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Title The transferability of drug resistances in fish pathogenic bacteriumPhotobacterium damsela subsp.piscicida isolated from yellowtail

Author(s) Bharadwaj, Manish Surendra

Citation (2002-03-31)

Issue Date 2002-03-31

URL http://hdl.handle.net/10069/7218

Right

NAOSITE: Nagasaki University's Academic Output SITE

http://naosite.lb.nagasaki-u.ac.jp

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Adissertationsubmittedinpartialfulfillmentoftherequirementsforthedegreeof

DoctorofPhilosophy

TheTransferabilityofDrugResistancesinFishPathogenicBacteriumPhotobacteriumdamselasubsp.piscicidaIsolatedfromYellowtail

by

BharadwajManishSurendra

Approvedbythedissertationcommittee:

eoeeeee--teo--"eeee-eoeo-e-beeo--ee"eee"eeeeeepee--e-e-eeeeeeeee-eeeeeeeeeeeeeeeee--eeeeeeeeeeeeeeeeeeeeoeooen

Prof.HideakiMoriiProf.TsuyoshiMuramatsuThesisAdviser&CominitteeMember,CommitteeMember,GraduateSchoolofMarine

GraduateSchoolofMarineScienceandEngineeringScienceandEngineering

eeoeeeebeeeeoeeeee-eee-eeeee-eeooe-e--e-eeeeeeee--eeeeeoee-ee-ee"eeoeeoee--ebeeeoee-oe-ebtb-eeoe-e-ee"-e-beeee

Prof.KazumaYoshikoshiProf.KenjiHaraCommitteeMember,CommitteeMember,GraduateSchoolofMarineGraduateSchoolofMariRe

ScienceandEngineeringScienceandEngineering

December,2001

AeckwagwEedigemxRessas

IwouldliketoexpressmydeepsenseofgratitudetomyresearchsupervisorDff.HideakiMorii,forhisexcellent

tkroKgghouttheperiodefifesearch,llagngratefugtoLectufferKentaroKasamaforgivingvalixablesuggestions,helpfu1commentsandextendingallpossiblehelpwheneverneeded.MysincerethankstoProf.TsuyoshiMkkfamatsu,Prof.KazumaYoshikoshiandProf.KenjiHarafortheirencouragementandcommentsduringmyresearch.Xamindebtedto.Dr.KiyoshiOsatomiforhisinvaluablesupportandhelpfulcomments.IwouldliketothankDr.KinyaKanaiforpresentingusmanybacterialstrains.IwouldiiketoextentmyveryspeciakhankstotheMinistryofMucation,Science,SportsandCukureofJapanfortheschoaarshipassistance.IwouLldliketothankaglmyfriendsandaabmatesfortheirhelpandco-operationwithoutwhichthisworkwouldhavebeendiffic"k.

Lastb"tnottheleast,Iwouldliketoacknowiedgemypareptts,sistefsandwifefortheirmoffaisupportandencoRsragementduringthecourseofthisstudy.

BharadwajManishSurendra

Pedieated tomyparents

kgSrecewABBREWMACMrKONS

Ap A]gnpgciggin

Apr Amapicillinresisrkawace

Cp Chgeramephenicol(Cpr ChleratwphereiÅíeAresistance

Cex CephaaeximChff ChrcemosomalDNADox DoxycycAine

Em EffyShffofitsxycin

Errgr EffythffomayÅíinffesistanee

Kgxk KamamycinKmotr KanamycinffesistanceMAC MinimumaiRhibigoryconcentwationNa NaMidixicacidNaff Nalidixicacidffesistaymce

Nf FuxazeiidgneOa OxolinicacidOtc Oxytetffacycaine

Rp RifampicinRpr RifaxKxpicinresistapmce

Sa SutphamenoxxxeehexineSar Sulphamaomeemethoxinexesistancesme Strepto]mycipm

Smr Strepterrma'yciptresistapmce

ssp. SubspeciesTc TeSxacycAineCfiI"cr EretwacycEineresistanceTjewP TrimethopffimTxEfupr TrimaethopifimffesisSancegeggpm1w1 MicregrampermikliXitergegkg-i MierogramperkiRogram

Åë@ntreewaas

Ckapter1.Ge"eralintroduction 1

Ckapter2.TheoccuxenceandtransferabilityofdrugresistanceinPhotobacteriumdamselasubsp.piscicidaisolatedfromyellowtail. 9

IntroducSion 9

Materialsandmethods 11

Resul&s 16

Discussion 26

Chapter3.Rplasmidtran'sfefofdrugresistancesinPhotobacteriumdamselasubsp.piscicidaisolatedfromyellowtail. 33

IRtroductioR 33

Materiaisandmethod 34

Results 36

Discussion 52

Chaptef4.CloningandnucleotidesequenceanalysisofaP-lactamasegenefrornPhotobacteriamdamselasubsp.pisciciaa. S9

ffntroduction S9

Materiaisandmethods 60

6S

Discussion 79

ChapterS.Drugresistanceofbacteriaisolatedfromtheintestineof

yellowtailcukuredwithfeedscontainingvariousdrugs. 87

Introduction 87

Materialsandmethods 88

Resuks 91

Discussion 10S

Chapter6.Generaldiscussion 112

Chapter7.Finalconclusions 116

References 119

Åëkthptrefffi.Gewaeeffntfiwatw$dieeectrfigwa

Fishformsaveryimportantpartofthejapanesefood

andisrelishedbythejapaneseasalsobyothernations.The

reasonforthepopularityoffishiRJapanisthatjapanbeing

aRislandco"ntryandalsohavingbeenbRessedwithmany

rivers,providesabundantsupplyoffish.Duetothis,

variousfisheshavebeencukuredinJapansinceaverylongtime.

Fishcultureinjapandevelopedrapidlyfromabout195O.Duetotheincreaseinfishculturefishdiseasesalsop

increasedinaparallelway.Thecausativeagentswere

bacteria,virusesandfungi.Thebacteriathatcausediseases

infishincludeAeromonassalmonicidathatcausefurunculosisinsalmonids;Edwarasiellatarda,whichcauses

paracolondiseaseineels(Anguillajoponica)sVibrio

angtsillaram,whichcakisesvibriosiSinayu\(Plecoglossess

altivelis\)andPhotobacteriumdamselasubspeciespiscicida

[Gauthieretal.,199S;formerly,Pasteecrellapisciciaa

\(Japtssen&Surgalla,1968\)\)whichcausespseudotuberculosisinye11owtail(Seriolaquingueradiata).

P.aamselasubsp.piscicida\(L.n.piscis,afish;L.suff.-

eida,fromL.v.caedo,tocutortokill;M.L.n.piscicida,fish

1

kilicr)coinpriseefGffam-negative(Kusuda&Yamaoka,

g972\),nonfiagellatedandstraightrodsofO.Sx1.Sgemin

size,andarefacugtativelyanaerebic.Negrowthoccuffsin

theabsenceofsodiumions(Gauthiex,etai.,199S).Eariier

Shimidu&Eg"sa(1972)consideredthatcellsdisplayed

Gram-variabilitywheRyoungi.e.ina2-18hcukuresincubatedat2O-2SOC.Pseudotuberculosis/pasteurellosis

\(describedbelow\)whichiscausedbythebacteriaisa

septicemiainwhichinternallygranuiomatous-likedeposits

maydeveloponthekidmeeyandspleen.Thesedeposits

comprisemanygreyish-whitebacterialcoloniesofO.S-1.e

mm2insize(Kusuda&Yamaoka,1972).Aiso,p"fulent

materialmayaccumuiateiRtheabdominalcavity\(X.ewiset

ale,19'l7O\)e

P.damselassp.piscicidawasdiscoveredduringthe

summerof1963,inwhiteperchandstripedbassinthe

upperffegionofChesapeakeBay,USA\(Snieszkoetal.,

1964\).Fremthediseasedfish,theorganismswhichwere

ffecoveredpossessedsomeofthesalieittfeaturesof

Pessteurellaandhencetheeonditionwastermaed"Pasteufelaosis".InJapanthediseaseisalsoffeferredteas

"Pseudotuberculosis"andhasbecomeofconsidewabRe

2

economicimportance,causipmgsignificantlossesinfarmed

ye11owtail.Economicaigessesduetodiseasesincugtuffed

xERarinefishingapamehavebeeAestimatedtobeabout2eO

]rxkiRRionU.S.doElarsannually(KusrcEda&Kawai,i998).Since

itsinitialffecognitioninyellowtails,pasteurellosisappearsto

keavespifeadtootherfishspeciesinckudimegtfedseabream

\(Yasunagaetal.,1983\)andblackseabream\(MurogaetaR.,19'tai7:7\)e

Antimicrobialcompo"ndshavebeenwidelyusedfor

thepreventionand/ortreatmentoffishdiseasesinJapan.

Someofthecommonlyusedantimicrobiagcontpouftdsare

ampiciAlin(Ap),chlovamphenicol(Cp),erythremycin(Em),

kanamycin(Km),nalidixicacid(Na),oxaiifticacid(Oa),

sulfaRilicacid(Sa),streptomyciR(Sm),tetracycline(Tc)and

tfimethoprim(Tmp),tonameafew.Thesecompoundshave

beenwidelyusedasfeedadditivestofarmedfisherhave

aasob,eenapplieddirectlyintofish-pondwaterfor

preventingfishiitfections.Duetotheextensiveuseef

antimicrobiaicompounds,maflystrainsofthefish

pathogenicbacteriamentionedabovehavedeveloped

resistamecetovariousdrugs,andthishasbroughtaboast

infectieptwithmaanltipledrug-resistametstraipmsofpathogens

incuItuffedfish(]Kime&Aeki,1993a).rrchesedrugresistant

3

pathegenicbacteriahavebecomaeacauseofseriousconcern

forthefishculturefaffmsthroughoastgapan.Sigxiilargy,the

esseofdffeegsasfeedadditivesforlivestockafidpoultryhas

resugtedinanincreaseofdrug-resiskantentericbacteriain

theseanimals(ARderson,E.S.,g968).

Antibioticresistantbacteriainthegeneraiscenariois

Rothingdifferent,somuchsothattheappearanceofmultipAe-drug-resistantpathogeRicbacteriahasbeen

recognisedbytheWHOasaseriousprobiemthathascomplicatedthemedica1treatmentofbacteria1infections.

Therefore,theWHOhascailedforasrgentniteasurestofight

againstthespifeadofpathogenicstrainsthatareresistaitt

toseveralantibiotics.thesemeasurescoRcernappropriate

aRtibiotictherapy,hygieneaftdcefitrolofbacterial

infectioftsaitdtheextensionofexistingsurveillance

systemstoaniRternationainetworkinordertocoilect

epidemiologicalinformationonaRtimicrobialresistance.

RecentreportshavealsoshownamarkedincreaseofaittibioticresistaftceiRfood-poisoniRgbacteriadaxeto

irrationalandexcessivempsesofantibieticsastherapewatic

agentorasgrowthpromoterinlivestock.Afietheraspectef

theantibioticffesistanceissueistheasseofantibiotic

resistaRcegenesasseIectionmarkeringeneticaIlyxxxodified

4

organisms(GMOs).rrifhemaainsafetyconcerRsrelatestothe

escapeortransferoftheantibioticresistaymcegenesto

sensitivebacterialstrainswhentheseGMOsareintroduced

intotheenviroRmeRt.

TransferabledrugresistanceseffishpathogeRic

bacteriahavebeendetectedinmukipledfugffesistant

strainsandacloserelationshipbetweentheincreasing

numberofresistantstrainsandtheexteRsiveuseof

chemotherapeuticsinfishfarmshasbeensuggestedby

Aokiandhiscoworkers(Aoki,etal.,197g,1972,1973).

llhedrugifesistaRceisoftenattributedtothepresenceof

tfansferableRplasmidsthathavebeenregardedtobe

responsibleforthedrugresistanceofvario"sfishpathogenicbacteria\(Aekietal.,g974,1977,1981,1983,

1984,1987,199O;Aoki&Kitao,1981,198S;Takashimaet

al.,198S;Akashi&Aoki,1986;Zhaeetal.,1992;Kitm&

Aoki,1993a\).TransferableRplasmidshavepiayedan

importantroleinthedisseminaSionofantibioticresistance

genesinthebacterialpopulationwithhorizontalgemee

transferbyconjugation\(Amabi]e-Cuevas&ChicuxeR,

1992\).Theevolutionofmultipledrugresistanceshasmade

itdifficulttopreventbacteriagimefectionbychemaoprophylaxisaxxdchemaotherapy,ffkeaccumulatiopmof

5

severalresistancegenesinasingiepgasmidisasseciated

withtxanspositionorsitelaptspeeificrecombinationofan

ifitegffepm(Stokes&Hall,1989).

Ekeoccurrffenceandtransferabilityofdrugffesistances

inP.aamaselassp.piscicidahavebeenperformedimgreat

details\(Aoki&Kittao,1985;Takashimaeta1.,1985;]Kim&

Aoki,(1993a).TheyinferredfromtheirwoffkthatR

pEasmidswereresponsibleforthetransfefofmostofthe

drugresistancegenesinP.damselassp.piscicida,exceptthe

P-lactamasegenethatconfefsresistancetoAp.Aspertheir

resuks,thetransferabledrugresistaitcesweretransferfed

fromamulti-drugresistantdoftorP.damselassp.piscicida

tothefecipientE.coliwithsamefrequenciesirrespectiveof

theresistances.Also,theypresumedthattheApresistance

wasfton-transferableasitwaschromosomebouitdandwas

notlocatedontheRplasmid.fihiswasbecause,intheir

experimentstheyfoundAp-resistancetobeconjugally

non-transferablegoE.coli.Howevero"rresultsare

differentfxomthemwithrespecttotransferabilityofAp-

resistanceandthetransfexfrequencyofdrugresistances

inP,aaneselassp.piscicida.Wealsohavepfesentedsome

otherftewinformatioftregardingtheoccuffreneeand

traRsfeifabigityefdifugresistancesinthebacterialstrains

6

\(inthesecondchapter\).

Ipmthethirdchapter,westncgdiedthecovregatiome

begweenRpRasgnidanddrugresistanceintheffespective

transcoftjugantsthatwereselectedwithindividkxaldfugsin

matingwitheachofthedonersdifferiRgindrugffesistance

patterns.Also,plasmidsinthedonorsthatexpressed

differentresistancepattefnsandtheRplasmidstransferredtotherecipientwereanagysedinanattemeptto

.findacorrelatioRbetweenthem.Also,wefoundthat,unlike

theresuksofAokiaRdcoworkerswhohavesceportedthe

Apresistancetobenofl-tifafisferable,thetransferabilityof

Apffesistaflcewasuniqueandisdiscussedindetailsinthe

secondaRdthirdchapters.

Enthefo"rthchapter,thecioningaxxdseqasepmcingof

theAp-resistancegene(P-lactamasegene)hasbeeft

discussedindetails.Thischapteralsodiscussestheanalysis

ofthefi-lactamasegefteafiditNspossibleorigin.Thisstudyis

veryiitkportantbecause,tilldatetherehavebeenhardgy

anyreportsenthecloningandsequencingoftheP-lactamasegenefremaaRyofthefishpathogexxicbacteria

studiedsofar,thoughmostoftheotherdrugffesistance

geneshavebeenclonedandsequencedinP.aamselassp.

ptscicidaandotherfishpathogenicbacteria.

7

Thefifthchapterdealswiththeoocurrenceofdrug-

resistanceinbacteriaintheintestinaltractofyeRlowtail

upontreatmentwithvaxiousdfugsasfeedadditivesfor

fish.ItaisoinvestigatesthequestionwhethexRplasmids

aretransferredfromonebactefi"mtoanother\(including

differentspecies\)intheintestinaltraetofyeliowtail.Ihis

studyisimportantasthebacteriaintheintestineof

yeilowtailareusuailybathedwithdifferentkindsofdrugs

thatareusedasfeedadditives.Hence,thepressufeofdrug

maycreateanenvironmentthatisseleetiveforbacteria

havingordevelopingdrugfesistanceandmuitiplyin&

eitherthroughverticakransferwhereamutantresistant

foradrugmaybeataselectiveadvantageandwillmultiply

andrapidlydominatetheenvironmentgorthroughthe

horizontaRtransferwhere-intheresistanceistransferred

fromonebacteriatoanotherinaDNAmoleculebytheprocessesofconjugation,transductionortraitsformation.

8

Åëkapdeff2.Wdaee$ececeeffrempceeewaditrgeeewasleffthlvfigfitry$ff

diffwsgffeesfiseeegeeceekwaPhotiobesctieriesmeaesnesegesswatosge.

pgssieutesfis$MntediffffgewmayeeMkowtafilj\(Semiola

qesimeqeresaiestres\)

KNPffSRODWÅë[ff'MON

EnjapaR,fishcukuredevelopedrapidlyfromabout

19SOandfishdiseasesalsoincreasedwiththedevelopment

offishcugture.Chemotherapeuticstudiesforfishdiseases

startedfromabout19SS.Presently,variousantimicrobiag

compoundshavebeenwideiyusedasfeedadditivestofarmedfishoradministereddirectlyintofishpendwater

forpreventionortffeatmentofbacterialdiseases.the

exteRsiveuseofdr"gshasres"ltedinanincreaseofdrug

resistantstrainsofbacterialfishpathogens\(Watanabeetag.,

1971;Aokietal.,1971,1972,1973;Aoki&Wataptabe,

1973a,b;Aoki,1974\).Drugresistanceismestlyattributed

tethepresenceoftransferableRplasmidsthathavebeefi

regardedtobefespoRsiblefoffthedrugresistanceof

variousfishpathogeflicbacteria\(Aokietai.,1974,1977,

198i,a983,1984,a987,g990;Aoki&Kitao,1981,198S;

9

Takashimaetal.,198S;Akashi&Aoki,a986;Zhaoetal.,

1992;Kim&Aoki,1993a\).fikeevolutionofmultidrug

resistanceshasmadeitdifficulttoprevefitbacterial

infectionbychemoprophylaxisandchemotherapy,which

inturithasca"sedseriouseconomicdanitageinfishfarms.

StudiesoftdrugresistanceanditXstransferinfish

pathogeRicbacteriaareveryimportantfortheprudent

useofantimicrobialcompouRdsforcontrollingbacterialfish

diseases.

Photobacteriesmdamselassp.pisciciaacomb.nov.

\(formerly,Pasteeerellapiscicida\)iswellkfiownasthe

causativeagentofpseudotwberculosisincultuifedyelgowtail

inJapan.Aoki&Kitao,1985;Takashimaetal.,198S;Kim&

Aoki,1993a,haveperformedextensiveexperiments

concerniRgtheoccurrenceandtransferabilityofdrug

resistancesinP.aamselassp.pisciciaaisolatedfrom

culturedyellowtailindifferentyearsfromvariousareasin

Japan.Theyhavereportedthatresistancestochloramphenicol(Cp),kaptamycin(Km),sulfamonomethoxine(Sa)andtetracycline(Tc)were

transferredbutampiciliin(Ap),nalidixicacid(Na)and

trimethoprim(Tmp)ffesistanceswerenottfansitraittedin

thebacteriagstrains(Takashimaeta].,Kim&Aoki,1993a),

10

thoswghApffesistancewasSransferredfromonlyoiteofthe

]gtsanystrainsstudied(Takashimaaetal.,g98S).Moreover,

thetransferfrequeRciesofdrRptgtfesistarmceswexealfiEkost

thesameamaongrespectiveresistancesofindividasaldonors

\(Kim&Aoki,1993a\).EEowever,ourresultsweredifferent

fromthemwithffespecttothetransferabilityofresistances

toApaRdTmpandthetfansferfrequenciesofdrugresistaRcesinP.damselassp.pisciciaa.Besides,these

resultscaoselycoincidedwithdrugresistancepatternofthe

bacteriaistrains.Moreoverwehavefewothernew,

informatioftwithregardstotheoccurrenceandtransferabilityofdrugresistancesiitthebacterialstrains,

Therefore,theoccurreRce,patternandthetransferability

ofdr"gresistaRceswereinvestigatedinP.aamselassp.

pisciciaastrainsisolatedfromcukuredyeglowtailin

differentyearsfromvariousareasinjapan.

MATERKAkSANpmMEwaODS

Bthecdeerta,meediRttaswadigifaswwTtrlaec$wadifiafi*mes.Oigxe

huandredeighty-threestraiftsofP.damselassp.pisciciaa

wereisolatedfromdiseasedyelRowtftilSeriolaqesingeeeraaiataipmvariousaxeasefJapanbetweefta984

11

and1994(Table1).ThebacterialstrainswereRormally

recoveredfroxvefishcgslturedindiffefentnetpe"sbuteight

straiRsisolatedin1994wereobtainedfromfishc"lturedin

thesameone.EscherichiacoliK-12x1O37Rprmutant

strain[arifampicin-resistantmutantstrainof\(1037

galK2galT22hsdRiacYZmetBZrelAsttpE44)](Iyobeetal.,

1981,1994\)usedastherecipiefttforeonjugaltransferof

drugresistancewasobtainedfromtheLaboratoryofDrug

ResistanceinBacteria,GunmaUniversitySchoolofMedicine,Japan.P.aamselassp.piscicidawasincubatedwith

BHIbfoth(Difco;Becton-Dickinson,Sparks,MD,USA)

containing29oNaClat28OCaxxdE.coliwithL]Bbroth\(Miller,

1972)[19obactotryptone(Mfco),O.S9obactoyeastextract

\(Difco\),1%NaCl,pH7.S]at37OCMueller-HiRtoRmedium

\(Difco\)coRtaining2%NaCland1.S9\(oagarwasusedfordrug

susceptibilitytest.BTTB-lactosenutrientagar[1%bacto

peptone(Difco),19obeefextract(Difco),1%lactose,O.OO459o

bromothymolblue,O.S%NaCl,1,5%agar.pH7.S]wasused

formaatingassay.

DffeegseesÅëepafibfiMfirkyaesa*ffP.desmeselassp.

pgseieiaes.Drugsuseeptibiiitytestwasperfofmedusing

12

-fi-abtsfl

ctffm$e/a

$eurceanctftug"g"gberofPhototseseterium

$ub$p,piseicictastratin$isewgatectfrem

yglllewtaiiofgeographicaliyckspersectarea$inJapan

Numberofstrain$i$elatediR:Yeear Naga-$agaOitaKesma-Kage-TetaE

$akimoto$hima198419851g8es

re87198819891990fi991

f992re93re94Tetag

Muelger-Hintonagarcontainiftgindividualdrugsthakwere

preparedbyaserialtwo-fogddikutionmethod,ffecomemefidedbythejapaneseSocietyefChexmeotherapy

\(198X\).ThecuelturewasgrowRovernighrkimMaxeller-

Hintoftbffoth,anddiiutedtoabout106ceglsmlqiusing

buffefedsalinewiehgelatin(BSG)selution[O.8S%NaCl,

O.O3%KE{2P04,O.O69oNa2HP04,O.O1%gelatin]andthe

dilutedeultuffewasiitoculaSedenMue11er-Hintormagarby

usingamicroplanter(ToyosokkiCo,Ltd.,Japan).TheMICs

werereadastheiowesteoncentrationresuitiRginconkplete

inhibitionofgrowthafter24and48hincubationsat28OC.

lheresistancetoeachantimicrobiaicompoundwasdefined

bythedistributionoftheMICs(Fig.1).rffhefollowing

antibioticsandchemotherapeuticswereempioyedfordrug

susceptibilitytest:Ap,cephalexin(Cex),Cp,doxycyelifie

\(Dox\),erythfomycin\(Em\),furazoiidofie\(Nf\),Km,Na,

oxolinicacid(Oa),oxytetraeycline(Otc),Sa,stxeptomycin(Sxn),TcamedTmp.

ÅëowajeeggeMkffawasbeerasssthy.Eachdonorstrainwas

incubatedinBHIbrothundershakingcenditionsfox9-12h

aitdE.colirecipientstraiRin]Bbrothfor8h.O,Smageachof

i4

donorandffecipientcukaJgresweremixedipmSO-mlEr]enrrilteyerfKaskand4m1ofequagvolluxneseachofBHlland

LBbrothswerceaddediRtotheflask.Matingwasperfoffxxxed

at28OCfor2hwithverygepmtleshaking.O.amgofgO-fold

serialdiiutionsofmatingmixturewasspreadoftBrMB-

lactoseagarwithSOpsgrifampicin(Rp)xxx1diiaitdeach

sellecteddrug:a2,SgegApmlani;2SpagCpmltii;3.g3gegDox

midii;aoongEmmgmi;1oougKmmlei;2oogegNamgdii;2Sng

Oamlmoi;2Sgegorcm1tii;64OOngSam1dii;2SngSmnxl"ig2S

pagTcmlluaiand4OOptgTmpmgdii.IRaddition,therecipientE,

coliK-12xaO37RprexptessedMICvaguesasfoilows:3.13

gagApm1dii;a2.SptgCpm1pti;3.13gegDoxmgmi;2SgegEmmaani;

12.Sgeg]Kmm1tai;SOptgNamlmi;3.13psgOamI-i;6.2SugOtc

mlani;640eptgSamiqi;12.SpsgSmml"i;3.13gegTcmlmi;

12.SptgTmpmlmi.Coloniesgrowinginthisdouble-

inhibitor-supplementedmediumafter24to48hof

incubationat370Cwerescoredaspresumaptivetrraitscenjugants,alldthefrequencyoftifaflsferwas

calcanlatedasthenumberoftransconjugantsperinitial

numbefofdonors.Tenormoretransconjugantsfrommaatingwerepickedandtestedforantibioticresistance

accordtwgtothemethodsxifxentionedabove.

15

t

RESaLi]LF]firS

MgÅëdifisgfffibeegfi$n$ffeempgfigwRfiÅëff*bfieefiecowagegeewads

7thedistributiofioftheMICsof14antimierobiai

compoundsagainst183strainsofP.damselassp.pisciciaa

isolatedfromyellowtaiiareshowninFig.1.TheMffC

distributionofTmpexhibitedthreegroupswhichwere

sensitive,intermediate-levegresistantandhighlevel

resistantwithvaluesrangingfromO.2to3.1,12.Sto1OO

and>8OOptgmlmi,respectively.TheMICdistributionsofAp,

Cp,Dox,Em,Km,Na,Oa,OtcandTcexpressedtwogroups,

whichweresensitiveandresistant.EheMICvaluesforAp

rangedfromO.02StoO.1ptgmlmiagainstthesextsitive

strainswhichshowedthehighestantimicrobialactivityin

thestudieddrugsbutwerewidelydistributedfrom6.2Sto

2OOptgmlmiagainsttheresistafitstrains.ThevaluesforOa

raftgedfromO.OSto12.Sptgmlmiagainstthetestedstrains

whichhadthesecondhighestactivityinthestudieddrugs

andtheactivitywasveryhighascompaxedtothechemicaliyrelateddrugNa.ThevaluesforDoxrangedfrom

16

$oo 1OOgo6040

Ap 8ew

604e

Cex

20 20

cp Dox

20 20osoo806040

Em Km

40sco'X80 o120

as Nen 1eo M60gbuoth40ptA20

8ew

6040

s 20

1OOgo6e40

Oa

o1eo80604e

Otc

20 20

$en $mfi

4ew 20o1OO806040

Tc

o6050403e20

Trnp

20 10oo

ut emigr Qan en oo o Me-dsumauc mmoo oNo e"oo w-.eq Nooetmee

oQ Noo" ci di eqQ NoowmaevoQ

ci @ vwwma 6 gtu'

Mgc\(pagS,/Flllfiig/g'eeX-:,'i'ill14o/i,,/iY./iM//vagsffi.\"/\h,\,/jb61Rl.'il,l/Iteg#,,/R,[S13..c",i.4,/?Ep,ecl'liiil14,,?xg,egla,.'etffey/ltc,.18f16r,Rh,/dS//V,,c.a/f"V'El:gseigf

'

O.1to12.Spsgmamuiforthetestedstfainswhichhadthe

thirdhighestactivityinthestudieddrugsandtheactivity

wasextremeayhighascomparedtotherelateddrugsTcandorc,especiaRyagainsttheresistantstraiRs."lhevalues

forEmrangedfrom0.4to400pagmlmiagainstthetested

strainsandcouidnotbeclassifieddistinctlybetweentwo

groupswhichweresensitiveandresistant.theMICdistyibutionsofCex,Nf,SaandSmshowedasinglegroupin

whichallthetestedstrainsweresensitivetoCex,NfandSm

andresistanttoSa[theMICvaluesofthesensitivestrains

\(toSa\)inP.damselassp.pisciciaawerebelow2Sugmluai

accordingtothepreviousreports\(Aoki&Kitao,198S;

Takashimaetal.,198S;]Kim&Aoki,1993a\)].TheMIClevels

ofSawere16OOptgml-iagainstallthestrainstested.

Drwwgncesfistawaece"ffP.damsegesssp.piseieidessetrakans

']thedrugresistanceofP.damselassp.pisciciaastrains

isolatedfromyeilowtailisshowninTable2.Algeralmostall

ofthestrainswereresistanttoKmorSa(9S%),respectively.Mostofthestrainsexhibitedresistancetothe

chemicallyrelateddfugsTc,OtcandDox(90%).The

18

evemaengee2 Drngre$i$tanceofPhotobactetiumdamselasco$p.ptsciclda $train$i$oeatectfuog"g"RyeElewtaig

frgtr'n1984tofi994injapan

Year Nasrg"igberofNecmberefisoeatesthatofferedresi$tancete :

iseiate$ApCpEg"nKg'rRNa Sa Tc Tg"ifep

1984 fio(o)1(roo)o(o)o(o)e(o) 1(toO) sctoo\) o(o)1985 2815(54)26(93)O(O)26(93)6(21) 28\(1OO> 25(89) o(o)fl986 85(63)8(100)O(O)8(roO)5(63) 8(1OO) 8(10@) e(o)fi987 25O(O)25(100)O(O)25(100)O(O)25(1Oe) 25(fiOO) o(o)1988 3734(92)36(97)O(O)36(97)8(22) 37(100) 36(97) 2(5)fi989 144(29)8(57)O(O)g2(86)8(57) 14(1OO) 1O(71) 7(50)fi990 Z67(44)8(50)O(O)fi6(100)11(69) S6(1OO) 12(75) fi2(86)

fi99a 74(57)4(57)2(29)5(71)5(71) 7(1OO) 4(57) 5(7fi)

fi992 142(14)9(64)2(fl4)$4(100)7(50) fi4(fiOO) 14(1oo) 2(Sag)

fi993 2515(60)17(68)1(ag)24(95)2e(80) 25(100) 2fi(84)2e \(80\)

te94 8O(O)8(100)8(100)8(100)O(O) 8(tOO) 8(100) 8(fi@O)

Tetag fi8386(47)150(82)13(7)fi74(95)70(38) fi83(1OO) 164(90) 56(31)

Nur#iberigmbrackgts$hewperceRtageofthel$ogate$thatefferectresi$tanceteeachctrng.

The$trerain$whichgrewiRconcentrantioRsrreoreahage6.3torAp,6.3 forCp,50 forErrg16OO' forKe"re,

25torNa,1600forSa,12.5torTc,emct12.5 gLg/g"g'gMtorTg"npareexpre$$ecta$resi$tantstrain$.

Tbe resuMtsofre$di$tancestoctexycyciineandexytetracyclijgegorexoeliticacictwere sagtsteeastho$eof

re$i$tance$tothechgg"g'#icaeiyreeatect-g-cgrNa,ife$pectivelyandthsreforeorr#itted.

Abbreviabon$:Ap,ae'riplciiMin;Cp,chegrampheRieeM;Eg"g'#,erythrog"giycgn; Krrgy kanarnycfiit;Na, naiidixic

acectISa, suttarg"kenog'g"teeth#xine;Tc,#earacyceine;Triftep,trirrgethoprirrg.

ifesistancetoCpwascarriedbyalmostalloftheisolates

before1988buteniyinabout60%ofthgseafeerg989exceptferthestrainsisoaatedipta994.Abeuthalfofthe

strainsexpressedresistancetoAp(47%).(intheether

hand,agreatpartofthestrainsweresusceptibletoErit

\(939o\),Tmap\(699o\)andtheifelateddrugsNaafidCta\(629o\).inadditioxxtheresistancetoEmwasnotshownimthe,

strainsisolatedbefore199e.ThestraiRsresistaflttoTmp

werenotobservedbeforea987andthehigh-levelresistantstrainswerefoundin1994.

llitaddition,significaRtdifferencesofdffug

susceptibiaityiRtheisoiateswerenotobservedamong

geographicagorigins.

ÅëtwwajeegeezagecaemsffeemegffdieceegffesfisaawaÅëee

ghedrugxesistancepatternsof183strainsofP.

damselassp.piscicidaisolatedfromyeilowtailanddreeg

resistancestrapmsferredfromthedoftorP.aaueselassp.

piscieidastraixxstotheffecipientE.coliK-a2xae37Rpr

strainareshowRiRgrable3\(iwaadditiorm,thedraxgffesistance

determinationtestswereperfewmedforeightbasicgrowaps

2e

esffeeetsEew3Drugresi$tancepatternofF?hotobacterium ctam$e/a$ubsp.piscieictex $train$

isolatectfromyellowtailancictrugree$istancetran$terrectfromP.ctamse/a $ubsp,"iseieida

strain$teg$cheriehiaco/iK-12xi037Rpr$train

Donor Recipient

Drugresi$tancepatternNumberof Drugre$i$tance Nurr\)berof

strain$ tran$ferreci $train$

t Nottran$fereect 6

2g Nottran$ferect a

3. Sa 2

4. $aTcCp a

5e $aKmTc 15

6. SaKm 9'e7'` $aKmTcTmpfi $aKmTc 1

8. $aKrnTcCp39 SaKmTcCp 39

9. $aKmTcCpNa" $aKrnTcCp "1O. $aKrnTcCpAp37 $aKmTcCp 37

" ,$aKmTcCpffrn2 $emKmTcCpim 2

12 $aKmTcCpNaTmpi SaKrnTcCp ptfi

13. $aKmTeCpApTrnp2 $aKmrcCp 2ti4. $aKmTcCpewmTmp9$aKmTcCpimTmp 9

t5. SaKmTcCpApNai5 $aKmTcCpAp 10

SaKmTcCp 5

t6. $aKmCpApNaTmp1$aKmCpAp s

i7. $aKraTcCpApNaTmp29 $aKmTcCpAp 29

18. $aKmTcCpApimNaTmp2$aKmTcCpApeern 2

Totala83 Total a83

Aboreeviation$:Ap,ampicillin;Cp,chloramphenicol;wam,erythromycin;Km ,kanamycln;Nem, malictixicacict;$a,$ulfamonomethoxine;Tc, tetracycline;Tmp,trimethoprim.

ofdffugs\).Sixofthea83strainswereffesistanttoonlySa

armdtheffemainingstrains(97%)weremkxkiplledifwag

resistantfrom3te8drangs.TRtepercepmtagesofthestffaiRs

cgassifiedwerehighfoffthepatternsofSa/KmTc/Cp/Ap/

Na,andTmp(pattexnsS,8,10,gSandW).

TheresistancestoSa,Km,Tc,Cp,Ap,EmandTmpwere

transferffedfromthedonorstotherecipiepmt.However,the

transferabilityofdrugresistanceswasdifferentwiththe

patternandievegoftheresistances.Hhatis,theresistaRces

toSa,Km,TqCp,ErnafidTmpweretransferredfromthe

dofierscarryingresistaRcesteTcorNaTxEkp.However,the

transferofTmpresistancewasrestrictedtostxainsbearing

high-Xevelffesistance(pattern14)."rcheresistancetoAp

wastransffwaittedfromthedonorscontainiRgresistapmcesto

NaTcerNaTmp(patterns1S-18).However,Apresistance

couldnottransferinalithestraiRscarryingresistaRcesto

NaTc(patterRIS).Ontheotherhand,theresistancestoNa

andTmpthatexhibitediAtermediate-level-ffesistamacewere

nottramesmaittedfromanyoftheffesistantstrains.Moreover,

itoReoftheresistapmcestransferredfromathosedonersthat

didfiotpossessresistaftcesSoTc,NaTcorNaTinp\(patterns

1and2\).

Numberapmdpercentageofstraiftsthattrampsferifed

22

drxggffesista"cefffomP.aamselassp.pisciciaatoE.coliK-a2

x1e37RpraxeshowninTable4.Thepercentagesofthe

sssainsthattrasmittedeachdrugresistanceweregOO,

99e3p96elp94.3,48.8,16.0andO%inEm"Tc;Cp;Sa;Kgn;

Ap;Tmp;andNaresistances,respectively.

Terasmasffeerffffeqwaewaecyoffdifftwgffeskstrasenece

lhetransferfrequencyofdrugresistancesfromShe

donorP.aamselassp.piscicidastrainsthatexhibited

differentresistaRcepatterxxstotherecipientE.coliK-12x

1037RprstrainisshowninTableS.Infact,theexperiment

wasperformedfofallstrainsstudiedaRdtheresultsare

presentedforenestraineachintheresistancepatterns

sincetheresuksweresimilaramongstrainsineachpattern.

Thedrugresistancesinthedonorsthatdidnotcarry

\(patterns3-9,gl,12,aftd14\)ortransmitApresistawace

\(patternsaO,13,andapartofstrainsinpattermeIS\)were

transfeffffedwithalmostthesamefrequencies\(1Om3-geg4,

mostly1Om4\),ffegardlessofthetfansmittedresistancesafid

thetestedstrains.Ontheothexhandthetrapmsfer'

23

wento ajTran$ferofctmagresiskancefrDrnPhotobactetiesmdamselasmbsp.ptscicida $itraiitsto

EschenchlacofiK-12x1037Rprstraiit

Dreeg r$$i$tanceNumberof$train$thaaNasmberof$train$thatPercentage 6f $trainsthat

gfferectre$i$tancetwait$ferrectresdistance traRsferrectre$fi$tance

es`g8.8

99e3

1OO94.3

o96efi

1OOfi6

Abbreviation$:Ap,ae'rtepicielfin;Cp,chMorarrgphendicoE;Egxfita,eryahrerifgycin;Kif"g"te, kanargeycirt; Naitailctixic'

acict] Sa,sutfarrggnog'rteethgxiRe;Tc,tetracyciine;Trg"igp,trlmethoprfig"n.

"dimaestots5Thefreqeseneyofctvagresfistakcegran$ferfrog"re Photobactertumdam$ela $ub$p.ptscic/da $train$exhibitiRg

ctifterentresistagecepatterststoEscherichiacofi K-12x1037Rp'strain

Dasrgre$iseaRcepaSterst -g-raR$ferfrequencyofctrugre$i$taitce$

eff#hectgRorstrains Sa Kg'rts Tc cp Ap Eg'rR Tff'g'sp

3e SaNaTrnp(PP9301) 2 .6x1o-4 1 1 / / 1 d

4e SaTcCp(PP84el) fi .6xfie'4 1 1.9x1o'` 2.5x1g4 1 / f

5. $aKrnTc\(PP9212> 7.3xfio'` 7.1x1e-` 7.8x1g4 1 / 1 1

6e SaKrnNa-ifwtp(PP9g05) 4.fix1o'` 5.9x1e-` / 1 1 1 -

7. SaKrxt-ifcTrgep(PP9e17) fi .9x1o-4 2 .5x1o-4 3.`fix1o-` / 1 1 -

8. SaKg'g"ts-g-cCp(PP87O2) 1 •6x1o'3 9.7x1e-` 2.`g-x•go-3 9.2x1o-4 1 / 1

g. SaKrxtTcCpNa(PP9214) 3 .3xfio'4 5.9 xfio'4 5.4x1o-` 2."gx1o-4 1 1 1

fiOi SaKwtTcCpAp(PP8g08) 3 .2x1o-3 2 #6x1o-3 2.3x1o'3 2.2x1o-3 - / 1

fifi .$aKmTcCpErn(PP9202) 3 .Ox1o-4 3 .fix1o-4 5.4x1o'` 2.8x1o-` -8x1o-4 1

fi2 .SaKg"g'sTcCpNa1-g"rip(PP89O6) fi .3xso-4 fi .Ox1e-4 2.1x1e-` 2.fix'go-4 / 1 -

fi3 .SaKmTcCpApTmp(PP8836) 3 .Ox1o-4 1 .8x1o-4 3.2x1o-` 3.4x1e-4 - 1 -

14. $aKrnTcCpErR1-wtp\(PP9400 1 .fix1o-4 fi -5x1o-4 2.4x1o-4 1.9x1e-` / 5.8x1e-` 2.2x1e'`

fi5. $aKrnTcCpApNa(PP8517) 6.2x1o-` 3 .8x1e-` 6.6x1o-` 3.0xfie-6 4.3x1o'6 1 1

aE(PP8608) 8.2x1e-4 5.1x1o""" 3.6x1e-4 6.7xfio-4- 1 1

S6e $aKrxtCpApNaTwtp(PP8912) 1 .2xio-4 2.0x1a-` / 6.7xfio'6 6.2xfio-6 1 -

17 .SaKer'gtetcCpApNaTg"np(PP901O) 2.5xfio-4 1 .2xfio-4 6.7xso-6 fi.4xfio-6 ag.4.xao-6 1 -

18e $aKmTcCpApErnNaTmp(PP9105)2e4X1O-3 3.`gx1o-3 1.4x1o-6 3.5x1o'6 5.4xfio-6 7.6xfio"'6m

$tralnmarnberi$$hownfiRbrackets.SyrrgboPs: -,stgaitraitsferrgcti/, notctegerg'e"teiRect.

Abbrgviation$:Ap,ary"kplciilift;Cp,cheorawtphenicei;Eg'E"e, erythromycixt;Km 'kanaryeycin;Na,nailctlxgc acictiSa, $eegtag'vkgnorifiettho-

xine;-g-c,aetracycgine;-E-rrgp,trimethoprlrri.

frequenciesofdrugffesistancesinthedonorsthattransmittedApffesistance\(patterns16-a8andpartefthe

strainsinpatternIS\)differedwithdffugffesistance

patterftsinthedonorsandamaoRgrespectiveresistaRcesin

individualdonors.Thatis,inthedenorscarrcyingresistancestoNaTc(patterft1S),Sa,KmandTeresistafices

weretraftsferredatthefrequencyofaboutleew4andCp

andApresistancesatthatofabout10ti6.Knthedonors

bearingresistancestoNaTmp(patterns16to18),Saand

Kmresistancesweretransmittedatthefrequencyofabout

1Om3-1Om4andTc,Cp,ApandEmresistancesatthatofabout

aeo6.

ptSÅëWSSroN

MffCdistributionsofeachdrugsweresimilartothose

reportedpffevio"sly\(Aoki&Kitao,198S;Takashimaetal,,

198S;Kusudaetal.,1988,199O;Kim&Aoki,1993a\)in

mostofthechemotherapeasticagentstested,Moreover,in

anticipatiomeofthepreviousreport(Kim&Aoki,1993a)Km,

SaandTcwereineffectivefewP.aesmselassp.pisciciaa

therapy,becauseofhighincidenceofresistapmtstffaiRsand

26

hightransferabilityoftheirffesistameees.However,KKiRhas

neveffbeenusedfortreatmentofanybacterialinfectiensin

maffiRefarms.TRRereisnolegtcalexpgainationfofthe

appearanceofKmffesistanceinP,aamselassp.piscicida

strains.However,Cpresistantstrainsisollatedafter1989

appaxeRtiydecreasedthanthoseisolatedbefore1988

exceptfortheisolatesin1994\(inaddition,alkheisolatesirm

1994exhibitedCpresistancesincethefishfromwhichthestrainswereisolatedwerecollectedfromfishcukuredinp

thesamenetpen\).SimiiarfactshavebeeRobservedbyKim

&Aoki(1993a).Thismightbeduetothefactthattheuseof

CpwasrestrictedagainstbacteriaAinfectiensofcultured

fishinjapansince1983,asdiscussedbyKim&Aoki\(1993a\).dntheotherhand,ourresultsweredifferent

fromthepifeviousreports\(Aoki&Kitao,198S;Takashima

etal.,198S;]Kim&Aoki,1993a\)inthefactthattheMgCsof

EmandTmpexhibitedbimodalandtrimodaidistributions,

respectively.

theMgCdistxibutionofTmpexhibitedthreegroups

namely,sensitive,intermediate-Ievelifesistantandhigh-

levelffesistant.Moreoverthesefisigivestffainsweres

isolatedbefore1987theifiterxxxediate-geveltfesistafttp

strainsfrom1988to1993,andthehighaevellffesistaRt

27

stffainsina994.lhesereswsksmaaysuggestthatTxEkp

resistaftcehasinereasedwitheverypassingyeaif.Thisis

thefirstrepertontheclassificationinSothffeegroups

aocordingtotheMffCvalaxesforTmpinP.aamselassp.

piscicidastffains,b"tithasalreadybeenobservedfer

variokgsdrugsinStreptococcesssp.isolatedfromcukured

yellowtail(Aekietal.,1990).Moreever,intheStreptococcassp.theresistancemechanismhasbeen

consideredtodifferbetweenintermediate-gevelandhigh-

levelresistances(Aokietal.,g990).Thefefore,the

differenceinresistaftceievelofTmpthatwasfoundinP,

damselassp.pisciciaaniayaisobeconsideredasmerelythe

differenceinthegroupofthestrains.kkeffesistance

mechanismofintermediate-gevelandhigh-levelresistancesneedstobedeterminediAthefuture.ElheMIC

distributioRofEmexhibifedtwogroupsthatweresefisitive

andresistantandalso,Emresistancewastransferabge.

ThesearethefirstfindingsinP.aamselassp.pisciciaes.

TR}etramesferabilityofdrugresistanceswasdiffereftt

withtheresistancepatterRsiRthedonors.rffhatis,thedffug

resistancetraftsferoccurredwhenthedonorscarified

resistancestoTc,NaTcorNaTmp.Previousay,ithasbeen

reportedthatdrugffesistaRcesinP.aamselassp.piscicida

28

strainswerepmottffaftsferredfromthestrainsbearing

resistancestocombinataonofAp,CpandTc\(Kim&Aoki,

1993a\)butweretraRsmittedfromthestifainscarrying

resistancestocombinatioRofCpandSa\(Kirri&Aoki,

g993a\).ThisreportmayseenocorrelationbetweenresistaftcestoTc,NaTcandNaTmpandthetraitsferability

ofdrugresistances.Furtherstudyneedstobedoneonthe

structureaftdfunctionofthewholeassemblyofresistaRce

genewhichisattachedtoothersegmentscontaininggene

forplasmidreplicationandtraRsferfunctiome,andonthe

mechanismoftranspositioneftheresistancegenes.

AlioftheresistancestoCp,Em,Kkme,SaamedTcwere

transfefredfremthedonofscarryingTcresistanceaspart

ofmugtiresisSafteebutSheresuEtsofKitm&Aoki(1993a)

weredifferentfromourresults.Moreover,Apresistance

containiRgthesetraftsferableresistancesweretraRsmitted

frommostpartoftkedonorscarryingresistancetoNaTc

andalgbearingresistaflcestoNaTmp,butaspeiftheresuats

ofKitm&Aoki(1993a)resistancestoAp,CpandTchadnet

beenrkransfexredfromalmostallofthedonorswhich

showedthesarrkeffesistaRcepatternasours;hence,Aoki

andcewerkers,TakashimaetaR.(198S)andKim&Aoki

\(1993a\)haveconsideredthattheApresistapmeesmightbe

29

residingonthechromosome.Aspertheseffesuks,the

trampsferabigityofresistancestoAp,CpaymdTcwexe

copmsiderabgydifferentbetweenoasrffesaxksaendthoseof

Kim&Aoki(g993a).Theffeasomeferconsiderabgedifference

isincoxnpffehensible.However,thedifferenceofthe

recipieRtsco"gdpossibiybeonereason.inaddition,ifthe

resistancestoNaTmpareconRectedwiththetraptsfer

fuRctionofdifugresistance,thefltheseresistaneesmaybe

presumedtobeeftcodedonaplasmid.

Regardingthe'donorsthatdidnotcarfyoftransfer

Apifesistance,aiftefdrugresistancesineachofthedoftors

weretrapmsmittedwithaimostehesamefrequenciesregardlessofthediffereRcesinthetransferredresistances

aftdthestudieddonors.Theseresultssuggestthatall

resistancesinthesedonorsarecodedonasingleplasmid.

Ontheotherhand,regardingthedonoffsthattransmitted

Apresistance,thetransferfrequeficiesofdrugresistance

differedamongeachdrugresistancethatweretransferred

frornthesamedonews,thoughthefrequenciesofthesame

ifesistancewereaisodifferentwiShthepattermeofdrug

resistancescarriedbythedofiors.lhesefaetssuggestthat

dffugresistancesinthesedenorsaretransferredbytwo

plasgEkEds,Moreover,thefrequencieswerealmostsame

30

atwongKnt,Saand/orTeresistaneesewAp,Cp,Tcand/ox

Exncltffesistances,besidesthefrequeRciesofresistancestethe

formeexwerehigherthantkoseofthelatter."flheseresaxks

suggesSthatplastwidencodingresistancestothefermeris

transferffedwithhighfrequencythaptthosecoding

resistancestothelatter.ffhecorrelationbetweenthe

traRsferofdrugresistaRcesaRdRplasmidswilibereported

inthenextchapter.

Inaddition,thetraRsferfrequenciesofdrugresistancesweredifferentwithgrowthcoRditionsfor

matingassay.Fgr,example,aiftthetransferabledrug

resistamecesinPP9401strain(pattern14)weretransferredinthefrequenciesofabout10tii,andin

PP91OSstrain(pattern18)KmafidSaresistanceswere

traRsmittedinthefrequenciesofabout1OO,andAp,Cp,Em

andErcresistancesinthoseofabout1Om4,iRmatingassayat

differentincubationtime(9hinP.aamselassp.pisciciaa).

SWMMARY

Theeccurrcenceandtransferabanityefdrugffesistarmce

withrespecttoa4antimicxobialcomopoexndswere

31

investigatedagainst183strainsofP.damselesssp.piscicides

afishpathogenghatwereisolatedfromceelturedyeRXowtail

Sertolageeinqeseraaiatainvarioassaffeasefgapanbetween

g984aftd1994.MECdistributionofTmpexpressedthree

growapswhichwereseRsitive,ingermediate-leveiresistant

andhigh-levelresistant;thoseofEmexhibitedtwegroasps

whichwexeseftsitiveandresistantuitliketheresultsp

repoiftedpreviously;thoseofAp,Cp,Km,Na(Oa),armdTc

\(Dox,Otc\)exhibitedtwogroupswhichweresensitiveand

resistant;andthatofSashowedoniyasinglegroupin

whichalltheteststraiRswerefesistant.AEmostallofthe

strainsweremultipledrugresistaxxtrangimegfrom3to8

drugs.TheresistancestoAp,Cp,Eni,K#n,Sa,TcandTmpin

thestrainsweretransferredtoEscherichiacoli]K-12

xaO37Rpr.ff{owever,thetransferabiiityoftheresistances

differedwiththepatternandRevelofdffugresistances,

especiallyTmpresistaRcewastransferredonlyinhigh-

tevelresistancestrain.11I}etransferfrequenciesofdifug

ffesistancesweredifferewatdependingasponwhetheroritot

tffansferableApresistapmcehasbeeflcarriedbythedonors

andtheresistancepatterninthedonorscarryingtransmissibEeApresistaftce.

32