Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Bacterial

and

Fungal

Pathogenesis

Inactivation

of

MarR

gene

homologs

increases

susceptibility

to

antimicrobials

in

Bacteroides

fragilis

Clara

Maria

Guimarães

Silva,

Déborah

Nascimento

dos

Santos

Silva,

Scarlathe

Bezerra

da

Costa,

Juliana

Soares

de

Sá

Almeida,

Renata

Ferreira

Boente,

Felipe

Lopes

Teixeira,

Regina

Maria

Cavalcanti

Pilotto

Domingues,

Leandro

Araujo

Lobo

UniversidadeFederaldoRiodeJaneiro,MedicalMicrobiologyDepartment,RiodeJaneiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24November2016 Accepted6May2017

Availableonline9August2017 AssociateEditor:RodrigoGalhardo

Keywords:

Bacteroidesfragilis

Anaerobicbacteria Multi-drugresistance Oxidativestressresistance

a

b

s

t

r

a

c

t

Bacteroidesfragilisisthestrictanaerobicbacteriamostcommonlyfoundinhuman

infec-tions,andhasahighmortalityrate.Amongothervirulencefactors,theremarkableability toacquireresistancetoavarietyofantimicrobialagentsandtotoleratenanomolar con-centrationsofoxygenexplainsinparttheirsuccessincausinginfectionandcolonizingthe mucosa.Muchattentionhasbeengiventogenesrelatedtomultipledrugresistancederived fromplasmids,integronsortransposon,butsuchgenesarealsodetectedinchromosomal systems,likethemar(multipleantibioticresistance)locus,thatconferresistancetoarange ofdrugs.RegulatorslikeMarR,thatcontrolexpressionofthelocusmar,alsoregulate resis-tancetoorganicsolvents,disinfectantsandoxygenreactivespeciesareimportantplayers intheseevents.Strainsderivedfromtheparentalstrain638R,withmutationsinthegenes herebyknownasmarRI(BF638R3159)andmarRII(BF638R3706)wereconstructedbygene disruptionusingasuicideplasmid.Phenotypicresponseofthemutantstrainstohydrogen peroxide,cellsurvivalassayagainstexposuretooxygen,biofilmformation,resistanceto bilesaltsandresistancetoantibioticswasevaluated.Theresultsshowedthatthemutant strainsexhibitstatisticallysignificantdifferencesintheirresponsetooxygenstress,butno changeswereobservedinsurvivalwhenexposedtobilesalts.Biofilmformationwasnot affectedbyeithergenedisruption.Bothmutantstrainshowever,becamemoresensitive tomultipleantimicrobialdrugstested.Thisindicatesthatasobservedinotherbacterial species,MarRareanimportantresistancemechanisminB.fragilis.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:lobol@micro.ufrj.br(L.A.Lobo). https://doi.org/10.1016/j.bjm.2017.05.005

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Bacteroidesfragilis,ananaerobicGramnegativebacteriumthat

colonizes the gastrointestinal tract of humans, isa minor componentofthehumanfecalmicrobiota.1_Despiteits ben-eficialrole asamember ofthegut microbiota,B.fragilis is thestrict anaerobic bacteriamostcommonlyisolated from humaninfection.2_{Thisdualityintheinteractionwiththehost} iswell recognized and several virulencefactors have been describedtoaccountforthisbehavior,including acapsular polysaccharidecomplex(CPC),3_{adhesinsandtheBacteroides}

fragilis toxin (BFT).4 _{The high toleranceto oxidative stress}

(OS)observedinthisspeciesisregarded asamajor advan-tage when invading oxygenated host tissues, such as the peritonealcavity5_{andresistingtheoxidativeburstimposed} by macrophages.6 _{An intense modulation of gene} expres-siontakesplaceinB.fragilisduringoxidativestressasshown by microarray studies,7 _{wherethe expression of over 45%} ofthe total genomeof the bacteriais affected.Several OS detoxifyingenzymesarefoundinthisspecies,suchas cata-lase (KatB), superoxide dismutase(SOD), Dps, thioredoxins and alkyl hydroperoxide reductase (AhpC), but the regula-tionoftheOSresponseinB.fragilisiscomplexandnotfully understood.ThetranscriptionalregulatorOxyRhasreceived attentioninthepastdecadesandisrecognizedasan impor-tantplayerduringOS.8_{StudieswithOxyRmutantsinB.fragilis} however,haveshownthatdespiteitsimportance,OxyRisnot thesolemediatoroftheOSresponseandotherregulatorsare involved.9

Recently,wedescribedtheroleofatranscriptional regu-latorhomologtothemultipleantibioticresistanceregulator (MarR)familyintheOSresponseofB.fragilis.10_Membersofthe MarRfamilyoftranscriptionalregulatorshavebeendescribed aswingedhelixproteinsthatbinddirectlytotheDNAto con-trolawiderangeofbiologicalprocessesinbothbacteriaand archaea.11,12 _{Sofar,the bestknown modelofregulationby} MarRfamilymembersistheregulationofmultipleantibiotic resistanceinEscherichiacolibytheprototypicalmemberMarR, describedbyGeorgeandLevyin1983.13_{Sincethen,several} members ofthisfamily have beencharacterizedas having animportantroleinmicrobialpathogenesisandresistanceto antimicrobialsalthoughthemechanisminwhichthe regula-tionoccursmaychangeindifferentspecies.14_{Transcriptional} regulatorsoftheMarRfamilyareknowntoaffectboththe oxy-genstressresponseandtheexpressionofvirulencefactors inGram negativepathogens suchasSalmonellaentericaand

E.coli.15_{Accordingtoannotationsinthegenebankdatabase}

(NCBI)somestrainsofB.fragilismaycarryuptofourdifferent homologsofgenescodingforMarRproteinsinitsgenome, buttheirroleinthephysiologyofthisspecieshasnotbeen studiedindetail.Multidrugresistanceinbacteriaisregarded asamajorchallengeforcliniciansintheworld.Strict anaero-bicbacteriaareparticularlychallenging,sincemosthospitals andclinicalsettings,particularlyindevelopingcountries,lack offacilitiesand equipmentusedforcultivationunderstrict anaerobiosis.Researchlabshaveshownthatmultidrug resis-tance (MDR) in Bacteroides is on the rise for the past few decades.16–18_{Resistancetomostclassesofantimicrobialdrugs} usedinclinicalsettingswasalreadyreportedforBacteroides

species,includingcarbapenemsandmetronidazole.18,19 Pro-ductionofbroadspectrum␤-lactamasesinB.fragilisiswell documented,andatleastthreeenzymescodedbythegenes

cepA,cfiAandcfxAwerealreadydescribed,18_butthe

mecha-nismsregulatingtheexpressionofresistancedeterminantsin anaerobesarepoorlyunderstood.Toaddresssuchissues,in thepresentstudywedisruptedtheexpressionoftwogenes codingforproteinsoftheMarRfamilyinB.fragilisand inves-tigatedtheeffectinantimicrobialresistance,susceptibilityto oxidativestress andbiliarysalts,andformationofbiofilms

in vitro. As previously observed, interferingwith

transcrip-tionalregulatorsofthemarRfamily(Teixeira2013),leadsto alteredphenotypesandresponsetoenvironmentalstressin

B.fragilis.

Methods

Bacterialstrainsandplasmids

Bacterial strainsand plasmidsusedinthisstudy are listed in Table 1. The B. fragilis strains were routinely grown in brainandheartinfusionagarsupplementedwithmenadione (10␮g/mL) and hemin (0.5␮g/mL) (BHI) and in pre-reduced and anaerobically sterilized BHIbroth(BHI-PRAS). Cultures weregrownat37◦Cinananaerobicchamber(Coylabs, Michi-gan, USA) with an atmosphere of 80% N2, 10% CO2 and 10%H2.Theantibioticsrifampicin(25␮g/mL),erythromycin (10␮g/mL) and gentamycin (100␮g/mL) were added to the mediawhenrequired.TheE.colistrainswereroutinelygrown inlysogenybroth(LB),containing1%triptone(w/v,Difco,BD, New Jersey,USA),0.5%yeastextract (w/v)(Oxoid, Thermo-Fisher,Massachusetts,USA)and0.5%NaCl(w/v) orLBagar (1.5% agar-agar, w/v, Difco). Ampicillin (100␮g/mL), tetra-cyclin (10␮g/mL),spectinomycin(50␮g/mL) and kanamycin (50␮g/mL) were added to the mediumwhen required. For cloningandelectroporation,superoptimalbrothwith catabo-lite repression (SOC) mediumcontaining 2%triptone,0.5% yeastextract,0.05%NaCl,2.5mMKCl,10mMMgSO4·10MgCl2 and20mMglucose,pH7.0,wasused.Antibioticswere pur-chasedfromSigma–Aldrich,Missouri,USA.

ConstructionofB.fragilismutants

B.fragilis638Rmutantsweregeneratedbyinsertional

inactiva-tionusingthesuicideplasmidpFD516.20_{Briefly,afragmentof} 300bpofthe geneBF638R3159(For-5 GGATGGAATTCCGCC-AGGCCGTTCandRev-5 CATAGACGTCGTTGATCAGGGGTTTC-ACG) and 252bp of the gene BF638R3706 (For-5 ATCGGA-ATCCATACCGGAACTTCAGACAG and Rev-5 TTCCTGC-AGCTCTTTTCCTGTTCTGG) were amplified by polymerase chain reaction (PCR) and cloned into the cloning vector pGEM®-T Easy (Promega). The sequences of the genes were obtainedfrom Genbank under the accession number FQ312004.1.ThePstI/EcoRIfragments,generatedafter diges-tionofthecloningvector,wasclonedintothePstI/EcoRIsitesof thesuicideplasmidpFD516andtransformedin electrocompe-tentE.coliDH10bstrains.Theresultingplasmidwasmobilized

intoB.fragilis638Rbytriparentalmating.21_{Transconjugants}

Table1–E-testresultsofinvitroactivityoftenantibioticsagainstwildtypeB.fragilisstrain638Randmutantstrains LB64andLB65.

Antibiotic 638RMIC(␮g/mL) LB64MIC(␮g/mL) LB65MIC(␮g/mL)

Teicoplanin 24 12 16 Tetracycline 0.19 0.064 0.125 Imipenem 0.064 0.016 0.023 Meropenem 0.064 0.016 0.032 Cefotaxime 12 1.5 4 Levofloxacin 2 1 1.5 Cefepime 3 1.5 1.5 Metronidazole 0.25 0.125 0.16 Polymyxin >1024 >1024 >1024 Linezolid 3 1.5 3

erythromycin (2␮g/mL) and gentamicin (100␮g/mL).

Inser-tion of the plasmid in the correct loci was confirmed

by PCR using M13 primers (IDT, Iowa, USA) as described

above.

Oxygenstresssurvival

Topreparetheinoculum,bacterialstrainsweregrownfor48h

at37◦Cin BHI-PRAS and the cells were harvested by

cen-trifugationat16,000×gfor5min.Cellswereresuspendedin

phosphatebufferedsalinesolution(PBS)pH7.3toanOD600=1.

Aninoculum of10␮Lofdecimalserialdilutionsupto10−7

werespottedinBHIplatescontainingrifampicin(25␮g/mL)

andgentamycin (100␮g/mL) andthenincubated at37◦Cin

aerobiosis.Cultureplateswere transferredtotheanaerobic

chamberateachtime pointof24,48 and72hand further

incubatedfor48hinanaerobiosis.Platesnotexposedto

oxy-gen were usedas controls.Theresults were scored asthe

lowestdilutionwheregrowthcouldbeobservedineachtime

point.

Hydrogenperoxidesusceptibility

Bacterial strains were initially grown for 48h at 37◦C in

BHI-PRASadjustedtotheMcFarlandscale1(3×108_UFC/mL)

and spread with aswab inBrucellaagar plates toachieve

confluent growth. Sterile filter paper disks (6mm,

Labor-clin)containing10␮Lofhydrogen peroxidesolutions(0.3%,

1% and 3%) were placed on the agar surface and the

plates were incubated for 48h at 37◦C in anaerobiosis.

Diametersofthe growthinhibitionzone weremeasured in

millimeters.22_{Allexperimentswereperformedintriplicates}

and statistical analysis was performed as described above (ANOVA).

Susceptibilitytobiliarysalts

Theminimalinhibitoryconcentration(MIC)andminimal bac-tericidalconcentration(MBC)ofbiliarysaltswereperformed asdescribed before.23 _{Toprepare the inoculumthe strains} weregrowninBHI-PRASsupplementedwithantibioticsfor 18h at 37◦C and then inoculated in fresh BHI-PRAS until anOD600 of0.4was reached. Bacterial growth was diluted 1:10 and 20␮Lwere inoculated in wells of a96-well plate

containing180␮LofBHI-PRASwithbiliarysaltsdilutions ran-gingfrom0to20%.Plateswereincubatedfor48hat37◦Cand growthwasmeasuredbyReadingtheOD600inaplatereader (Multiskan,MolecularDevices,USA).TheMICwasdetermined as thelast concentrationwhere theOD reading was supe-rior to the blank well (non-inoculated well). For the MBC, threeconcentrationsabovetheMICwereplatedinBHIagar withantibioticsandincubatedfor48hat37◦C.Growthwas evaluatedbyvisualinspection.Threeindividualexperiments conductedintriplicateswereperformed.

Biofilmformation

Assaysforbiofilmformationweredonespecificallyfor anaer-obes asdescribed inthe literature.24 _{Bacterial strains were} inoculatedin96-wellplatesinPRAS–BHIsupplementedwith hemin(0.5mg/mL–Sigma–Aldrich,USA)and1%glucose(w/v) andincubatedfor24hat37◦Cinananaerobicchamber.The mediawasthenremovedandcarefullywashedwith200␮L ofphosphatebufferedsalinesolution(PBS–10mMsodium phosphate, 150mMNaCl,pH7.4).Thewells were driedfor 1hat60◦Candstainedwith2%Gram’svioletcrystal.Excess dye was washed with distilled water and driedfor 10min at60◦C.Intracellulardyewassolubilizedwith150␮Lof33% glacialaceticacidinwater(v/v)andtheopticaldensityatan OD570nmwasmeasuredforeachwell.Allexperimentswere conductedintriplicates.Non-inoculatedwellswereusedas background controls. Statistical analysis wasperformed as describedabove(ANOVA).

E-test

Antimicrobial susceptibility tests were performed using E-tests strips according to the manufacturer’s instructions (Biomerieux,France)andtheCLSIguidelines(Clinical Labo-ratories StandardsInstitute). Bacterial strainswere initially grown for 48h at 37◦C in BHI-PRAS and adjusted to the McFarland scale1(3×108UFC/mL)andspread withaswab in Brucellaagar plates supplementedwith5% defibrinated sheep blood to achieved confluent growth. E-test strips were placed in the plates with the help of sterileforceps and the plates were incubated in anaerobiosis for 48h at 37◦C.

Results

Survivalafterexposuretoatmosphericoxygenisreduced

inMarRIandMarIImutants

Our previous resultsshow that the inactivation of a MarR homolog(BmoR)inastrainofB.fragilisleadstoincreased sen-sitivitytooxygen.10_{Theexpressionoftwogenes(BF638R3159} and BF638R3706) coding proteinswith homology to trans-criptionalregulators oftheMarRfamilywasinterrupted by insertionofasuicideplasmidintheircodingsequence.Strains carryingtheplasmidinsertedintothelocusBF638R3159and BF638R3706are respectivelyreferredasLB64andLB65.To test if the single inactivation of the two remaining MarR homologsinB.fragilishasasimilareffect,10-foldserial dilu-tionsofthebacterialstrainswerespottedonagarplatesand exposedtoatmosphericoxygenforupto72h.Bacterial sus-ceptibilitywasevaluatedbygrowth recoveryafterexposure tooxygeninincreasingtimepoints.Asanticipatedthewild typestrain638Randbothmutantstrains,LB64andLB65 pre-sentedconfluentgrowth whengrowninstrictanaerobiosis, evenatthemaximumdilution(10−7).Differenceamongthe strainswasnotedassoonasafter24and 48hofexposure tooxygen.Growthwasreducedby2ordersofmagnitudefor bothmutantstrains(10−2)whencomparedtothewildtype strain(10−4).After72hofexposuregrowthwasonlyobserved inthe wild typestrain 638R.Thisresult indicatesthat the absenceofeitherofthetwoMarRhomologsaffectresistance toOS.

Susceptibilitytohydrogenperoxideisnotaffectedby

inactivationofMarRhomologs

Toconfirmthe role ofMarRhomologsinthe OSresponse, susceptibility to hydrogen peroxide was evaluated by disk diffusion in Brucella agar plates. The diameters of inhibi-tionhaloswerecomparedbetweentheparentalandmutant strains.Allstrainswere susceptibletothethree concentra-tions of H2O2 tested (0.3%,1% and 3%) and no significant changes were observed in growth of the mutant strains (Fig.1).

Susceptibilitytobiliarysaltsandbiofilmformation

Resistancetobiliary saltsisimportantforintestinal bacte-rialresidents. Mechanismsofresistanceincludealterations of the permeability of the external membrane and efflux pumps.25_{SinceMarRisknowntoregulatetheexpressionof} outermembraneproteins,weevaluatedwhetherinterrupting geneshomologstomarRwouldcompromisesurvivalinhigh bileconcentrations. After48hofincubation,all thestrains wereinhibitedbyconcentrationsabove10%ofbiliarysalts. ThreeconcentrationsabovetheMICwereplatedtodetermine theMBC.Again,allthreestrainswerekilledby12%ofbiliary saltsindicatingthattheMICandMBCwasidenticalamong thethreestrainstested.

Productionofbiofilminpolystyrenesurfaceswasobserved inthewildtypestrainaspreviouslydescribed.24_Astrainof

3% 1% 0.3% H2O2 0 2 4 6 Hala diameters (mm) 638R LB 64 LB 65

Fig.1–Inhibitionofbacterialgrowthbydiskdiffusionof hydrogenperoxideinsolidmedia.Growthofwildtype strain638RofB.fragiliswascomparedtostrainsLB64 (BF638R3159)andLB65(BF638R3706)carryingmutations ingenehomologstomarR.Inhibitionhaloindiametersis shownwithstandarddeviations.

Staphylococcusepidermidis(ATCC35984)wasalsousedasa

pos-itivecontrolfortheassay.Capacitytoformbiofilminvitrowas notaffectedinthemutantstrainswhencomparedtothewild type(Fig.2).

AntimicrobialsusceptibilityevaluatedbyE-test

Antibioticsusceptibility testingwas performedusingE-test strips in accordance to CLSI guidelines. The inability to expressMarRhomologsinB.fragilisinfluencedantimicrobial susceptibility and rendered both mutant strains more

0.8 0.6 0.4 0.2 0.0 35984 638R LB 64 LB 65 Strains OD 570

Fig.2–Invitrobiofilmformationbywildtypestrain638Rof

B.fragilisandisogenicmutantsformarRhomologs.Bars

representstandarddeviationsofthreeindependent experiments.

susceptible to teicoplanin, tetracyclin, imipenem, meropenem,cefotaxin, cefepime,levofloxacinand metron-idazol(Table1).TheMICforpolymyxinremainedunchanged andstrain LE64wasmoresusceptibletolinezolidthanthe wildtypestrain.

Discussion

It isestimated that only 1% ofall Bacteroides spp. present inthegastrointestinaltractbelongtotheB.fragilisspecies. Despitebeingaminorcomponentofthemicrobiota,it has importantrolesinthegut,particularlyinthedevelopmentof theimmunesystem.26_{Incasesofdisbiosisorruptureofthe} integrityoftheintestinalmucosaitalsoemergesasan oppor-tunisticpathogenanditisthemostcommonlyisolatedstrict anaerobicbacteria fromhumaninfection.2 _{Thisambiguous} interaction withthehumanhostmaybeexplainedinpart bytheexpressionofseveralvirulencefactors,includingthe CPC,adhesinsand secretedhydrolytic enzymes.4 _Although the complete details of the transitionfrom asymbiotic to apathogenic statearenotfullyunderstood,thecapacityto survivethehostileoxygenatedenvironmentoftheperitoneal cavityandresisttoantimicrobialdrugscertainlyplaya deci-siverole.

Ithasbeendemonstratedthatuponexposuretooxygen,B.

fragilismountsastressresponsethatmayaffectup to45%

ofits genome.7 _{Interestingly,the oxidative stress response} alsoaffectsthe expressionofvirulencefactors27 _indicating thatitmayworkasanenvironmentalsensorforthe transi-tionfromtheanaerobicgutlumentotheoxidizedperitoneal cavity.RecentlywedemonstratedthattheinsertionofaDNA fragmentinthegenelocusBF638R0571disruptedthe expres-sionof aMarR homologin B. fragilisleading to analtered responsetooxidativestress.10_{Thisgeneproductwasrenamed} BmoR,andmutantstrainsunabletoexpressthisproteinwere moresusceptibletooxidativestressthantheirparentalstrain. The638Rstrainusedintheaforementionedstudycarriesat leasttwootherhomologsofMarRproteinswhose function are notknown.We constructedtwosingle mutantstrains, eachcarryingadisruptioninoneofthetworemainingMarR homologsandanalyzedthesubsequentphenotype. Suscepti-bilitytooxygenwasincreasedafterlong-termexposure(72h) inthemutantstrains,butnottohydrogenperoxide.Similarly, BmoRwas showntorespondtooxygenstress,withonlya modestresponsetohydrogenperoxide.10_{Sincehydrogen} per-oxideisoneofthemajorreactivespeciesgeneratedbyoxygen exposure,thelackofresponsetothiscompoundcouldmean thatMarRhomologsinthespeciesarenotassociatedwiththe detoxificationofoxygen,but ratherwithametabolic path-wayrelatedtoredoxbalanceinthecell.Anaerobicbacteria areknowntocontainseparateresponsemechanismsfor dif-ferent sourcesofoxidativestress.8,28,29 _{Theregulator OxyR}

inB.fragilisforexampleregulatesacomplexglobalresponse

thatleadstodetoxificationofhydrogenperoxide. Although OxyRmutantsare moresusceptibletoatmosphericoxygen than wildtype strains,the stress responseorchestratedby OxyRisdirectedatpreventingacollateraldamagecausedby oxidativestress,theaccumulationofH2O2inthecell.5,8 Unfor-tunately,besidesOxyRtherearefewstudiesassociatedwith

regulationofoxidativestressresponseandseveralredox pro-teinsthathavenoknownregulatorassociatedwith,likethe thioredoxinsthatcontributefortheoxidativestressresponse byprovidingreducingpower,whichcouldbecontrolledby,for example,MarRhomologs.

MarRfamily proteinsare implicatedinresistanceto bil-iary salts in Gram negative pathogens such as Salmonella

enteritidis.30 _{Direct binding ofthe bile salt deoxycholate to}

MarRwas demonstratedtoinhibitDNA bindingand to de-repressthemarRABoperon,butsurprisingly,thiseffectdoes notincreaseresistancetobilesaltsinS.enteritidis.Similarly, wedidnotobserveanychangeinsusceptibilitytobilesaltsin

theB.fragilismutantstrains.SinceB.fragilisiswelladaptedto

theintestinalenvironment,itisnotsurprisingthatredundant mechanismsofregulationforbilesaltssusceptibilityarein place.InGramnegatives,regulationofvirulencegenesisalso affected byMarRhomologs.15 _{Thisglobalregulationprofile} reflectstheneedtocoupleenvironmentalsensingand viru-lencegeneexpressioninbacterialpathogens.MarRhomologs regulatetheexpressionofsurfacestructuressuchas polysac-charidecapsuleinBordetellabronchiseptica31 _{andfimbriaein}

E.coliwithaconsequentimpairmentinformationofbiofilm.32 Again,inB.fragiliswedidnotobserveanyeffectonbiofilm for-mation.Invitrobiofilmassaysarelimitedbytheinconvenience ofreplicatingtheconditionsfoundinthegutepithelium.Even with the mutationin marR homologs, artificial media and physicalsupport(polystyrenemicroplates)maybepermissive forbiofilmaccumulation.

Despitemodestresultsfoundintheprevious phenotypi-caltests,theeffectoftheabolishmentoftheexpressionof MarR homologs in antimicrobial susceptibility was notice-able.MarRwasfirstdescribedastherepressorofanoperon relatedtomulti-drugresistanceinE.coli,and mutationsin MarRinE.colileadstoanincreasedresistanceprofile.33,34_The increaseinresistanceinthisspeciesisassociatedwiththe expressionofeffluxsystemsnegativelyregulatedbyMarR.35 Conversely,inourexperiments,disruptionoftheexpression ofMarR homologsleadsto decreasedresistance toseveral antimicrobial drugs, including drugs from different classes andwithdifferenttargetsinthecell.Thisresultpointstoa defectonageneralmechanismofresistance,suchasefflux pumpsormembranepermeability.Severalexplanationsmay accountforthiscontradictoryeffect.First,despitethe homol-ogybetweenmarRinB.fragilisandE.coli,thegeneticcontexts aredifferentinthesespecies.ThereisnohomologtoMarA orMarBdownstreamMarRhomologsinB.fragilisforexample. Thus,thegenescontrolledbythisregulatormaybeina differ-entlocationinthechromosome.AlthoughMarRwasshown toregulatethe expressionofeffluxsystemsinsome bacte-rialspecies,35_{inothersitcontrolstheexpressionofvirulence} genesandoutermembraneproteins15_{aswellasproteinsthat} modify the architecture ofthe cell envelope.31 _{It is} possi-blethatoutermembraneporinsinvolvedintheentranceof antibioticsinthecellareupregulatedinourmutants, allow-ing moredrugstoreach theirtarget.Andfinally,thereisa possibleepigeneticeffectcausedbytheinsertionofa plas-midinthebacterialchromosomeleadingtodownregulation ofgenesdownstreamofourtargetgenes,anoppositeeffect towhatwouldbeobservedintheeventofalackofa repres-sor.Thislatereffectiscurrentlybeinginvestigatedinourlab,

butthelackofappropriatemoleculartoolssuitableforstrict anaerobicspeciessuchasB.fragilisisfrustrating.

Althoughmanyanswersstillremainelusive,ourworkis thefirsttostudytranscriptionalregulatorsoftheMarR fam-ilyinthestrictanaerobeB.fragilisinrelationtoantimicrobial resistance.Thereisacleareffectobservedinantibiotic resis-tanceassociatedwithdisruption oftheexpressionofMarR homologs. This indicates a participation of these proteins in the regulation of resistance factors.The change in the resistanceprofile was notspecific for a particularclass of antibiotics,andasmentioned,ageneralmechanismsuchas membranepermeabilitymaybeinvolved.Wedidnotobserve significantalterations inotheraspectsofthephysiology of thebacteriumsuchasbiliarysaltsandbiofilmformation.We hypothesizedthat,asobservedwithotherbacterialspecies, genesinvolvedinmembranepermeabilityaremodulatedby MarR homologs in B. fragilis. Understanding these mecha-nismsisparamountforthe developmentofnewstrategies tofightmulti-drugresistantinfectionscausedbythis oppor-tunisticpathogen.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswanttothankMr.JoaquimdaSilvafor techni-calsupport.ThisworkwasfundedbyFundac¸ãodeAmparo a Pesquisa do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenac¸ãodeAperfeic¸oamentodePessoaldeNívelSuperior (CAPES).

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