5ml
CSPD/100sqcmofmembrane.Wipeoffoutersurfacesofacetate,putincassettewithXray
film.Expose(usuallynolongerthan1h)anddevelopfilmaspermanufacturer's
specifications.
11.MultiplexPCRidentificationofV.parahaemolyticus(8)
V.parahaemolyticusmultiplexPCRanalysis,analternativeconfirmationstepforsuspectisolates.
Prepareculturetemplatesbygrowingovernightat352CinTSB2%NaCl.Centrifugeoneml
ofcultureinamicrocentrifugetubefor3minat15,000g.Washthepellettwicewith
physiologicalsaline.Resuspendthepelletin1.0mldH20andboil10min.Storetemplateat
20Cuntiluse.Thefollowingprimersetsshouldbeused:
Threeprimersets(8)
tlhgenespeciesspecific
(450bp)
LTL
[sameasabove].
RTL
5'gctactttctagcattttctc
tgc3'
VPTRHL
trhgene(500bp)sameas
above.
5'aaagcggattatgcagaagca
ctg3'
5'ttggcttcgatattttcagtatct3'
5'cataacaaacatatgcccatt
VPTRHR
tccg3'
VPTDHL
5'gtaaaggtctctgacttttggac
3'
tdhgene(270bp)
VPTDHR
5'tggaatagaaccttcatcttc
acc3'
ThefollowingPCRreagentsarerecommended:
Reactionvol.
Reagent
(finalconcarethesameasabove)
dH2O
28.2l
10Buffer.MgCl2
5.0l
dNTPs
8.0l
primermix(6primers)
7.5l
template
1.0l
Taqpolymerase
0.3l
Totalvol
50.0l
ThefollowingPCRconditionsshouldbeused:
PCRconditions:
denature
94C
3min
94C
1min
anneal
60C
1min
extend
72C
2min
finalextend
72C
3min
hold
8C
indefinite
25cycles
12.AgarosegelanalysisofPCRproducts.
Mix10lPCRproductwith2l6loadinggelandloadsamplewellsof1.5to1.8%agarosegel
containing1g/mlethidiumbromidesubmergedin1TBE.Useaconstantvoltageof5to10
V/cm.IlluminategelwithaUVtransluminatorandvisualizebandsrelativetomolecularweight
markermigration.Polaroidphotographscanbetakenofthegelfordocumentation.Positiveand
negativeculturecontrolsandreagentcontrolshouldbeincludedwitheachPCRrun.
V.vulnificus
IdentificationandEnumerationMethod
TwoanalyticalschemesforisolatingandenumeratingV.vulnificusaredescribed.Thefirstisamost
probablenumber(MPN)analysiscoupledwithidentificationofsuspectisolatesusingbiochemicalprofiles,
DNAprobecolonyhybridizationorPCR.Thesecondschemeincludestwodirectplatingmethodsemploying
hybridizationwithDNAprobesforcolonyidentificationthathavebeenusedinseveralstudiesandhavebeen
showntobeequivalenttotheMPNmethod(29,134).Agaschromatographictechniquethatcomparesfatty
acidprofileshasalsobeensuccessfulforidentifyingV.vulnificus(68).
Seafoodsamples
1.Enrichment,isolation,andenumeration
1.Prepareaninitial1:10dilutionofsampleinPBSfollowingtheprocedureforV.parahaemolyticus.
PreparedecimaldilutionsinPBS.Preparea1:10dilutionoftheoysterhomogenateasfollows:Weigh
20gramsofthehomogenateintoasterilebottleandaddPBStodilutetoafinalweightof100g.Mix
byshaking.Additional10folddilutionscanbepreparedvolumetrically(i.e.1mlof1:10to9.0mlofPBS
fora1:100dilution).
2.Inoculate31mlportionsofthedilutionsinto3tubescontaining10mlAPW.Iflownumbersare
expected2gportions(1gofoyster)directlyfromtheblendercanbeinoculatedinto3100mlAPW.
Incubatetubes18to24hat352C.
3.Streaka3mmloopfulfromthetop1cmofAPWtubeswithgrowthontomCPCorCCselectiveagars.
IncubatemCPCandCCagarsovernightat3940C(3537Cif3940Cnotavailable).Oneither
agar,coloniesareround,flat,opaque,yellow,and1to2mmindiameter.
4.UponidentificationofV.vulnificus,refertotheoriginalpositivedilutionsofAPWandapplythe3tube
MPNtables(Appendix2)forfinalenumerationoftheorganism.
2.Biochemicalidentificationofisolates.
Unlessotherwisespecified,allmediainthissectionarepreparedtocontainaminimumof0.5%NaCl.
Note:ifDNAprobeorPCRisusedforconfirmation,steps57arenotneeded.
1.TransfertwoormoresuspiciouscolonieswithaneedlefromCCormCPCagarplatestoTSAwith2%
NaClandstreakforisolation.
2.Inoculatebiochemicalmediausingasinglecolony.Screeningreactions,AGS,oxidasereaction,
motility,andsalttolerance,areusedasforV.parahaemolyticus..
3.API20Ediagnosticstripscanalsobeused.Prepareculturesuspensionin2%NaClsoln.Biochemical
reactionstodifferentiateV.vulnificusfromV.parahaemolyticuscanbefoundinTable1.
3.DNAprobeidentificationofspeciesspecificcytolysingene,vvhA(29,134)
Theoligonucleotidesequenceforalkalinephosphataselabel:
5'Xgagctgtcacggcagttggaacca3'
SourceofthisprobeisthesameasforV.parahaemolyticus,
1.SamplepreparationanddilutionsaresameaswithMPNprocedureandthatpresentedfortheAP
probehybridizationforV.parahaemolyticusexceptdryVibriovulnificusagar(VVA)istheplating
medium.
2.Weigh0.20gofoyster:PBShomogenatedirectlyfromblender(0.1gofoystertissueand1dilution)
ontoVVAplateusingbalancewith0.01gsensitivitytotareplate.
3.Pipet100lof1and2dilutionsontolabeledVVAplates.FromDecemberthroughMarchplatingthe
1and2dilutionsisadequateandfromMaythroughOctober,summermonths,1,2,and3dilutions
areadequate.
4.UsesterilehockeystickstospreadoysterinoculumevenlyontoVVAplates.Dryplatesanduniform
distributionofinoculumareessentialforadequatecolonyisolation.Incubateplates1824hat35
2C.Relativelylarge(12mm)yellowopaquecolonies(friedeggappearance)aretypicalofV.
vulnificusonVVA.
5.Spottingcontrolcultureswillaidcolonycountingtoselectplatesforcolonyliftsandtoselectisolates
foridentificationandstorage.
6.Plateswith25250typicalcoloniesshouldbeusedforcolonyliftsandisolateselectionifavailable.
Additionaldilutionscanbeusedifuncertain.Colonyliftsfromplateswithconfluentgrowthornotypical
colonieswillprobablybeunproductive.
7.Whatman#541filtersofcolonyliftsarelysedandneutralizedasdescribedpreviously.
Themicrotiterplatesystemformultiplecultureretentioncanbeusedatthispoint.
8.FilterpreparationandProteinaseKtreatment:followtheproceduresoutlinedinsectionforV.
parahaemolyticus.
4.EnumerationofV.vulnificusbyDNAgeneprobe.
1.ThehybridizationstepsarethesameasforV.parahaemolyticus,exceptthetemperaturefor
hybridizationandwashingoffiltersis55C.Allotherstepsincludingcolorimetricdetectionarethe
same.
5.ConfirmationofV.vulnificusbypolymerasechainreaction(41).
1.IsolatesobtainedbytheMPNprocedurecanbeconfirmedbyPCR.
2.PrimersforPCRvvhAordiglabelingofprobeforenumeration(519baseamplicon)arefrombase785
to1303ofthecytolysingene.Thefollowingprimersshouldbeused:
Vvh785F
5'ccgcggtacaggttggcgca3'
Vvh1303R
5'cgccacccactttcgggcc3'
3.Thefollowreagentsarerecommended:
Reactionvolume
Reagent
(finalconcentrationsare
thesameasabove)
dH2O
28.2l
10Buffer.MgCl2
5.0l
dNTPs
8.0l
primermix(6primers)
7.5l
template
1.0l
Taqpolymerase
0.3l
Totalvol
50.0l
4.ThefollowingPCRconditionsshouldbeused:
PCRconditions:
denature
94C10min
denature
94C1min
anneal
62C1min
extend
72C1min
finalextend
72C10min
hold
8Cindefinite
25cycles
5.AgarosegelanalysisofPCRproducts.Mix10lPCRproductwith2l6loadinggelandloadsample
wellsof1.5to1.8%agarosegelcontaining1g/mlethidiumbromidesubmergedin1TBE.Usea
constantvoltageof5to10V/cm.IlluminategelwithaUVtransluminatorandvisualizebandsrelative
tomolecularweightmarkermigration.Polaroidphotographscanbetakenofthegelfor
documentation.Positiveandnegativeculturecontrolsandreagentcontrolshouldbeincludedwith
eachPCRrun.
6.Culturetemplatesarepreparedbygrowingovernightat352CinTSB2%NaCl.Onemlofcultureis
centrifuged15,000gfor3min.Thepelletiswashedtwicewithphysiologicalsaline.Thepelletis
resuspendedin1.0mldH20andboiled10min.Templatecanbestoredat20Cuntilused.
7.ThegeneprobeenumerationofV.vulnificususingdigvvhfollowsthesamedirectplatingprocedure
outlinedforV.parahaemolyticus,exceptVVAisused.Thehybridizationandwashtemperaturearethe
same,65C.
Interpretationofmicrobiologicalfindings
1.ContaminationoffoodorwaterwithenterotoxigenicV.cholerae,orV.mimicus(althoughrarely
encountered)constitutesanimportantfindingfromthestandpointofpublichealth.Theentirelotof
contaminatedfoodshouldbewithheldfromdistributionuntiltheappropriatehealthauthoritiesarenotified
andanepidemiologicinvestigationcanbeundertaken.Theserogroup,biotypeandenterotoxigenicity
resultsshouldbereportedforeachsample.
2.TheisolationofV.parahaemolyticusfromseafoodisnotunusual.V.parahaemolyticusisanormal
saprophyticinhabitantofthecoastalmarineenvironmentandmultipliesduringthewarmsummermonths
(27,52).Duringthisperiodtheorganismisreadilyrecoveredfrommostoftheseafoodspeciesharvested
incoastalareas.ThevirulentstrainsareseparatedfromtheavirulentstrainsofV.parahaemolyticusby
meansoftheKanagawatestortdhgenedetection(120).Inmostinstances,V.parahaemolyticus
Kanagawanegativeseafoodstrainsdonotcausehumangastroenteritis.ThepresenceofKanagawa
positivestrains,ortdhand/ortrhcontainingstrains,constitutesapublichealthconcern.Aheatprocessed
productshouldnotcontainviableV.parahaemolyticusandifso,wouldindicateasignificantproblemin
manufacturingpracticesorpostprocesscontamination.
3.Duringthesummermonths,GulfCoastandMidAtlanticshellfishnormallywillcontainV.vulnificusand
highlevelshavebeenisolatedfromwarmestuarineareas(118).Theorganismisrareinshellfishfrom
theWestCoast.Moststrainsisolatedhavebeendemonstratedtobepotentiallyvirulent.Clinical,
environmentalandfoodisolateshavebeenfoundtobehighlyvirulenttomice(60,90,113,124),but
infectionsarerelativelyrareevenamongthoseofincreasedrisk(liverdisease).However,those
individualsatriskshouldbecautionedtonotconsumerawshellfishduringcertainperiodsoftheyear
whenlevelsofV.vulnificusareincreased,normallyMaythroughOctober(84,104).AswithV.
parahaemolyticus,aheatprocessedproductshouldnotcontainviableV.vulnificusanditsisolationisa
significantfinding.
Acknowledgment
TheauthorswishtothankpreviousFDAcontributorstothischapter:RobertM.Twedt(retired),JosephM.
Madden(retired),ElisaL.ElliotandMarkL.Tamplin.
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HypertextSource:Vibriocholerae,V.parahaemolyticus,V.vulnificus,andOtherVibriospp.,Bacteriological
AnalyticalManual,8thEdition,RevisionA,1998.