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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
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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