LaboratoryMethods>BAM:<i>Vibrio</i>

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
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