Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

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Veterinary

Microbiology

Virulence

genes

and

antimicrobial

resistance

of

Pasteurella

multocida

isolated

from

poultry

and

swine

Thales

Quedi

Furian

,

Karen

Apellanis

Borges,

Vanessa

Laviniki,

Silvio

Luis

da

Silveira

Rocha,

Camila

Neves

de

Almeida,

Vladimir

Pinheiro

do

Nascimento,

Carlos

Tadeu

Pippi

Salle,

Hamilton

Luiz

de

Souza

Moraes

CentrodeDiagnósticoePesquisaemPatologiaAviária,FaculdadedeVeterinária,UniversidadeFederaldoRioGrandedoSul, PortoAlegre,RSCEP:91540-000,Brazil

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t

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o

Articlehistory:

Received20October2014 Accepted24June2015

AssociateEditor:NiltonErbet LincopanHuenuman Keywords: Pasteurellosis Virulencefactors Multiplex-PCR Antimicrobialsusceptibility

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b

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Pasteurellamultocidacausesatrophicrhinitisinswineandfowlcholerainbirds,andisa sec-ondaryagentinrespiratorysyndromes.Pathogenesisandvirulencefactorsinvolvedarestill poorlyunderstood.Theaimofthisstudywastodetect22virulence-associatedgenesbyPCR, includingcapsularserogroupsA,BandDgenesandtoevaluatetheantimicrobial susceptibil-ityofP.multocidastrainsfrompoultryandswine.ompH,oma87,plpB,psl,exbD-tonB,fur,hgbA, nanB,sodA,sodC,ptfAweredetectedinmorethan90%ofthestrainsofbothhosts.91%and 92%ofavianandswinestrains,respectively,wereclassifiedinserogroupA.toxAandhsf-1 showedasignificantassociationtoserogroupD;pmHASandpfhAtoserogroupA.Gentamicin andamoxicillinwerethemosteffectivedrugswithsusceptibilityhigherthan97%;however, 76.79%ofpoultrystrainsand85%ofswinestrainswereresistanttosulphonamides. Fur-thermore,19.64%and36.58%ofavianandswinestrains,respectively,weremulti-resistant. Virulencegenesstudiedwerenotspecifictoahostandmaybetheresultofhorizontal transmissionthroughoutevolution.Highmultidrugresistancedemonstratestheneedfor responsibleuseofantimicrobialsinanimalsintendedforhumanconsumption,inaddition toantimicrobialsusceptibilitytestingtoP.multocida.

©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:thales.furian@ufrgs.br(T.Q.Furian).

http://dx.doi.org/10.1016/j.bjm.2015.11.014

1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Pasteurellamultocidaisanormalinhabitantoftherespiratory tractofhealthyanimals1_{;however,itisanenigmaticpathogen}

knownforbeingassociatedwithadiversityofrespiratory syn-dromesthat can affecta rangeofhostspecies.2 _Moreover,

itiseithertheprimarycausativeagentorpresentsamajor roleintheevolutionofcertaindiseasesofeconomicimpact, suchasatrophicrhinitisinpigsandfowlcholera(FC)inavian species.2

Thevariabilityoftheclinicalsignsincasesofpasteurellosis ispossiblyexplainedbythealterationofthecommensal rela-tionshipbetweenhostandbacteria,aswellasbythepresence ofvirulencefactorsthatdifferseveralvariantsofonespecies ofthispathogenicmicroorganism.1,3_{Somemolecularstudies}

havebeendevelopedtoidentifygenesrelatedtovirulenceinP. multocida,butlittleisknownregardingthevirulencegene con-tentbetweenP.multocidastrainsindifferentanimalhosts.4–6

Thecapsuleisoneofthemajorvirulencefactorsidentified inP.multocida,7_{andthegenesrequiredforthesynthesisand}

transportofthesecapsulartypesareencodedwithinasingle regionofthegenome.8_{Allstrainscanbeclassifiedintofive}

dif-ferentcapsulartypesorserogroups(A,B,D,EandF)according tothepresenceofcapsularantigens.7

Although the strains are generally susceptible to the majority of antimicrobials,9 _{the increased incidence of}

multidrug-resistant pathogenic bacteria has been widely reportedinthelastseveraldecades.6_{Comparedwithhuman}

specificpathogens,evenlessdataareavailableonresistance patternsin pathogensexclusivelyfound infoodanimals.10

However,theknowledgeandthecontrolofantibiotic treat-mentinP.multocidaareimportant,becausetheantimicrobial usemayselectresistantstrains,increasingtheirprevalence.11

Moreover,the useofantibioticsinthecontrolof pasteurel-losis,aswellasthedevelopmentofserotype-specificvaccines, requires surveys in each geographical area.12 _{But studies}

relatedtotheperformanceofantimicrobialsusceptibility pat-ternsinP.multocidastrainsofavianoriginarerareinBrazil.13

OurobjectivewastodeterminewhetherstrainsofP.multocida isolatedfromcasesofFCandfrompigswithrespiratory prob-lemsinsouthernBrazilvaryintheirrepertoireof22virulence genes,andtoassessthesusceptibilityofthestrainstoeight antimicrobialagents.

Materials

and

methods

StrainsofP.multocida

Ninety-sixstrainsofP.multocidapreviouslyisolatedofclinical casesinsouthernBrazilwereselected.Afterthediagnosiswas confirmed,twocoloniesofeachstrainplatedonbloodagar wereselectedandstoredin1mLofdefibrinatedsheepblood atatemperatureof−80◦C.Thereactivationandpreliminary teststoconfirmthepresenceofpuresampleswereconducted accordingtoGlissonetal.14_{Ofthestrainsselected,58%(56/96)}

wereisolatedfromtheliverorheartofchickens/turkeysand 42%(40/96)fromthelungofpigsthatwerebeingraisedfor humanconsumption.

Detectionofvirulence-associatedgenes

An aliquot of BHI after an overnight incubation (1mL) was selected for DNA extraction using the commercial kit NucleoSpin®_{Tissue(MachereyNagel}®_{,Düren,Germany).}

Ini-tially,aPCRprotocolforspecies-specificamplificationofthe kmtgenewasperformed,asdescribedbyTownsendetal.15

Fif-teengenesassociatedwithvirulence(ompH,oma87,sodA,sodC, hgbA, hgbB,exBD-tonB,nanB,nanH,ptfA,pfhA,toxA), includ-ing the genes forcapsulemolecular typing ofserogroupA (hyaD-hyaC),B(bcbD)andD(dcbF),weresurveyedaccordingto themultiplexPCRprotocolsstandardizedbyFurianetal.16,17

Anadditionalsevengenes(hsf-1,pmHAS,fur,psl,ompA,plpB, tadD)weresurveyedaccordingtoprotocolsdescribedbyEwers etal.4_andTangetal.6_{withmodifications.Referencestrains}

of P. multocida (ATCC 15742, ATCC 12945 and ATCC 12946) wereselectedaspositivecontrols.Membersofthe Pasteurel-laceaefamily(RiemerellaanatipestiferATCC11845,Mannheimia haemolyticaATCC29694andPasteurellagallinarumATCC13360) wereusedasnegativecontrols.

The amplification reactions were performed in a ther-mocycler (SwiftMaxPro Thermal Cycler –ESCO Technologies®_,

Hatboro, Pennsylvania, USA). Electrophoresis ofthe ampli-fied products was carried out in a 1%or 1.5% agarose gel (InvitrogenUltrapureTMAgarose®,Carlsbad,California,USA) stained with ethidium bromide, after which the amplified products were photo documented (AlphaDigDoc Pro – Alpha Innotech®_{,SanLeandro,CA,USA).Allprotocolswererepeated}

threetimesinparallelwiththerelevantpositiveandnegative controls.

Antimicrobialsusceptibilitytesting

All of the strains were tested for their antimicrobial sus-ceptibility by the disk diffusion method according to the performance standards M31–A3 of the Clinical and Labo-ratory Standards Institute (CLSI).18 _{Each sample was tested}

against8antimicrobialagents(Oxoid®_{):erythromycin(15␮g),}

gentamicin (10␮g), amoxicillin (10␮g), sulphaquinoxaline (300␮g),tetracycline(30␮g),sulfamethoxazole-trimethoprim (1.25–23.75␮g), ceftiofur (30␮g) and enrofloxacin (5␮g). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923wereselectedasqualitycontrolstrains.The interpre-tationofresultswasbasedonthebreakpointsprovidedbythe CLSIguidelines.19,20

Statisticalanalysis

Descriptivestatisticswereusedtocalculatetheabsoluteand relativefrequenciesofthevirulencegenesandantimicrobial susceptibilityprofiles.Chi-square(2_{)andFisher’sexacttest}

wereusedtoevaluatethegivenpairofgenesandto deter-minetheassociationbetweenthegenesandthehostspecies ofisolation.Comparisonsofgeneproportionswithingroups ofthesamefunctionweremadebyMcNemar’stest.Statistical testingwasperformedwithSPSSsoftware(StatisticalPackage forSocialSciences),andpvaluesof<0.05wereconsidered sta-tisticallysignificant.

Table1–Thedistributionof22virulenceassociated

genesofPasteurellamultocidadetectedbyPCRaccording

tohostspecies.

Processor enzyme

Gene Absoluteand relative frequency(%) –avian strains(n=56) Absoluteand relative frequency(%) –swine strains(n=40) Capsule biosynthesis hyaD-hyaC 51(91%) 37(92%) dcbF 3(5%) 3(8%) bcbD 0(0%) 0(0%) Outermembrane protein ompH 54(96%) 39(98%) oma87 56(100%) 40(100%) ompA 34(61%) 38(95%) plpB 55(98%) 39(98%) psl 54(96%) 39(98%) Adhesins ptfA 54(96%) 39(98%) pfhA 35(63%) 21(53%) tadD 21(38%) 34(85%) hsf-1 28(50%) 8(20%) Sialidases nanH 53(95%) 34(85%) nanB 55(98%) 40(100%) Superoxide dismutases sodA 54(96%) 40(100%) sodC 54(96%) 40(100%) Hyaluronicacid synthetase pmHAS 49(88%) 37(92%) Dermonecrotic toxin toxA 0(0%) 1(3%)

Ironmetabolism exbD-tonB 55(98%) 39(98%) fur 54(96%) 39(98%) hgbA 55(98%) 37(92%) hgbB 52(93%) 23(58%)

Results

Distributionofvirulencegenes

The absolute and relative frequencies of the virulence-associatedgenesarepresentedinTable1.Themajorityofthe virulence-associatedgenesareregularlydistributedamongP. multocidastrains.Thegenes associatedtooutermembrane proteins(ompH,oma87,plpB,psl),adhesion(ptfA),iron meba-tolism(exbD-tonB,fur,hgbA),sialidases(nanB)anddismutases (sodA,sodC)weredetectedinmorethan90%ofthestrainsof bothhosts.

ThegeneshyaD-hyaC,dcbFand bcbDareassociatedwith capsularbiosynthesisofP.multocidaspecifictoserogroupsA, D and B,respectively. Over90% of the strainsbelonged to serogroupAinbothhostspecies,andtherewasastrong sig-nificantdifferencecomparedtodcbF(p<0.001).Twostrainsof avianoriginwerenotidentifiedinanyofthethreecapsular types.Morethan90%ofthestrainswerepositiveforgenes encodingoutermembraneproteins(ompH,oma87,psl,plpB)in bothhostspecies.However,ompAwasdetectedin61%ofavian strainswithasignificantlylower occurrence(p<0.001)than thatobservedfortheothergenesinthesamefunctionalgroup. Similarly,hgbBshowedalowerpercentage(58%)thanother genesinvolvedinironmetabolismamongtheswinestrains

(p<0.001). No significant difference was observed between exbD-tonB, furandhgbA(p>0.05).Thegenesencoding siali-dases(nanH,nanB),dismutases(sodA,sodC)and hyaluronan synthase(pmHAS)alsoshowedfrequenciesclosetoorhigher than90%anddifferenceswithinthegroupswerenot signifi-cant(p>0.05).

Ahighervariationoffrequencieswasobservedamongthe adhesins.ThegeneptfAencodingasubunitoftypeIV fim-briaewassignificantlyhigherinbothhostspecies(p<0.001) compared to pfhA and hsf-1, which encode a filamentous hemagglutinin and an autotransport adhesin, respectively. Additionally,thefrequencyofpfhAexhibitedasignificant dif-ference(p<0.05)comparedtotadDinbothhosts.Ontheother hand,asignificantdifferencebetweentadDandhsf-1 frequen-cies(p<0.001)wasonlyobservedamongstrainsisolatedfrom pigs.

The

association

between

virulence

genes

and

serogroups

Theassociationbetweenvirulencegenesand serogroupsis presentedinTable2.Somevirulencegenesexhibited distinc-tiveassociationswithserogroupAandserogroupD.Thegenes toxAandhsf-1arepositivelyassociatedwithserogroupD,and the genespmHASandpfhA withserogroupA.Onthe other hand,anegativeassociationofpmHAS,pfhAandtadDwith serogroupDwasobserved,aswellasofhsf-1withserogroupA. ThecombinationofgenesamongP.multocidaregardlessof hostspeciesofisolationisshowninTable3.Inadditiontothe highfrequenciesexhibitedbythemajorityofselectedgenes,

Table2–Theabsoluteandrelativefrequencies(%)of

virulenceassociatedgenesdetectedbyPCRaccordingto

serogroupsAandDofPasteurellamultocida.

Gene SerogroupA(gene hyaD-hyaC)(n=88) SerogroupD(gene dcbF)(n=6) ompH 85(97%) 6(100%) ompA 65(74%) 6(100%) plpB 86(98%) 6(100%) psl 86(98%) 5(83%) exbD-tonB 86(98%) 6(100%) fur 85(97%) 6(100%) hgbA 84(95%) 6(100%) hgbB 67(76%) 6(100%) nanH 79(90%) 6(100%) nanB 87(99%) 6(100%) sodA 86(98%) 6(100%) sodC 86(98%) 6(100%) pmHAS 86(98%)** _0(0%)** toxA 0(0%) 1(17%)* ptfA 85(97%) 6(100%) pfhA 56(64%)* _0(0%)* tadD 53(60%) 0(0%)* hsf-1 28(32%)** _6(100%)*

oma87:constantinbothserogroups. ∗ _{Significantassociation(p<0.05).} ∗∗ _{Significantassociation(p<0.001).}

Table3–Thepercentageofvirulence-associatedgenesinpairsamong96strainsofPasteurellamultocidaanalyzed.

ompH toxA ptfA nanH exbD-tonB sodA pfhA hgbA sodC nanB hgbB oma87 hsf-1 fur pmHAS psI ompA plpB tadA

ompH 100 toxA 1 100 ptfA 97 1 100 nanH 93* _{100 90 100} exbD-tonB 100* _{100 98 100}* ₁₀₀ sodA 98 100 98 99 98 100 pfhA 58 0 57 60 59 59 100 hgbA 96 100 96 99* _{96 96 100}* ₁₀₀ sodC 99 1 98 99 99* _{98 100 98 100} nanB 100* _{100 99 100 100}* _{99 100 99 100}* ₁₀₀ hgbB 78 100 78 80 78 78 77 82* _{78 78 100} oma87 100 100 100 100 100 100 100 100 100 100 100 100 hsf-1 36* _{100 38 33}* _{36 37 38 35}* _{36 37 42 38 100} fur 99* _{100 97 99}* ₉₉* _{97 96 97 98 98}* _{96 97 92}* ₁₀₀ pmHAS 90 0 90 89 89 89 98* _{90 89 90 87 90 78}* _{90 100} psl 99* _{100 97 99}* ₉₉* _{97 98 97 98 98}* _{96 97 92}* ₉₉* _{98 100} ompA 77* _{100 76 76}* _{44 75 63}* _{74 77 76 68}* _{75 39}** ₇₇* _{73 76 100} plpB 100* _{100 98 100}* ₁₀₀** _{98 98 98 99 99}* _{97 98 94 100}* _{98 100}* _{100 100} tadD 60 0 58 57* _{59 56 48}* _{55 59 58 45}** _{57 28}* ₅₉* ₆₂* _{59 74}* _{59 100}

TheassociationofhyaD-hyaCanddcbFwithothergenesislistedinTable2. ∗ _{Significantassociation(p<0.05).}

∗∗ _{Significantassociation(p<0.001).}

therewasasignificantassociationinthecombinationof viru-lencegenesin45situations.ThecombinationsbetweenplpB andexbD-tonB,tadDandhgbB,andompAandhsf-1indicatea strongsignificantassociation(p<0.001),asshowninTable3. Eachofthegenes,excepttadD,presentedasignificant associ-ation(p<0.001)forbothhostspeciesandinthiscasecannot beselectedasspecies-specificmarkers.

Antimicrobialsusceptibility

TheantimicrobialsusceptibilitypatternsofP.multocidastrains are described in Table 4. No drug was effective in inhibi-tingthe growth of100%of the poultrystrains, but strains ofthis group showeda higher susceptibility to80% ofthe antimicrobialstested,withtheexceptionofenrofloxacinand sulphaquinoxaline.Onlyamoxicillinandgentamicininhibited thegrowthofmorethan80%ofporcinestrains.Forthe pur-poseofthisstudy,multidrug-resistance(MDR)wasdefinedas acquirednon-susceptibilitytoatleastoneagentinthreeor moreantimicrobialcategories.21_{36.58%oftheporcinestrains}

weremulti-resistant,while19.64%ofthepoultrystrainswere resistanttothreeormoredrugsindifferentcategories.The highestpercentagesofresistanceobserved,regardlessofthe hostorigin,wererelatedtosulphaquinoxaline.

Discussion

TheserogroupclassificationofP.multocidaaccordingthe cap-sular typing is used for studies on the pathogenesis and epidemiologyoftheagent.Thebiologicalbasisofthis phe-nomenon is unknown, but it suggests that the capsule is relatedtothepathogenesisoftheindividualdiseasesandto hostpredilectionofparticularserogroups.22_{Itisknownthatin}

additiontothespecificrelationtocertaindiseasesandspecies,

thereisadominantgeographicaldistributionofthe capsu-lartypesinsomecases.However,theserelationshaveshown variationsinrecentyears.23 _{Serogroupclassificationisrare}

inBrazil,especiallywithstrainsofP.multocidaisolatedfrom poultry.TypeA,identifiedin91%oftheavianstrains,isthe majorserogroupfoundinthishost,andsimilarresultswere observedinother studies.24,25 _{Only5%ofthe avianstrains}

wereclassifiedincapsulartypesDandnoneintypeB,which isalsopresentinbirds,aswellastypeF.Theoccurrenceof theseserogroupsisconsideredrare.26_{Likewise,capsulartype}

Awasalsoprevalentamongstrainsfromswine(92%),which demonstratesavariableprevalenceoftheserogroupsincases ofrespiratorydiseaseaccordingtothegeographicregion.6,27

Althoughthemajorityofvirulencegenespresenteda sim-ilardistributionbetweenserogroups,someweresignificantly associatedwithaspecificcapsular type,suchas pfhAwith serogroup A.Tang et al.6 _{reported the presenceofpfhA in}

25% ofstrains from serogroup A, and onlyin 3.1% of the strainsfromtypeD.Similarresultswere obtainedbyEwers et al.4 _{thatanalyzed289strains isolatedfrom varioushost}

species. The positive association of pfhA and capA is not likelyduetoaphysicallinkage,asthegenesareseparated byapproximately839kbinthegenomeofP.multocidastrain Pm70.Hypothetically,acapApositiveclonemayhaveacquired pfhA by horizontal gene transfer and achieved an advan-tage to survivewithin the host.5 _{The toxA gene, encoding}

adermonecrotic toxin(PMT– P. multocidatoxin),presented asignificantassociationtoserogroupD(p<0.05).Thistoxin inducesosteolysisintheturbinatebonesandplaysan impor-tantroleinthepathogenesisofprogressiveatrophicrhinitis.27

AlthoughtoxAisassociatedwithserogroupD,28_other

stud-ies havedetecteditinstrainsofserogroupAisolatedfrom differenthosts.29_Tangetal.6_{alsoobservedthepositive}

asso-ciationofhsf-1withserogroupDandthenegativeassociation ofpmHASwiththesameserogroup.Ontheotherhand,pmHAS

Table4–TheantimicrobialsusceptibilitypatternsofPasteurellamultocidastrainsbydiskdiffusiontestaccordingtohost species.

Antimicrobial Hostspecies

Poultry Swine

Relativefrequency(%) Relativefrequency(%) Susceptible Non-susceptible Susceptible Non-susceptible

Amoxicillin 98.21 1.79 100 – Ceftiofur 98.21 1.79 77.5 22.5 Enrofloxacin 76.79 23.21 77.5 22.5 Erythromycin 94.64 5.36 60 40 Gentamicin 98.21 1.79 97.5 2.5 Tetracycline 87.5 12.5 60 40 Sulphaquinoxaline 23.21 76.79 15 85 Sulfamethoxazole-trimethoprim 80.36 19.64 67.5 32.5

wassignificantlyassociatedwithserogroupA.Thehyaluronan synthasePmHASisadual-actionsynthasethattransfers alter-natingunitsofd-glucuronicacidandN-acetyl-d-glucosamine intheformationofhyaluronicacid,themajortypeAcapsular material.30

Thehighfrequencyofvirulencegenesanalyzedwasalso observedinotherstudieswithstrainsfrombothhosts.4–6 _A

similardistributionofcertaingenes,regardlessofhostspecies orP.multocidaserotype,maysuggesttheselectionoffactors thatpresentcross-protectionascandidatesforvaccine devel-opment.Someoftheseantigenspotentiallyserveasvaccine candidates.31,32_{Asanexample,porinsaregenerallyconserved}

amongspecies and have high immunogenicity.33 _However,

studieswithexperimentalinfectionfrequentlyshowvariable resultsinanimalprotection.33,34

Theabilityofpathogenstoobtainironisakeyfeature dur-ingthe infectionprocess.35 _{ThefrequencyofhgbB inavian}

strainswashigherthantheoccurrenceof85%observedfor Ewersetal.,4_{theonlystudythatinvestigateditspresencein}

thishost.Ontheotherhand,thegenewasdetectedinonly58% ofthestrainsfromswine.Betheetal.5_{detectedhgbBinless}

frequencyindiseasedthaninhealthyanimals,aresultalso foundbyShayeghetal.,29_{whoanalyzedstrainsfromovine.In}

bothstudies,thereisnotthoughttobearelationshipbetween thepresenceofhgbBandpasteurellosis.

Themajorityofcommensalandpathogenicbacteriathat interactwith eukaryotic hosts express adhesive molecules on their surfaces to promote interaction with host cell receptors.36_{WiththeexceptionofptfAthatencodesafimbrial}

subunittypeIV,37_{theotheradhesin-relatedgenespresented}

afrequencyinferiorto65%.Studiesshowavariationinthe frequencyofadhesinsanddifferininterpretationregarding therelationshipbetweentheadhesinandthedisease.Ewers et al.4 _{observed a variation between 7 and 100% of pfhA}

accordingtothehostspeciesandrelateditspresencetothe occurrenceofpasteurellosisincattle.Similarly,Betheetal.5

connectedpfhAwiththeoccurrenceofrespiratorydiseasein swine.However,Shayegh et al.29 _{detectedalow frequency}

(18%)ofthegeneamongovinestrainsanddidnotrelateit withpasteurellosisinthisspecies.

Antibiotictherapyisstillaneffectivetoolinthetreatment ofinfectionscausedbyP.multocida,asthestrainswere sus-ceptibletothemajorityofdrugscommerciallyavailable.The

highsusceptibilityofstrainsfrombothhoststoamoxicillin, gentamicinandceftiofurwasalsoobservedinotherstudies indifferentcountries,6,9,38,39_{aswellasinBrazilwithstrainsof}

porcineorigin.40,41_{Incontrasttootherstudiesthatreportlow}

activityofaminoglycosides,38_{gentamicinwaseffectiveinthe}

currentstudy.Likewise,resistancetoerythromycingenerally observed42_{wasnotfoundamongthestrainsfrompoultry.}

However,excessiveandunjustified useofantimicrobials may accelerate the emergence of resistant strains. Among theconventionaldrugsinveterinarymedicine,tetracyclines are widely employed,11,12 _{which explains the high}

per-centage of resistance in this study and the literature.11,43

Another example isthe high frequencyofnon-susceptible strainstoquinolonesandthelowefficacyofsulphonamides, chemotherapeuticagentsindicatedforthetreatmentofFC,43

alsofoundinseveralstudies.6,42,44

Increasingmultidrugresistanceisattributableatleastin parttotheuseofantibioticadditivesinanimalfeedandthe extensiveuseofantimicrobialagentsinveterinarymedicine.6

Similarly,thehorizontaltransferofgenesbymobilegenetic elementsfrom differentbacterialgeneraandspeciesfavors thedevelopmentofmultidrug-resistance.45_{Thepresenceof} P. multocida strains in a polymicrobial environment offers theopportunitytoacquireresistancegenesthathave devel-opedinotherbacteriabuthavebeenrefinedunderselective pressure.10

Conclusion

Thisstudyprovidedinformationregardingthedistributionof virulencegenesinstrainsofP.multocidaisolatedfrompoultry andswineinBrazil.Thesegenespresentedahighfrequency inthemajorityofthecasesandtheywerenotspecifictoa host.Similarly,highmultidrugresistanceevincestheneedof restrictiveand responsibleuseofantimicrobialsinanimals intendedforhumanconsumption,inadditiontoantimicrobial susceptibilitytestingtoP.multocida.

Conflicts

of

interest

r

e

f

e

r

e

n

c

e

s

1.SamuelMD,BotzlerRG,WobeserGA.Aviancholera.In:

ThomasNJ,HunterDB,eds.InfectiousDiseasesofWildBirds.

12thed.Ames,IA:BlackwellPublishing;2007:239–269.

2.WilkieIW,HarperM,BoyceJD,AdlerB.Pasteurellamultocida:

diseasesandpathogenesis.CurrTopMicrobiolImmunol.

2012;361:1–22.

3.VieiraMAM.Ilhasdepatogenicidade.OMundodaSaúde.

2009;33:406–414.

4.EwersC,Lübke-BeckerA,BetheA,KieblingS,FilterM,Wieler

LH.VirulencegenotypeofPasteurellamultocidastrains

isolatedfromdifferenthostswithvariousdiseasestatus.Vet

Microbiol.2006;114:304–317.

5.BetheA,WielerLH,SelbitzHJ,EwersC.Geneticdiversityof

porcinePasteurellamultocidastrainsfromtherespiratory

tractofhealthyanddiseasedswine.VetMicrobiol.2009;139:

97–105.

6.TangX,ZhaoZ,HuJ,etal.Isolation,antimicrobial

resistance,andvirulencegenesofPasteurellamultocida

strainsfromswineinChina.JClinMicrobiol.2009;47:

951–958.

7.HarperM,BoyceJD,AdlerB.Pasteurellamultocida

pathogenesis:125yearsafterPasteur.FEMSMicrobiolLett.

2006;265:1–10.

8.TownsendKM,BoyceJD,ChungJY,FrostAJ,AdlerB.Genetic

organizationofPasteurellamultocidacapLociand

developmentofamultiplexcapsularPCRtypingsystem.J

ClinMicrobiol.2001;39:924–929.

9.HuangTM,LinTL,WuCC.Antimicrobialsusceptibilityand

resistanceofchickenEscherichiacoli,Salmonellaspp.,and

Pasteurellamultocidaisolates.AvianDis.2009;53:89–93.

10.WassenaarTM,SilleyP.Antimicrobialresistanceinzoonotic

bacteria:lessonslearnedfromhost-specificpathogens.Anim

HealthResRev.2008;9:177–186.

11.BabetsaM,SandalakisV,VougidouC,etal.Tetracycline

resistancegenesinPasteurellamultocidaisolatesfrombovine,

ovine,caprineandswinepneumoniclungsoriginatedfrom

differentGreekprefectures.AfrJMicrobiolRes.

2012;6:3917–3923.

12.KehrenbergC,Schulze-TanzilG,MartelJL,Chaslus-DanclaE,

SchwarzS.AntimicrobialresistanceinPasteurellaand

Mannheimia:epidemiologyandgeneticbasis.VetRes.

2001;32:323–339.

13.RigobeloEC,BlackallPJ,MalutaRP,deÁvilaFA.Identification

andantimicrobialsusceptibilitypatternsofPasteurella

multocidaisolatedfromchickensandJapanesequailsin

Brazil.BrazJMicrobiol.2013;44:161–164.

14.GlissonJR,SandhuTS,HofacreCL.Pasteurellosis,

avibacteriosis,gallibacteriosis,riemerellosisand

pseudotuberculosis.In:Dufour-ZavalaL,ed.ALaboratory ManualfortheIsolation,IdentificationandCharacterizationof AvianPathogens.5thed.Athens,GA:AmericanAssociationof

AvianPathologists;2008:12–18.

15.TownsendKM,FrostAJ,LeeCW,PapadimitriouJM,Dawkins

HJS.DevelopmentofPCRassaysforspecies-and

type-specificidentificationofPasteurellamultocidaisolates.J

ClinMicrobiol.1998;36(4):1096–1100.

16.FurianTQ,BorgesKA,PilattiRM,AlmeidaC,NascimentoVP.

IdentificationofthecapsuletypeofPasteurellamultocida

isolatesfromcasesoffowlcholerabymultiplexPCRand

comparisonwithphenotypicmethods.BrazJPoultrySci.

2014;16:31–36.

17.FurianTQ,BorgesKA,RochaSLS,etal.Detectionof

virulence-associatedgenesofPasteurellamultocidaisolated

fromcasesoffowlcholerabymultiplex-PCR.PesquiVetBras.

2013;33:177–182.

18.CLSI(ClinicalandLaboratoryStandardsInstitute).

PerformanceStandardsforAntimicrobialDiskandDilution SusceptibilityTestsforBacteriaIsolatedfromAnimals–Document M31-A3.Wayne,PA:ClinicalandLaboratoryStandards

Institute;2008.

19.CLSI(ClinicalandLaboratoryStandardsInstitute).Methods

forAntimicrobialDilutionandDiskSusceptibilityTestingof InfrequentlyIsolatedorFastidiousBacteria–DocumentM45-A2.

Wayne,PA:ClinicalandLaboratoryStandardsInstitute;

2010.

20.CLSI(ClinicalandLaboratoryStandardsInstitute).

PerformanceStandardsforAntimicrobialDiskandDilution SusceptibilityTestsforBacteriaIsolatedfromAnimals;Second InformationalSupplement−DocumentVET01-S2.Wayne,PA:

ClinicalandLaboratoryStandardsInstitute;2013.

21.MagiorakosAP,SrinivasanA,CareyRB,etal.

Multidrug-resistant,extensivelydrug-resistantand

pandrug-resistantbacteria:aninternationalexpertproposal

forinterimstandarddefinitionsforacquiredresistance.Clin

MicrobiolInfect.2012;18:268–281.

22.ChungJY,ZhangY,AdlerB.Thecapsulebiosyntheticlocus

ofPasteurellamultocidaA:1.FEMSMicrobiolLett.1998;166: 289–296.

23.BorowskiSM,BarcellosD,MorésN.Pasteurelosepulmonar.

In:SobestianskyJ,BarcellosD,eds.Doenc¸asdossuínos.

Goiânia:CânoneEditorial;2007:177–181.

24.ShivachandraSB,KumarAA,GautamR,SinghVP,Saxena

MK,SrivastavaSK.Identificationofavianstrainsof

PasteurellamultocidainIndiabyconventionalandPCRassays.

VetJ.2006;172:561–564.

25.JabbariAR,EsmaelzadehM,MoazeniJulaGR.Polymerase

chainreactionofPasteurellamultocidacapsulesisolatedin

Iran.IranJVetRes.2006;7:50–55.

26.DaviesRL,MacCorquodaleR,BaillieS,CaffreyB.

CharacterizationandcomparisonofPasteurellamultocida

strainsassociatedwithporcinepneumoniaandatrophic

rhinitis.JMedMicrobiol.2003;52(Pt1):59–67.

27.DaviesRL,MacCorquodaleR,CaffreyB.Diversityofavian

PasteurellamultocidastrainsbasedoncapsularPCRtyping

andvariationoftheOmpAandOmpHoutermembrane

proteins.VetMicrobiol.2003;91:169–182.

28.DzivaF,ChristensenH,vanLeengoedLA,MohanK,OlsenJE.

DifferentiationofPasteurellamultocidaisolatesfromcasesof

atrophicrhinitisinpigsfromZimbabwebyRAPDand

ribotyping.VetMicrobiol.2004;102:117–122.

29.ShayeghJ,AtashpazS,HejaziMS.Virulencegenesprofile

andtypingofovinePasteurellamultocida.AsianJAnimVetAdv.

2008;3:206–213.

30.DeAngelisPL,GunayNS,ToidaT,MaoWJ,LinhardtRJ.

IdentificationofthecapsularpolysaccharidesofTypeDand

FPasteurellamultocidaasunmodifiedheparinand

chondroitin,respectively.CarbohydrRes.2002;337:1547–1552.

31.KastenRW,CarpenterTE,SnipesKP,HirshDC.Detectionof

Pasteurellamultocida-specificDNAinturkeyflocksbyuseof

thepolymerasechainreaction.AvianDis.1997;41:676–682.

32.BoschM,GarridoME,PérezdeRozasAM,BadiolaI,BarbéJ,

LlagosteraM.Pasteurellamultocidacontainsmultiple

immunogenichaemin-andhaemoglobin-bindingproteins.

VetMicrobiol.2004;99:103–112.

33.LeeJ,KimYB,KwonM.OutermembraneproteinHfor

protectiveimmunityagainstPasteurellamultocida.JMicrobiol.

2007;45(2):179–184.

34.OkayS,ÖzcengizE,GürselI,ÖzcengizG.Immunogenicity

andprotectiveefficacyoftherecombinantPasteurella

lipoproteinEandoutermembraneproteinHfromPasteurella

multocidaA:3inmice.ResVetSci.2012;93:1261–1265.

35.RatledgeC,DoverLG.Ironmetabolisminpathogenic

36.KlineKA,FälkerS,DahlbergS,NormarkS,

Henriques-NormarkB.Bacterialadhesinsinhost–microbe

interactions.CellHostMicrobe.2009;5:580–592.

37.HatfaludiT,Al-HasaniK,BoyceJD,AdlerB.Outermembrane

proteinsofPasteurellamultocida.VetMicrobiol.2010;144:

1–17.

38.YoshimuraH,IshimaruM,EndohYS,KojimaA.

AntimicrobialsusceptibilityofPasteurellamultocidaisolated

fromcattleandpigs.JVetMedB:InfectDisVetPublicHealth.

2001;48:555–560.

39.HabrunB,KompesG,CvetnicZ,SpicicS,MiroslavB,MitakM.

AntimicrobialsensitivityofEscherichiacoli,Salmonellaspp.,

Pasteurellamultocida,StreptococcussuisandActinobacillus pleuropneumoniaeisolatedfromdiagnosticsamplesfrom

largepigbreedingfarmsinCroatia.VetArhiv.

2010;80:571–583.

40.BorowskiSM,IkutaN,LungeV,FonsecaA,MarquesE,

CardosoM.Caracterizac¸ãoantigênicaefenotípicadecepas

dePasteurellamultocidaisoladasdepulmõesdesuínoscom

pneumoniae/oupleurite.PesquiVetBras.2002;22:97–103.

41.SoaresTCS,MottaRG,PaesAC,ListoniFJP.Perfilde

sensibilidadedeamostrasdePasteurellamultocidae

MannheimiahaemolyticaisoladasnaregiãodeBotucatu,São

Paulo,Brasil,noperíododejaneirode2000anovembrode

2007.VetZootec.2009;16:173–179.

42.ShivachandraSB,KumarAA,BiswasA,RamakrishnanMA,

SinghVP,SrivastavaSK.Antibioticsensitivitypatterns

amongIndianstrainsofavianPasteurellamultocida.TropAnim

HealthProd.2004;36:743–750.

43.MohamedMA,MohamedMW,AhmedAI,IbrahimAA,

AhmedMS.Pasteurellamultocidainbackyardchickensin

UpperEgypt:incidencewithpolymerasechainreaction

analysisforcapsuletype,virulenceinchickenembryosand

antimicrobialresistance.VetItal.2012;48:77–86.

44.JonasM,MorishitaTY,AngrickEJ,JahjaJ.Characterizationof

ninePasteurellamultocidaisolatesfromaviancholera

outbreaksinIndonesia.AvianDis.2001;45:34–42.

45.WalshC,FanningS.Antimicrobialresistanceinfoodborne

pathogens−acauseforconcern?CurrDrugTargets.