Expression of the marA, soxS, acrB and ramA genes related to the AcrAB/TolC efflux pump in Salmonella entérica strains with and without quinolone resistance-determining regions gyrA gene mutations

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The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

Expression

of

the

marA,

soxS,

acrB

and

ramA

genes

to

the

AcrAB/TolC

efﬂux

pump

in

Salmonella

enterica

strains

with

and

without

quinolone

resistance-determining

regions

gyrA

gene

mutations

Rafaela

Gomes

Ferrari

a,∗

_,

_Antonio

_Galiana

b

_,

_Rosa

_Cremades

b

_,

_Juan

_Carlos

_Rodríguez

b

_,

Marciane

Magnani

a

_,

_Maria

_Cristina

_Bronharo

_Tognim

c

_,

_Tereza

_C.R.M.

_Oliveira

a

_,

Gloria

Royo

b

a_Agricultural_Sciences_Center,_Department_of_Food_Science_and_Technology,_Universidade_Estadual_de_Londrina,_Londrina,_PR,_Brazil b_Microbiology_Department,_University_General_Hospital_of_Elche,_Universidad_Miguel_Hernández_de_Elche,_Elche,_Spain

c_Department_of_Basic_Health_Science,_Universidade_Estadual_de_Maringá,_Maringá,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11April2012

Accepted13September2012

Availableonline28February2013

Keywords: Salmonellasp. Fluoroquinolone Efﬂuxpump Antibioticresistance

a

b

s

t

r

a

c

t

Several studieshave been conducted in recentyears to elucidate the structure,

func-tionandsigniﬁcanceofAcrB,MarA,SoxSandRamAinSalmonellaenterica.Inthisstudy,

therelativequantiﬁcationofacrB,soxS,marAandramAgenesexpressionwasevaluated

in 14 strainsofS. enterica,with or without accompanying mutationsinthe quinolone

resistance-determiningregionsofthegyrAgene,thatwereexposedtociproﬂoxacin

dur-ingtheexponentialgrowthphase.Thepresenceofciproﬂoxacinduringthelogphaseof

bacterialgrowthactivatedthegenesmarA,soxS,ramAandacrBinallS.entericastrains

ana-lyzedinthisstudy.ThehighestexpressionlevelsforacrBwereobservedinstrainswith

gyrA mutation,andmarA showedthe highestexpressioninthe strainswithout

muta-tion.Considering onlythestrainswithciproﬂoxacinminimuminhibitoryconcentration

values<0.125␮g/mL (sensitivetociproﬂoxacin),themostexpressed geneinthestrains

both withandwithoutmutationswasacrB.Inthestrainswithciproﬂoxacin minimum

inhibitoryconcentrationvalues≥0.125␮g/mL(lowsusceptibility),withandwithout

muta-tionsingyrA,themostexpressedgenewasmarA.Inthisstudy,weobservedthatstrains

resistant tonalidixicacidmayexpressgenesassociatedwiththeefﬂuxpumpandthe

expressionoftheAcrAB-TolCpumpgenesseemstooccurindependentlyofmutationsin

gyrA.

∗ _{Corresponding}_author_at:_Centro_de_Ciências_Agrárias,_Departamento_de_Ciência_e_Tecnologia_de_Alimentos,_Universidade_Estadual_de

Londrina,Av.CelsoGarciaCids/n,Londrina,PR,Brazil.

E-mailaddress:rafaelaferrari@yahoo.com.br(R.G.Ferrari).

(2)

Introduction

The resistance of Salmonella enterica to quinolones is

usu-allymediatedbymutationsintopoisomerasegenes.However,

the presence of plasmids and active efﬂux pumpscan be

determinantfactorsindecreasedantibioticsusceptibilityand

increasedresistance.1

Efﬂuxpumpsactinresponsetobacterialstressandthe

expressionof pumps reducesthe accumulationof organic

compoundsorantimicrobialagentswithinthecellandcan

alsobecausedbychromosomalmutations,resultingin

mul-tiple antibiotic resistance (MAR).2 _According _to _Baucheron

etal.,3_efﬂux_pumps_play_an_important_role_in_resistance,

espe-ciallywhenﬂuoroquinolonesareinvolved.

The AcrB efﬂux pump belongs to the

resistance-nodulation-cell division (RND) superfamily and acts in

associationwithtwotypesofproteins:theoutermembrane

channel (TolC)and a periplasmic ‘adaptor’ protein(AcrA).4

Regulators belonging to the AraC/Xyls family of

transcrip-tional activators,5 _i.e. _marA,6 _soxRS,7 _rob,8 _and _ramA9 _have

beenfoundtoactivateacrABtranscriptionbybindingtothe

marboxsequencecharacterizedinitspromoter.10

TheMarAand SoxS proteinscanactivate acrAB

expres-sion. The marRAB operon isresponsible for producing the

MarAtranscriptional activator protein,6 _which _can_be

syn-thesizedinresponsetothe presenceofantibioticsandcan

resultinthemulti-drugresistancephenotype(MDR).Inturn,

thesoxRSoperonisactivatedinresponsetooxidativestress,

withtheSoxSproteinsubsequentlyactingastranscriptional

activator.11 _The_{overexpression}_of_soxRS_also_contributes_to

increasedantibioticresistanceinGram-negativebacteria.

TheproductoftheramRgeneregulatesthegeneramA,12

whichproducestheproteinRamA.RamAishomologous to

MarAandSoxS.13_Bacteria_{overexpressing}_ramA_may_exhibit

theMDRphenotypebyinducingexpressionofacrABandtolC.9

S.entericastrainswiththetolCgeneexperimentallydeleted

show anincreased susceptibility toantimicrobial agents.14

Similarly,strainsofS.entericashowedhighersusceptibilityto

ciproﬂoxacinwhenramAwasinactivated.15

Althoughseveralstudieshavebeen conductedinrecent

yearstoelucidatethestructure,functionandsigniﬁcanceof

AcrB,MarA,SoxSandRamA,16–18_their_regulation_in_S.

enter-icaisnotfully understood.In thisstudy,the expressionof

acrB,soxS,marAandramAwasevaluatedinfourteenstrains ofS.enterica,withorwithoutaccompanyingmutationsinthe

quinoloneresistance-determiningregions(QRDR)ofthegyrA

gene,thatwereexposedtociproﬂoxacinduringthe

exponen-tialgrowthphase.

Materials

and

methods

Strains

We analyzed 14 strains of S. enterica that are resistant to

nalidixic acid (NAL). These strains were divided into two

groups:sevenstrainswithout mutationintheQRDRregion

ofthegenesgyrA,gyrB,parCandparEandsevenstrainswith

mutationsin the QRDRof gyrA,but withno mutations in

theQRDRofothergenes.Amongthestrainswithmutations

inthe gyrAQRDR,onewaspositive forthe qnrB19plasmid

gene. Theciproﬂoxacin minimum inhibitory concentration

(CipMIC)ofeachstrainwasdeterminedusingE-testgradient

strips(ABBiodisk,Solna,Sweden).OncetheCipMIChadbeen

determined, each strainwas cultivated induplicatefor2h

inMueller-Hintonbroth(MHB)andciproﬂoxacinwasadded

tooneofthetwoculturesataconcentrationcorresponding

tohalfofthecorrespondentlyCipMICofeachstrain.19_After

30minofexposuretotheantibioticat37◦C,theculturewas

centrifugedforsubsequentRNAextraction.

RNAisolation

RNAisolationwasperformedaccordingtoChomczynskiand

Sacchi20_by_the_{guanidinium-thiocyanate–phenol–chloroform}

method, with some modiﬁcations. After centrifugation at

14,000×gfor10min,thesupernatantwasdiscarded,200␮L

of lysis solution was added and the samples were

incu-batedfor1hat37◦C.Afterthistime, 50␮Lof1%SDS was

added. Aftervortex agitation,25␮Lof2MNaCl and200␮L

phenol:chloroformweremixedintothematerial;whichwas

placedonicefor10min.Thematerialwasthencentrifuged

at 14,000×g for 20min at 4◦C, and the supernatant was

recovered.Anequalvolumeofisopropanolwasaddedtothe

supernatant;mixturewasincubatedat−70◦_C_for₂_h_and_then

centrifuged at14,000×g for11minat4◦C.Afterthe

super-natantwasdiscarded,thepelletwasdriedunderairﬂuxand

thenvortexedin750␮Lof75%ethanol.Anewcentrifugation

wasconductedat14,000×gfor11minat4◦C;thesupernatant

wasdiscarded;thepelletwasdriedfor1handresuspendedin

30␮Lofnuclease-freewater(InvitrogenTM_)._The_RNA_samples

were treatedwithRQ1 DNase(AmbionAM 1906),following

themanufacturer’s instructions.RNApurityand

concentra-tionwasmeasuredusingtheNanodropND-1000.

Reversetranscriptase(RT)

RT reactionswereconductedasdescribedbyChicoetal.,21

using1␮goftotalRNA,90ngofrandomhexamers(QIAGEN®₎

and 0.5mMdNTPs (InvitrogenTM_)._Prior _to_cDNA _synthesis,

denaturationwasperformedfor5minat65◦C.Afterthisstep,

200UofMMLVreversetranscriptase(InvitrogenTM_),₁₀_mM_of

DTT (InvitrogenTM),and 20unitsofRNaseinhibitor (RNase

OUT,Invitrogen)wereaddedtoeachreactiontoaﬁnal

vol-ume of20␮L.Thereactionconditionswere 10minat25◦C,

15minat42◦Cand5minat99◦C.

Relativequantiﬁcationofgeneexpression

After obtaining the cDNA, quantitative real-time reverse

transcriptase PCR(qRT-PCR) wasperformed using the7500

Real-Time PCR System, SDS software version 1.4 (Applied

Biosystems). The reactions were performed in a ﬁnal

vol-ume of20␮L,containing300nM ofeach primer,100nMof

each probe (Table 1), 2␮L ofcDNA and 1× TaqMan®

uni-versal master mix (Applied Biosystems). Standard curves

wereconstructedtocalculatethegene-speciﬁcPCRefﬁciency

from10-foldseriesdilutionofthemixedcDNAtemplatefor

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Table1–TaqMan®_primers_and_probes._Sequences_of

primersandprobesusedforqRT-PCR(quantitative real-timereversetranscriptasePCR)reactions,designed withthehelpofthePrimerExpressTM_2.0_software

(AppliedBiosystems).

Primersandprobesa _Primers_sequences₅_→₃

16s-F CGTGTTGTGAAATGTTGGGTTAA

16s-R CCGCTGGCAACAAAGGATAA

probe16s TCCCGCAACGAGCGCAACC

acrB-F TGAAGACCAGGGCGTATTCCT acrB-R TTTTTGCGTGCGCTCTTG

probeacrB ACAATGGTCCAGCTCCCCGCG

soxS-F CGGAATACACGCGAGAAGGT soxS-R GAGCGCCCGATTTTTGATATC

probesoxS TGCTGCGATACATAGCCCAGGTCCA

marA-F GACCCGGACGTTCAAAAACTAT marA-R TCGCCATGCATATTGGTGAT

probemarA TGATGTGCCGCCACACAAATACCG

ramA-F CCAGAAGGTGTATGATATTTGTCTCAAG ramA-R GGTTGAACGTGCGGGTAAA

proberamA TTGATTCGCAGCAAACCTTTACGCG

a _Probe _used: _{6-carboxyﬂuorescein} _(FAM); _quencher _used: 6-carboxytetramethylrhodamine(TAMRA).

calculated using the formula E=[10(−1/S)_]₋_1, _where _E

rep-resentsthe calculated efﬁciency, and Sis the slope ofthe

standardcurve.22 _To_check_all_primers _{speciﬁcity,}_real-time

PCRwas performed on cDNA and productswere analyzed

byelectrophoresison2%agarosegelandethidiumbromide

staining.NegativeqRT-PCRcontrolwithnotemplateswas

per-formedforeachprimerpair.22

Statisticalanalysis

TheresultswereanalyzedusingSPSS18.0software.The

geo-metric means (GM) of the CipMICs were calculated using

thefollowing

n _y1y2y2·_·_·yn_formula:_where_y_represents_the

individualCipMICofeachstrain,andnrepresentsthetotal

numberofCipMICvalues.23

Results

Thepresenceofciproﬂoxacinduringthelogphaseof

bacte-rialgrowthactivatedatleastoneofthefourgenestestedin

allS.entericastrainsanalyzedinthis study.Theexpression

levelsofthegenesmarA,soxSandramAwerehigherinthe

strainswithoutamutationingyrA,gyrB,parAorparCthanfor

strainswithmutations.However,thehighestexpressionlevels

foracrBwereobservedinstrainswithgyrAmutation(Fig.1).

The GM value for the four pump genes evaluated in

Salmonellastrainswithoutmutationswas2.70,andforstrains

withamutationingyrAitwas2.13.Amongthestrainsmutated

ingyrA,strain209-2showedhigherexpressionofthegenes

analyzed,witha5.99GM.Incontrast,thestrainwithout

muta-tioningyrAwiththehighestlevelsofgeneexpressionwas

238-2,witha15.38GM(Table2).

Afterassessingthelevelofexpressionforeachgeneinthe

strainswithoutmutation,marAshowedthehighest

expres-sion(GM=6.93),followedbysoxS(GM=2.72),acrB(GM=2.38)

andramA(GM=1.16).InthestrainswithmutationsingyrA,

the gene marA also showed higher levels of gene

expres-sion (GM=3.41) but was followed by acrB (GM=3.25), soxS

(GM=1.77)andramA(GM=1.05).

Considering only the strains with CipMIC

val-ues<0.125␮g/mL (sensitive to ciproﬂoxacin), the most

expressedgeneinthestrainsbothwithandwithout

muta-tionswasacrB.Inthethreemutatedstrains,theGMforthis

genewas9.28,andinthestrainwithoutmutationthelevelof

expressionofacrBwas11.8(Table2).

InthestrainswithCipMICvalues≥0.125␮g/mL(low

sus-ceptibility), withand without mutationsin gyrA,the most

expressedgenewasmarA.Forthefourmutatedstrains,marA

expressionrangedfrom0.98to11.96,andtheGMwas3.36.

In contrast,forthesixstrains withoutmutation, themarA

expressionlevelrangedfrom1.29to122,withaGMof6.62.

AmongthefourstrainswithgyrAmutationsandreduced

susceptibility to ciproﬂoxacin,strain 288-2, serovar

Heidel-berg,andstrain300-2,serovarEnteritidis,scarcelyexpressed

thepumpgenestested.Ontheotherhand,S.Enteritidisstrain

209-2,despiteaGMof5.99,presentedaCipMICvaluefour

timeslowerthanS.Enteritidisstrain216-2,whichshoweda

GMof1.66(Table2).

Fourofthesixstrainswithoutmutationsandreduced

sus-ceptibilitytociproﬂoxacinshowedsigniﬁcant expressionof

theefﬂuxpumpgenesevaluated,withGMvaluesbetween4.25

and15.26.Incontrast,strain232-2,withnomutationingyrA

andthelowestlevelofgeneexpressionlinkedtothepump

(GM=0.23),presentedaCipMICvalueof0.5␮g/mL.Strain

297-2,serovarCorvallis,positivefortheqnrB19plasmidgene,also

presentednoexpressionofanyofthefourgenesstudiedthat

arelinkedtothepumpcomplex(GM=0.65).

Discussion

There are reports that AcrAB pump expression in

Escherichia coli can increase the MIC for ciproﬂoxacin and

norﬂoxacin uptoﬁve times.24 _In _this_study,_all _the _strains

evaluated showed expression of at least one gene related

to efﬂuxpumpsinthe presenceofciproﬂoxacin.However,

the expression ofthese genes and their relationship with

reducedsusceptibilitytociproﬂoxacinwashighlydivergent,

independentofmutationsingyrA.TheS.Enteritidisstrains

250-2,229-2and213-2,withmutationsingyrAandexpression

of atleast 3 genes linked to the pump, presentedCipMIC

valuesoflessthan0.125␮g/mL.

However, other S. Enteritidis strains (209-2, 300-2 and

216-2),withgyrAmutationsandbothwithandwithout

expres-sionofthe genesstudied,showedreducedsusceptibilityto

ciproﬂoxacin.

Baucheronetal.3 _have_reported_a_reduction_of_up_to

64-foldinCipMICvaluesafterinhibitionoftheAcrABoperonin

strainsofS.Typhimurium,withorwithoutmutationsingyrA.

Morgan-Linnelletal.25_also_failed_to_correlate_AcrAB

expres-sionandthepresenceofmutationsintopoisomerasegenes

withtheMICforﬂuoroquinolones.Theincreaseinthelevels

ofAcrABexpressiondidnotalwaysincreasetheMICs,

(4)

Fig.1–DistributionoftheexpressionlevelsofthemarA,soxS,ramAandacrBgenesstudiedinrelationtoCipMIC(␮g/mL)

andgyrAgenemutations.WT,wild-typestrain;CIP,ciproﬂoxacin.

values.Furthermore,theexpressionoftheefﬂuxpumpwas

reportedasbeingresponsibleonlyforbaselineresistance.26

Inthisstudy,strainsofS.EnteritidiswithoutQRDR

muta-tionsinthe genesgyrA,gyrB,parCorparEshowedreduced

susceptibilitytociproﬂoxacin.Theexpressionofgenes

asso-ciatedwiththe efﬂuxpumpseemstohaveinﬂuenced this

reduction.Strains121-1,135-2,91-1and238-2ofS.Enteritidis

showedaGMof4.94,4.25,5.25and15.26,respectively.Inthis

Table2–DistributionofthemutationsobservedinQRDRofthegyrAgeneinSalmonellastrainsofdifferentserovarswith thecorrespondingminimalinhibitoryconcentrationforciproﬂoxacin(CipMIC)andgeometricmean(GM)data.

Straina _Serotype _CipMIC_(␮g/mL) _Mutation _Level_of_expression_in_the_presence_of_{ciproﬂoxacin}

soxS db _marA _db _acrB _db _ramA _db _GMc

250-2 Enteriditis 0.0156 Ser83-Tyr 1.39 0.11 7.26 0.51 7.67 0.38 2.46 0.26 3.71 229-2 Enteriditis 0.0156 Asp87-Asn 1.88 0.14 2.60 0.47 4.70 0.07 19.43 0.78 4.60 213-2 Enteriditis 0.0156 Asp87-Asn 0.40 0.30 2.26 0.20 22.16 0.16 9.78 0.33 3.74 209-2 Enteriditis 0.1250 Ser83-Tyr 9.65 0.13 11.96 0.30 37.27 0.54 0.30 0.85 5.99 300-2 Enteriditis 0.2500 Ser83-Phe 1.99 0.61 3.16 0.10 0.00 0.00 0.00 0.00 0.50 288-2 Heidelberg 0.5000 Ser83-Tyr 2.71 0.46 0.98 1.30 0.59 0.79 0.00 0.00 0.63 216-2 Enteriditis 0.5000 Ser83-Tyr 1.00 0.11 3.43 0.16 2.20 0.27 1.01 0.70 1.66 238-2 Enteriditis 0.2500 wt 42.2 0.29 9.65 0.40 3.43 0.22 38.85 0.19 15.26 121-1 Enteriditis 0.3800 wt 4.66 0.31 2.82 0.72 7.89 0.40 5.74 0.46 4.94 135-2 Enteriditis 0.3800 wt 2.90 0.45 122 0.20 9.19 0.24 0.00 0.00 4.25 91-1 Enteriditis 0.1250 wt 6.96 0.96 8.50 0.43 1.56 0.14 8.22 0.43 5.25 119-2 Enteriditis 0.0470 wt 2.27 0.18 9.19 0.38 11.80 0.36 1.25 0.30 4.19 232-2 Enteriditis 0.5000 wt 0.16 0.68 1.29 0.39 0.00 0.00 0.13 1.25 0.23 297-2d _Corvallis _0.5000 _wt _0.79 _0.33 _2.31 _0.13 _0.97 _0.19 _0.00 _0.00 _0.65

wt,strainwithoutmutationsingyrA,gyrB,parCandparE. a _All_strains_were_resistant_to_NAL.

b _Typical_deviation.

c _Geometric_mean_of_the_four_genes. d _Strain_positive_for_qnrB19.

(5)

group,itisimportanttonotethatstrain135-2presentedmarA

expressionof122,andstrain238-2ofS.Enteritidisincreased

expressionofboththesoxSandramAgenesaswellasmarA

(Table2).

TheexpressionofmarAisinduced byanumberof

sub-stances,including quinolones.27 _The_marA _gene _is_able _to

mediatedrugresistancebydecreasingexpressionoftheOmpF

porinandcausingoverexpressionoftheAcrBefﬂuxpump.6,16

StudieswithE. colireportedMICvaluesforoﬂoxacinup to

eightfoldhigherinstrainswithoverexpressionofthe marA

gene,in comparisontostrains that onlyhad mutationsin

gyrA.12,28,29_Kern_et_al.30_observed_that_the_rate_of_successful

treatmentofE. coliwithﬂuoroquinolones was signiﬁcantly

lower in strains that expressed marA. In clinical isolates,

increasedlevels ofAcrABpumpgeneexpressionmayarise

duetochangesinmarAortobactericidalprotectiongenerated

bytheregulatorsofthisgene.ThefactthatthemarAgeneis

associatedwiththemarRABoperonactivatorsuggestsitsuse

(marA)asatargetforstudiesonstrategiesagainstmicrobial

resistance.25

ThesoxSgenewasthemostexpressed,aftermarA,inthe

non-mutatedstrainsanalyzedinthisstudy.Thisgeneisthe

transcription activatorthat positively regulatesthe

expres-sionofmorethan20genesrelatedtotheincreasedsynthesis

ofproteinsassociatedwiththeAcrAB-TolCefﬂuxpump.31–33

TheSoxRSregulatorysystemprotectscellsagainstoxidative

stressgeneratedbyvariousantimicrobials.32_The_S._Enteritidis

strain238-2,withoutmutationsingyrA,showedexpression

ofthesoxSgeneandreducedsusceptibilitytociproﬂoxacin.

Kehrenbergetal.34_observed_an_eightfold_increase_in_the

Cip-MICvaluesofS.VirchowstrainswhensoxSwasexpressedbut

norelationshipwiththepresenceofgyrAmutations.

Theexpressionofefﬂuxpumpgeneswaspracticallyzero

fortheHeidelbergserovarstrain(GM=0.63),whichshowedthe

highestCipMICamongallthestrainsanalyzedinthisstudy.

Itsuggeststhatthisreductioninsusceptibilityisduetothe

intrinsicresistancecommonlyobservedinthisserovar.35

TheCorvallisserovarpresentedaCipMICof0.5␮g/mL,but

nomutationintheQRDRregionofthetopoisomerasegenes

thatcorrelateswiththelowexpressionlevelsforthestudied

genesrelatedtoefﬂuxpumps.However,thereduced

suscep-tibilitytociproﬂoxacinobservedmayrelatetothepresence

oftheqnrB19plasmidgene. AccordingtoLi,36 _the_qnr_gene

facilitatestheselectionofchromosomalmutationsrelatedto

quinoloneresistanceinS.entericabysigniﬁcantlyincreasing

thelevelsatwhichmutantscanbeselected.

The 232-2 S. Enteritidis strain presented a CipMIC of

0.5␮g/mLanddidnotexpressanyofthestudiedgeneslinked

totheefﬂuxpump.Itispossiblethatalternativemechanisms

ofgeneregulation,suchasacrR,thatareindependentof

mar-sox-robwereresponsibleforcontrollingtheexpressionofAcrB inS.enterica.37_Mutations_in_genes_controlling_the

transcrip-tionorexpressionofothergenes,suchasacrD,acrAortolC15

mayalsobeinvolved,inadditiontotheAcrEFefﬂuxpreviously

describedinS.enterica.38,39

Another possibility for the reduced susceptibility to

ciproﬂoxacin observed in strain 232-2 could be mutations

ingyrAoutside the QRDRregion.Capooret al.40 _have

sug-gested anincreaseto the regionalboundariesthat include

theQRDRofthegyrAgenebecausemutationsassociatedwith

resistancetoquinoloneswere observedbeyondthe current

domain.

In this study,weobserved thatstrains resistant toNAL

may express genes associated with the efﬂux pump as a

formofprimaryresistancetothisdrug.Thismechanismmay

perform a pre-selectionfor less sensitive strains, with the

subsequentemergenceofmutatedandresistantstrains.One

possiblemechanism toincrease thelife ofquinolonesand

ﬂuoroquinoloneswouldbethedevelopmentofinhibitorsof

theAcrAB-TolCsystem,whichcouldminimizetheselection

ofstrainsresistanttoNAL.TheexpressionoftheAcrAB-TolC

pumpgenesseemstooccurindependentlyofmutationsin

gyrA,becausestrainswithoutmutationsintheQRDRregions

ofthetopoisomerasegenesevaluatedinthisstudypresented

higher expression of the genes associated with the efﬂux

pumps.However,theresultsindicatetheneedforadditional

studiestobetterunderstandthecomplexinterplaybetween

theexpressionandregulationofefﬂuxpumpgenesandother

mechanismsofresistancetoquinolonesinS.enterica.

Conﬂict

of

interest

Allauthorsdeclaretohavenoconﬂictofinterest.

Acknowledgements

WewouldliketoacknowledgeCNPqandCapesforﬁnancial

supportintheformofPhDscholarshipsawardedtoRFand

MM.

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