A novel resistance mechanism to triclosan that suggests horizontal gene transfer and demonstrates a potential selective pressure for reduced biocide susceptibility in clinical strains of Staphylococcus aureus

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InternationalJournalofAntimicrobialAgents40 (2012) 210–220

ContentslistsavailableatSciVerseScienceDirect

International

Journal

of

Antimicrobial

Agents

jo u rn al h om epa g e :h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / i j a n t i m i c a g

A

novel

resistance

mechanism

to

triclosan

that

suggests

horizontal

gene

transfer

and

demonstrates

a

potential

selective

pressure

for

reduced

biocide

susceptibility

in

clinical

strains

of

Staphylococcus

aureus

Maria

Laura

Ciusa

a,1

_,

_Leonardo

_Furi

a,1

_,

_Daniel

_Knight

b,1

_,

_Francesca

_Decorosi

c

_,

_Marco

_Fondi

d

_,

Carla

Raggi

e

_,

_Joana

_Rosado

_Coelho

f

_,

_Luis

_Aragones

g

_,

_Laura

_Moce

g

_,

_Pilar

_Visa

g

_,

_Ana

_Teresa

_Freitas

f

_,

Lucilla

Baldassarri

e

_,

_Renato

_Fani

d

_,

_Carlo

_Viti

c

_,

_Graziella

_Oreﬁci

e

_,

_Jose

_Luis

_Martinez

h

_,

Ian

Morrissey

b,∗∗

_,

_Marco

_Rinaldo

_Oggioni

a,∗

_,

_the

_BIOHYPO

_Consortium

a_Dipartimento_di_{Biotecnologia,}_Università_di_Siena,_Siena,_Italy b_Quotient_Bioresearch,_Fordham,_UK

c_Dipartimento_di_{Biotecnologie}_Agrarie,_Università_di_Firenze,_Firenze,_Italy d_Dipartimento_di_Biologia_Evolutiva,_Università_di_Firenze,_Firenze,_Italy e_Istituto_Superiore_di_Sanità,_Roma,_Italy

f_Euroﬁns_Biolab,_Barcelona,_Spain

g_INESC-ID/IST_Technical_University_of_Lisbon,_Lisbon,_Portugal h_Centro_Nacional_de_{Biotecnologia–CSIC,}_Madrid,_Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received27February2012 Accepted24April2012 Keywords: Biocide Resistance Cross-resistance Horizontalgenetransfer FabI

Triclosan

a

b

s

t

r

a

c

t

ThewidelyusedbiocidetriclosanselectivelytargetsFabI,theNADH-dependenttrans-2-enoyl-acylcarrier

proteinreductase,whichisanimportanttargetfornarrow-spectrumantimicrobialdrugdevelopment.

Inrelationtothegrowingconcernaboutbiocideresistance,wecomparedinvitromutantsandclinical

isolatesofStaphylococcusaureuswithreducedtriclosansusceptibility.ClinicalisolatesofS.aureusas

wellaslaboratory-generatedmutantswereassayedforminimuminhibitoryconcentration(MIC)and

minimumbactericidalconcentration(MBC)phenotypesandgenotypesrelatedtoreducedtriclosan

sus-ceptibility.Apotentialepidemiologicalcut-off(ECOFF)MBCof>4mg/Lwasobservedfortriclosanin

clinicalisolatesofS.aureus.TheseshowedsigniﬁcantlylowerMICsandhigherMBCsthanlaboratory

mutants.Thesegroupsofstrainsalsohadfewsimilaritiesinthetriclosanresistancemechanism.

Molec-ularanalysisidentiﬁednovelresistancemechanismslinkedtothepresenceofanadditionalsh-fabIallele

derivedfromStaphylococcushaemolyticus.Thelackofpredictivevalueofin-vitro-selectedmutationsfor

clinicalisolatesindicatesthatlaboratorytestsinthepresentformappeartobeoflimitedvalue.More

importantly,detectionofsh-fabIasanovelresistancemechanismwithhighpotentialforhorizontalgene

transferdemonstratesfortheﬁrsttimethatabiocidecouldexertaselectivepressureabletodrivethe

spreadofaresistancedeterminantinahumanpathogen.

1. Introduction

Thereis growing concernworldwide regarding thepossible

effect of biocides on antibiotic resistance. The Food and Drug

Administration(FDA)andtheEnvironmental ProtectionAgency

∗ Correspondingauthor.Presentaddress:DipartimentodiBiotecnologia, Policlin-icoLeScotte(lotto5,piano1),UniversitàdiSiena,53100Siena,Italy.

Tel.:+390577233101.

∗∗ Correspondingauthor.Presentaddress:QuotientBioresearch,NewmarketRoad, Fordham,CambsCB75WW,UK.Tel.:+441638722960.

E-mail addresses: i.morrissey@ntlworld.com (I. Morrissey), oggioni@unisi.it

(M.R.Oggioni).

1 _These_three_authors_contributed_equally_to_this_work.

(EPA)intheUSA,thePanelonBiologicalHazardsofthe Norwe-gianScientiﬁcCommitteeforFoodSafety,theScientiﬁcCommittee

onEmerging and NewlyIdentiﬁed Health Risks (SCENIHR) and

theScientiﬁcCommitteeonConsumerSafety(SCCS)inthe Euro-peanUnion(EU),andtheAustralianMicrobiologicalSocietyhave, amongstothers,allexpressedconcernandhaveprogrammes run-ning to investigate theimpact of biocide use on antimicrobial resistance [1–5]. Bacterial resistance tobiocides has been well studied in vitro, but concrete evidence of clinicalresistance is lacking[6,7].In viewofthenewlicensing requirements, proto-colsareurgentlyneededtoprovideriskassessmentsontheuse of biocidalproducts, especially asthereis noconsensusonthe methodologiestobeused tostudybacterialresistancetowards biocides.

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M.L.Ciusaetal./InternationalJournalofAntimicrobialAgents40 (2012) 210–220 211

Thebiocidetriclosanhasreceivedmuchattentionbecauseit iswidelyusedandreportsindicatingemergenceoftriclosan resis-tancehavebeenpublished[8–11].Furthermore,incontrasttoother biocides,triclosanatlowconcentrationsactssimilarlyto antibi-oticsona speciﬁccellulartarget,the enoyl-acylcarrierprotein reductase(FabI),anessentialenzymeinbacterialfattyacid syn-thesis.TriclosanexhibitsexcellentactivityagainstStaphylococcus aureusandisusedtocontrolthecarriageofmeticillin-resistant S.aureus(MRSA)inhospitals[shampooorbathadditivewith2% (20g/L)triclosan][12].LaboratorystudieswithEscherichiacoliand S.aureushaveshownthatmutationsinFabIanditsoverexpression decreasebacterialsusceptibilitytotriclosan[9,13,14].The possi-ble selectivepressure exertedbytriclosan raisessomeconcern

asFabIis a promisingtarget for newnarrow-spectrum

antimi-crobialsagainstMycobacteriumtuberculosis,Plasmodiumfalciparum anddrug-resistantS.aureus[15–17].

Theaimofthisstudywastoanalysethemolecularnatureand phenotypesoftriclosanresistanceinS.aureus,withparticularfocus ontherelationshipbetweenin-vitro-selectedmutantsandclinical isolates.

2. Methods

2.1. Clinicalstrains

Acollectionof1388S.aureusstrainscollectedin2002–2003 from different geographical origins, representing hospital and community-acquired infections, werescreened to ascertain tri-closan susceptibility. Staphylococcus haemolyticus strains were fromacollectionofclinicalisolatesinSiena(Italy).

2.2. Bacterialsusceptibilitytesting

Minimuminhibitoryconcentrations(MICs)weredeterminedby brothmicrodilutionaccordingtoClinicalandLaboratoryStandards Institute(CLSI)guidelines,exceptforthewaytriclosanwasadded tothecultures[18].Stocksolutionsoftriclosan(Irgasan;Sigma, Steinheim,Germany)werepreparedat102400mg/Linmethanol. Owingtothehighhydrophobicityoftriclosan,serial16-folddiluted substocksinmethanolwherepreparedfromwhichtoprepare sub-setsofthreedilutionsinthemicrotitreplate.Thisapproachwas takentoavoidserialtwo-folddilutionsinmicroplatesinorderto minimiseabsorptionof triclosantothe plastic and todecrease thechancesof triclosanprecipitating outof solutionwhen

tri-closan inmethanol wasadded towater. Minimum bactericidal

concentrations (MBCs) were determined by subculturing 10␮L

fromeachwellwithoutvisiblebacterialgrowthonMueller–Hinton agarplates(Biotec,Grosseto,Italy).After24hofincubationat37◦C, thedilutionyieldingthreecoloniesorlesswasscoredastheMBC, asdescribedbytheCLSIforstartinginoculaof1×105_CFU/mL_[19]_.

Noneutralisationstep wasincludedin theMBCassayasinitial experimentsveriﬁedthattriclosancarry-overdidnotoccurwhen 10␮Lwasinoculatedontoagar(datanotshown).Thesensitivityto chemicalcompoundswastestedbyphenotypemicroarrayutilising BiologmicrotitreplatesPM11throughPM20asdescribed(Biolog Inc.,Hayward,CA)[20].

2.3. Biocideactivitytesting

Biocideactivitywastestedaccordingtothestandardsdeﬁned bytheEuropeanstandardEN1276[21].Inbrief,1.5–5×108_CFU

ofbacteriain1mLweremixedwith1mLofbovineserum albu-min(BSA) (Sigma)at0.03g/L (cleanconditions)asinterference substance.Afterwards,thisbacterialsuspensionwasmixedwith 8mLofatriclosandilutioncontaining1.25timesthedesiredtest concentration.Fortheactivityassay,preparationoftriclosanstock

wasperformedasfollows:300mgoftriclosanwasdilutedin1mL

ofdimethylsulphoxide(DMSO)andthismixturewasdilutedin

200mLofhardwater(compositiondeﬁnedinEN1276)[21]. Sub-sequentdilutionsoftriclosanwereundertakeninhardwater.A solutionofhardwatercontaining0.5%DMSOwastested accord-ingtoEN1276against S.aureustoensurethata solutionwith 0.5%DMSOdoesnothavebactericidalactivity.Theconcentrations oftriclosanutilisedfortheassaywere100,600and 1000mg/L. After5minofcontacttimebetweentriclosan,BSAandbacteriaat 20◦C,1mLofthetestsolutionwasmixedwith8mLofneutraliser (3g/Llecithin,30g/Lpolysorbate80,5g/Lsodiumthiosulfate,1g/L l-histidineand30g/Lsaponin)and1mLofwater.After5minofthe neutralisationstep,1mLoftheneutralisationmixand1mLof ten-folddilutionswereculturedontotrypticsoyagar(TSA)(Lioﬁlchem, RosetodegliAbruzzi,Italy)platesinduplicateandwereincubated at37◦Cfor48h.CFU/mLweredeterminedandlogCFU/mL reduc-tionwascalculatedforeachstrainagainsteachofthethreetriclosan concentrationstested.Theconcentrationof600mg/Lwas deter-minedasthelowestconcentrationtestedthatproduced a5log reductioninCFU/mLwithreferencestrainS.aureusATCC6538. 2.4. Invitroselectionoftriclosan-resistantmutants

Triclosan-resistantmutantswereselectedfromS.aureus refer-encestrains,includingthestandardlaboratorystrainRN4220,the referencestrainforbiocidetestingATCC 6538,and threeMRSA

clinical isolates (MW2, Mu50 and COL) for which the genome

sequenceswereavailable.Single-stepmutantswereselectedby culturingca.1×1011_CFU_of_S._aureus_cells,_harvested_from₃₀_mL

of liquid culture, on TSA with 0.5mg/L triclosan (plates con-tained<0.1%methanolfromthebiocidestock).Multistepmutants were selected by serial passageof strains in liquid tryptic soy broth(Lioﬁlchem)containingtwo-foldincreasingconcentrations oftriclosan(0.25mg/Lto4mg/L).Singlecolonieswererandomly selectedfromeachassayandweresubculturedforfurtheranalysis. 2.5. Statisticalcorrelationtest

Threedifferentstatisticaltestswereperformedtoassess poten-tialcorrelations betweenphenotypes and genotypesof clinical isolatesandlaboratorymutants.Fisher’sexacttestwasusedasa statisticaltestappliedtocontingencytablestodeterminewhether therewerenon-randomassociationsbetweentwocategorical vari-ables.Spearman’scorrelationcoefﬁcientwaschosenbecausewe hadunknownsampledistributionsandthetestedvariablesdidnot showalinearrelationship[22].Two-sampleKolmogorov–Smirnov testwasusedtocomparethefoldchangedistributionofthetwo typesofstrains(clinicalisolatesandinvitromutants)[22]. 2.6. Molecularanalysis

The central part of the fabI gene was ampliﬁed in isolates

showingreduced susceptibilitytotriclosan.DNA wasampliﬁed

with primers TAGCCGTAAAGAGCTTGAA and

ATATTTTCACCTG-TAACGCCA (Euroﬁns MWG Operon, Germany), controlled with

Vector NTI- software v.6 (Informax Inc., Bethesda, MD), using

standard PCR conditions and were sequenced by the Sanger

method(BMR Genomics,University of Padova, Italy).For some

selectedstrains,withoutmutationsinthecentralpartofS.aureus

fabI(sa-fabI),primersGATACAGAAAGGACTAAATCAAAand

TTTC-CATCAGTCCGATTATTATA were used to amplify and sequence

the whole gene. A selection of fabI allele sequences has been

depositedinGenBank(accessionnos.JF797286throughJF797303). Whole-genomesequencingoftheS.aureusclinicalisolate QBR-102278-1619wasperformedbytheInstituteofAppliedGenomics (Universityof Udine,Italy) usingan IlluminaGenome Analyzer

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212 M.L.Ciusaetal./InternationalJournalofAntimicrobialAgents40 (2012) 210–220

Fig.1.Minimuminhibitoryconcentration(MIC)andminimumbactericidalconcentration(MBC)distributionandfabIgenotypesofclinicalStaphylococcusaureusisolates. Triclosansusceptibilityof1388clinicalisolatesisreportedaccordingtotheir(A)MICand(B)MBC.Thegenotypeofthoseclinicalisolateswithreducedsusceptibilityto triclosan(highMBC)isshowninpanels(C)and(D)bysortingstrainsaccordingtotheirMICandMBC,respectively.Shadingdifferentiatestriclosan-resistantstrainswitha mutatedsa-fabI(grey),wild-typesa-fabI(openbars)andthoseheterodiploidforthesh-fabIgene(black).

IIplatform(Illumina, SanDiego,CA).Open-readingframe(ORF)

prediction was carried out using Prodigal software (Oak Ridge

NationalLaboratory, Oak Ridge, TN). Detectionof S. haemolyti-cusfabI(sh-fabI)wasperformedbyreal-timePCRusingprimers

TGGCGAAGAAGTAGGCAATATandGCAACAATACTACCACCGTT.The

sh-fabIinsertinQBR-102278-1619wasdepositedinGenBankwith accessionno.JQ712986.

3. Results

Analysisof1388clinicalisolatesofS.aureusrevealeda contin-uousdistributionoftriclosanMICsfrom≤0.015mg/Lto32mg/L, withasinglemodalMICof0.03mg/L(Fig.1A).Incontrast,triclosan MBCspresentedadiscontinuousdistribution(Fig.1B).After per-formingsamplingoftheMBCdatasetinordertobalancethescale ofobservations,wecanﬁtamixtureofnormaldistributions show-ingthatwehavetwodifferentpopulations,suggestingapotential epidemiologicalcut-off(ECOFF)[23]MBCof≤2mg/Lforthe sus-ceptiblepopulation and >4mg/Lfor ‘resistant’ strains(Fig. 1B). AlthoughstatisticalanalysisshowedthatMICandMBCvaluesof triclosanofclinicalstrainsweremoderatelycorrelated(=0.73; P<0.001),itwouldappearthattheMBCisbetterabletoseparate triclosan-non-susceptiblestrainsthantheMIC.Sixty-eightstrains presentingreducedsusceptibilityforthisbiocide(MBC>4mg/L) werechosenforfurthercharacterisation.Thebiocideactivityassay accordingtoEN1276conﬁrmsadecreasedactivityoftriclosanfor strainswithreducedsusceptibilitytothebiocide(Table1).

Toassessthemolecularbasisofresistancetotriclosan,mutant strainswereselectedinvitrofromﬁveS.aureusreferencestrains. Single-stepmutantswereselectedinfourofthemwith frequen-ciesof2.4×10−9 forMW2,3.4×10−10forMu50, 3.4×10−9 for COLand1.4×10−9forATCC6538.FromstrainRN4220,which pre-sentedintermediatesusceptibility(MBC=2mg/L),onlymultistep mutantscouldbeselected.Irrespectiveofthestrainsfromwhich theywereselected,themutantsshowedtriclosanMICsof1–8mg/L (modalMIC=4mg/L)andMBCsof4–32mg/L(modalMBC=8mg/L) (Fig. 2A and B). Unlike theclinical isolates, MICs and MBCs of

triclosan for in vitro mutantspresent a strongstatistically sig-niﬁcantnon-linearcorrelation(=0.90;P<0.001).Thedifference

betweentheMICandMBCoflaboratorymutantswasusuallyof

oneortwodilutions,whilstforclinicalstrainsthesedifferences weregenerallymuchhigher(Fig.2C).Thiswasthecaseevenwhen theinvitromutantsandtheclinicalisolatespresentedthesame sa-fabImutation(Tables 2and3 ).Thiswasfoundtobe

signif-icantly different using a two-sample Kolmogorov–Smirnovtest

(P<0.001).Phenotypemicroarrayforchemicalsensitivitytoover 300compounds[20]conﬁrmedthatthein-vitro-selected triclosan-resistantmutantsdidnotacquireanyfurtherresistancephenotype inadditiontotriclosan(datanotshown).

Toidentifythegenotypesconferringreducedtriclosan suscep-tibility,thefabIgenewassequenced.Amongthe68clinicalisolates withreducedsusceptibilitytotriclosan,30presentedamutation insa-fabI,whilst38strainshadawild-typesa-fabIallele(Table2;

Fig.1CandD).Ofthe30strainswithamutatedsa-fabI,22 car-riedpreviouslydescribedmutations,whilst8strainsshowedfour novelmutations,whichisinaccordancewithotherpublisheddata

[9,10](Table2;Fig.3A).ClusteringwasobservedfortheTTC611TGC mutation,onlyfoundinstrainsfromItaly(4of5)andFrance(2

of7)andthefourGCA593GGA-CTT622TTTdoublemutants,which

wereisolatedatdifferentcitiesintheUSAandCanada.Most in-vitro-selectedmutantshadpreviouslycharacterisedfabImutations

[9–11,17],withtheexceptionofRN4220mutants,whichallshowed

aGAC301TACmutation,andoneATCC6538derivative,whichhad

aTTC611TCCchange(Table3;Fig.3A).Onlytwoofsixmutations

selectedin vitro (GCA593GGA and TTC611TGC)matched

muta-tionsdetectedinclinicalisolates(Fig.3A).Twoclones(MO035and MO079)showednovariationinthesa-fabIgenedespitehighMICs andMBCstotriclosan(Table3).

Toidentifyfurtherthemolecularbasisofreducedtriclosan sus-ceptibilityofclinicalisolateswithawild-typefabIallele,thewhole genomeofonestrainwithatriclosanMICof4mg/LandMBCof

32mg/L(QBR-102278-1619)wassequenced.A3016bp

chromoso-malinsertcarryinganadditionalfabIgene,showing84%nucleotide and91%aminoacididentitytosa-fabI,andaninsertionsequence

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Table1

TestingoftriclosanactivityonStaphylococcusaureusstrainsfollowingClinicalandLaboratoryStandardInstitute(CLSI)andEuropeanstandardEN1276guidelines.

Strain MIC(mg/L) MBC(mg/L) EN1276(logreductionCFU/mL)a _Note

100mg/L 600mg/L 1000mg/L

ATCC6538 0.12 0.25 0.33 5.45 >5.48 Wild-type

QBR-102278-1177 4 32 0.18 4.04 5.48 Mutatedsa-fabI

QBR-102278-1219 4 32 0.27 3.96 4.01 Mutatedsa-fabI

QBR-102278-1619 4 32 0.41 4.67 5.45 sh-fabI

MIC,minimuminhibitoryconcentration;MBC,minimumbactericidalconcentration.

a_Values_report_logarithmic_reduction_(R)_of_bacterial_counts_within₅_min_contact_time_and_subsequent_{neutralisation}_(product_is_considered_active_if_log_R_>_5).

IS1272(Fig.3B)wasfoundinanintergenicregionoftheS.aureus

chromosome(MW2 position 141825)(Fig. 3B). Theintegration

occurredin theloop ofa hairpinwithan18bpinvertedrepeat stem,whichdeterminedaninsertbetweentwoshortdirectrepeats. DatabasesearcheswiththisadditionalfabIgeneshowedits pres-ence,with100%identity, inthechromosomeofS.haemolyticus (Fig.3B),whichdoesnothaveanyfurtherfabIgene.Thisstrongly suggeststhat the sh-fabI allelemost likely belongs to thecore genomeofS.haemolyticus.Supportingthisstatement,PCR anal-ysisdemonstrated thepresence ofsh-fabI in aselection of ﬁve S.haemolyticusclinicalstrains,irrespectiveoftheirsusceptibility totriclosan(MBCrange1–32mg/L).Furthersearchesforsh-fabI showedmultiplehitsindifferentstaphylococci,includingS.aureus andStaphylococcusepidermidis,wheresh-fabIwaslocatedon plas-midsthatalsocarrythemultidrugresistance(MDR)efﬂuxpump

forquaternaryammoniumcompoundsQacA(GenBankaccession

nos.FR821778andGQ900465)[24,25].Thefactthattheseplasmids carrythe3016bpinsertborderedbypartsoftheinvertedrepeat oftheS.aureuschromosomeindicatesthedirectionofhorizontal transfer.

PCRassaysofthe68clinicalisolateswithreducedsusceptibility totriclosanidentiﬁed sh-fabIin24 ofthe38 strainswith wild-typefabIand in4ofthe30strainswithmutatedfabI(Table2). Distributionofsh-fabIinS.aureusstrainswithreducedtriclosan

susceptibilityshowedgeographicalclustering,withpositivityin 9/10isolatesfromMexico,7/10fromCanada,5/10fromBraziland 4/8fromJapan,withnostrainsfromothercountriesincludingthe USA,Italy,SpainandGermany.Onlyoneofthesh-fabI-positive clin-icalisolateswaspositivefortheMDRefﬂuxdeterminantqacA(data notshown).Clinicalstrainswithdecreasedsusceptibilityto tri-closanhadastrongassociationwiththepresenceofamutatedfabI geneorthealternativesh-fabIgene(Fisher’sexacttest,P<0.001).

4. Discussion

FabIisthetargetofisoniazid,animportantagentforthe treat-mentoftuberculosis,andisoneofthedrugtargetsthathasbeen rediscoveredinrecentyearsforrationalantimicrobialdrug devel-opment[17,26]. Inthis context,careful analysisoftheeffectof triclosan,awidelyutilisedbiocideanddisinfectant,whichalso tar-getsFabI,onthesusceptibilityofstaphylococciisofprimeinterest. ToaddressthemolecularbasisoftriclosanresistanceinS.aureus, 68strainswithreducedsusceptibilitytothebiocideselectedfroma worldwidecollectionofclinicalandcommunity-acquiredS.aureus

were analysed. As FabI is the only known target of triclosan

[9,13,14],attention wasfocused onthe nucleotidesequence of fabI.Surprisingly,onlyapproximatelyone-halfofthestrains show-inghighMBCvaluestotriclosanhaddetectablemutationsinthe

Fig.2.Minimuminhibitoryconcentration(MIC)andminimumbactericidalconcentration(MBC)distributionandfabIgenotypesoflaboratorymutants.Triclosansusceptibility oflaboratorystrains,includingreferencestrainsandmutants,isreportedaccordingtotheir(A)MICand(B)MBC.Genotypicdataareshownbyshadingofthecolumns differentiatingsusceptiblereferencestains(wild-typesa-fabI,openbars)andtriclosan-resistantmutantswithmutatedsa-fabI(black)andwild-typesa-fabI(openbars).(C) DistributionoftheMBC/MICfoldchangeofstrainswithreducedsusceptibilitytotriclosanselectedinvitro(n=28)(openbars)andisolatedfromtheclinicalstraincollections (n=68)(black).

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214 M.L. Ciusa et al. / International Journal of Antimicrobial Agents 40 (2012) 210– 220 Table2

fabIgenesequencesofStaphylococcusaureusclinicalisolatesandreferencestrains.

Isolate

Polymorphic sites in fabI a

sh–fabI sa-fabI MIC (mg/L) MBC (mg/L) Comment b 122222223333333334444455556666677 3801256780133677883567947891126802 3446651241589338149081801330120783

COL CTAGGCTACGCGCTTATGTCCTCAGACTTCTTTT – wt 0.25 1 Reference strain

QBR-102278-1619 ... + wt 4 32 wt allele in 16 sequenced genomes

QBR-102278-1888 ... – wt 0.03 16 wt allele in 16 sequenced genomes

QBR-102278-2175 ... + wt 0.25 16 wt allele in 16 sequenced genomes

QBR-102278-2305 ... – wt 4 64 wt allele in 16 sequenced genomes

QBR-102278-2321 ... – wt 4 32 wt allele in 16 sequenced genomes

QBR-102278-1219 ...G. – Mutated 4 32 TTC611TGC known mutation

QBR-102278-1177 ...G. – Mutated 4 32 TTC611TGCknown mutation

QBR-102278-1865 ...G... – Mutated 0.5 16 GCA593GGA known mutation

QBR-102278-1970 ...G... – Mutated 0.5 32 GCA593GGA known mutation

QBR-102278-1917 ...G..T – Mutated 2 16 GCA593GGA, CTT622TTT known mutations

QBR-102278-1207 ...C..T.T.CTCT...C...T.... – Mutated 0.12 8 ACA583TCA new allele

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M.L. Ciusa et al. / International Journal of Antimicrobial Agents 40 (2012) 210– 220 215 Table2(continued)

QBR-102278-1883 ...C...T.T.CTCT...C....G..T – Mutated 2 8 GCA593GGA CTT622TTT known mutations

QBR-102278-2345 ...C...T.T.CTCT...C... – wt 1 2 wt allele in 4 sequenced genomes

QBR-102278-2363 ...T.CTCT... + wt 16 32 wt allele in 23 sequenced genomes

QBR-102278-1878 ...C..T.T.CTCT...C....G..T – Mutated 2 16 GCA593GGA, CTT622TTT known mutations

QBR-102278-2069 ...C..T.T.CTCT...C....G..T – Mutated 2 32 GCA593GGA,CTT622TTT known mutations

QBR-102278-1894 GT....C..T.T.CTCT...C....G..T – Mutated 2 16 GCA593GGA, CTT622TTT known mutations

QBR-102278-1651 ...C..T.T.CTCT...G... – Mutated 2 32 GCA593GGA known mutation

QBR-102278-1653 ...C..T.T.CTCT...G... – Mutated 2 32 GCA593GGA known mutation

QBR-102278-2019 ...C..T.TCCTCT...G.. – Mutated 0.25 16 TTC610GTC new allele

ATCC25923 ...A...C..T.T.CTCT....T...A.. – wt 0.06 1 Reference strain

QBR-102278-1097 ....TTC...T.CTCT... – Mutated 0.25 32 GGT226TGT,GGC255GGT new allele

QBR-102278-1203 T...T.CTCT... + wt 2 16 wt allele in 4 sequenced genomes

QBR-102278-1107 T...T.CTCT... + wt 4 32 wt allele in 4 sequenced genomes

QBR-102278-1052 T...T.CTCT...C... + wt 0.5 64 wt allele in 4 sequenced genomes

QBR-102278-1544 ...T.CTCT...G... – Mutated 2 64 GCA593GGA known mutation

QBR-102278-1144 ...T.CTCT...G. – Mutated 1 32 TTC611TGC known mutation, new allele

MW2 ...T.TTCT... – wt 0.5 1 Reference strain

QBR-102278-2311 ...T.C... – wt 1 64 wt allele in 4 sequenced genomes

QBR-102278-2212 ...T.C... + wt 2 32 wt allele in 4 sequenced genomes

QBR-102278-2221 ...T.C... + wt 0.5 16 wt allele in 4 sequenced genomes

QBR-102278-2605 ...C...T.T.C...C... + wt 32 64 wt allele in 4 sequenced genomes

QBR-102278-2546 ....TC....CT.C... + Mutated 1 64 GGC255GGT, GGC338GCT new allele

QBR-102278-2342 ...T..T..G... + Mutated 2 32 GCA593GGA known mutation

QBR-102278-2348 ...T..T..G... + Mutated 0.5 32 GCA593GGA known mutation

QBR-102278-2254 ...T...G... + Mutated 1 32 GCA593GGA known mutation

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216 M.L. Ciusa et al. / International Journal of Antimicrobial Agents 40 (2012) 210– 220 Table2(continued).

Mu50 ...T..T... – wt 0.25 0·5 Reference strain

QBR-102278-2346 ...T..T... – wt 2 32 wt allele in 23 sequenced genomes

QBR-102278-2222 ...T..T... + wt 1 32 wt allele in 23 sequenced genomes

QBR-102278-2205 ...T... + wt 1 16 wt allele in 19 sequenced genomes

QBR-102278-2204 ...T... + wt 1 16 wt allele in 19 sequenced genomes

ATCC6538 ...T..T...CAC. – wt 0.12 0·25 wt new allele

QBR-102278-1236 ...T..T...CAC. – wt 4 16 wt allele in 23 sequenced genomes

QBR-102278-1607 ...T..T...CAC. – wt 0.12 32 wt allele in 23 sequenced genomes

QBR-102278-2072 ...T..T...CAC. + wt 0.25 32 wt allele in 23 sequenced genomes

QBR-102278-1210 ...T..T... – wt 0.25 16 wt allele in 23 sequenced genomes

QBR-102278-2070 ...T..T... – wt 0.12 32 wt allele in 23 sequenced genomes

QBR-102278-1158 G...GT... – wt 2 8 wt new allele

QBR-102278-1969 ...A... – wt 0.25 32 wt new allele

QBR-102278-2018 ...A... + wt 0.5 16 wt new allele

RN4220 ...A... – wt 1 2 Reference strain

MIC,minimuminhibitoryconcentration;MBC,minimumbactericidalconcentration;wt,wild-type. a_Polymorphic_sites_are_indicated_with_respect_to_the_fabI_sequence_of_S._aureus_COL.

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M.L. Ciusa et al. / International Journal of Antimicrobial Agents 40 (2012) 210– 220 217 Table3

Genotypeandphenotypeofinvitromultistepandsingle-stepexposuremutants.

ID

Polymorphic sites in fabI a

FabI sa-fabI MIC (mg/L) MBC (mg/L) Comment

122222223333333334444455556666677 3801256780133677883567947891126802

3446651241589338149081801330120783

COL CTAGGCTACGCGCTTATGTCCTCAGACTTCTTTT wt 0.12 1 Reference strain

MO082 ...T... Ala95Val Mutated 8 16 SSM

MW2 ...T...T.TTCT... wt 0.12 0.12 Reference strain

MO075 ...T...T.TTCT... Ala95Val Mutated 4 16 SSM

Mu50 ...T..T... wt 0.06 0.12 Reference strain

MO079 ...T..T... wt 4 16 SSM

MO080 ...T...T..T... Ala95Val Mutated 4 4 SSM

ATCC6538 ...T..T...CAC. wt 0.12 0.25 Reference strain

CR001 ...T..T..G...CAC. Ala198Gly Mutated 4 8 SSM

CR002 ...T..T....G.CAC. Phe204Cys Mutated 4 8 SSM

CR003 ...T..T....G.CAC. Phe204Cys Mutated 2 8 SSM

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218 M.L. Ciusa et al. / International Journal of Antimicrobial Agents 40 (2012) 210– 220 Table3(continued).

d2 ...T..T....G.CAC. Phe204Cys Mutated 1 4 MSM

d7 ...C.T..T...CAC. Tyr147His Mutated 2 8 MSM

MO051 ...T...T..T...CAC. Ala95Val Mutated 4 8 MSM

MO052 ...T..T....C.CAC. Phe204Ser Mutated 8 16 MSM

RN4220 ...A... wt 1 2 Reference strain

MO034 ...T...A... Asp101Tyr Mutated 8 8 MSM

MO035 ...A... wt 8 8 MSM

MIC,minimuminhibitoryconcentration;MBC,minimumbactericidalconcentration;SSM,single-stepmutant;MSM,multistepmutant. a_Polymorphic_sites_are_indicated_with_respect_to_the_fabI_sequence_of_{Staphylococcus}_aureus_COL.

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Fig.3.SchematicmapofmutationsintheStaphylococcusaureusfabI(sa-fabI)andofStaphylococcushaemolyticusfabI(sh-fabI)genes.(A)Mutationsinsa-fabIarereportedon aschematicmap.Mutationsdetectedinclinicalisolatesaremappedabovethesequence,whilstmutationsselectedinvitroareshownbelowthesequence.(B)Schematic alignmentofthesh-fabIgeneregionofstrainQBR-102278-1619toS.haemolyticus(NC007168)andS.aureusMW2(NC003923).GenenumberingoftheQBR-102278-1619 open-readingframe(ORF)isasforMW2.ThealignmentshavebeenreproducedfromanalignmentperformedwiththewebversionoftheArtemisComparisonTool(Sanger Centre).Thethinlinerepresentsthe3016bpfragmentinsertedintheS.aureuschromosomeinstrainQBR-102278-1619.Overallnucleotideidentityintheshadedareasis giveninpercent.

codingregionofsa-fabI.Whole-genomesequencingofoneofthese strainsshowedthepresencea3kbgenomicisletcarryingan addi-tionalfabIgeneidenticaltothatbelongingtothecoregenomeof S.haemolyticussh-fabI.Bycloningsa-fabIontoaplasmidvector,it hasbeendemonstratedthattriclosanresistancecanbeachievedby increasingtheamountoftarget[14].Inasimilarway,thepresence ofsh-fabItogetherwithsa-fabIconstitutesacompletelynovel resis-tancemechanism,actingbyincreasingthetargetamountthrough

heterologoustargetduplication.Theonlyknownmechanismsof

triclosanresistanceatthetimeofwritingthisarticleweredueto

chromosomalmutations.Oneofthemostimportantobservations

inthisworkistheidentiﬁcationoflikelyhorizontaltransferofthis novelbiocideresistancemechanism.

Detectionoftheinvertedrepeatsequencesgainedbyinsertion intheS.aureusgenomeindicatesthatthedirectionoftransferis fromS.haemolyticustoS.aureusandfromtheS.aureuschromosome toplasmids[24,25].Furtheridentiﬁcationofsh-fabIinnumerous staphylococciinmetagenomeandmicrobiomedatabasesindicates thatthegeneisactivelyspreading.

Itisdifficulttounequivocallyestablishtheselectiveforcesthat causeselection ofaspecificmechanismofresistance, especially whendeterminantscanconfersimultaneousresistancetodifferent drugsorwhenseveraldifferentresistanceelementsareassociated inthesamegenetransferelement[27].Forbiocidesthatcan pro-ducecross-resistancetoantibiotics,itisdifficulttoknowwhether

theselectiveagenthasbeenthebiocideortheantibioticitself. InthecaseofFabI,thisenzymeistargetedonlybytriclosaninS. aureus.Identificationofaresistancemechanismtotriclosanacting byheterologoustargetduplicationexcludesotherantimicrobials asbeingselectiveforces.Thisfindingisadirectdemonstrationthat thebiocidetriclosanproducesaselectivepressureonS.aureusand otherstaphylococciandisthefirstclearevidencethatutilisation ofbiocidescandrivedevelopmentofbiocideresistanceinclinical isolates.

AgenciessuchastheFDArequestarisk–beneﬁtassessmentfor humanantibioticsthatincludesevaluatingtherisksofresistance generation.Forantibioticsusedinanimals,theseresistancerisks arean importantsafety issuethat isaddressed inall antibiotic submissions.Recently,theneedforsuchrequirementshasbeen raisedforbiocides.Forinstance,arecentEUproposalfor licens-ingofbiocidesasksthat‘compoundsshouldhavenounacceptable effectsonthetargetorganisms,inparticularunacceptable resis-tanceorcross-resistance’[28].Inviewoftherequirementsposed, thepossibilityofdevising aninvitroassayfor testingbacterial resistancetothebiocidetriclosanwasevaluated.Itisknownthat triclosan-resistantfabImutants canbeselected in vitro [9–11].

Theaimwastoassess whethersuchmutantshave any

predic-tivevalueforresistanceobservedinclinicalisolates[29].Mutants wereselectedbytwodistinctproceduresinﬁvedifferent refer-encestrains, but a mutation that was alsodetected in clinical

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220 M.L.Ciusaetal./InternationalJournalofAntimicrobialAgents40 (2012) 210–220

isolateswasfoundinonly5of28mutants,albeitthemostprevalent one.Asecondveryimportantaspectisthatallin-vitro-generated mutantstrainsshowsimilarMICandMBCvalues,indicatingthat triclosanremainedbactericidalforthesestrains.Thisisincontrast toclinicalisolateswhereMICsweremuchlowerthanMBCs, indi-catingamorebacteriostaticactionoftriclosanintheseresistant strains.Thisdifferencewasalsoobservedintheinvitromutants andclinicalisolatescarryingthesamemutationandsuggeststhat clinicalisolatesmighthaveaccumulatedcompensatingmutations thatmodifythephenotypeandallowareductionintheprobable ﬁtnesscostgivenbythemutationsgeneratedinvitro[27].Thus, boththephenotypicproﬁleandthegenotypeofmutationsdiffered invitrofromthosedetectedinclinicalisolates.Withrespecttothe requestbycurrentlegislationtoruninvitrotestsbeforeplacing

anactivecompound onthemarket,wecanconcludethat such

atestisfeasiblefortriclosan,butthatsuchatestdoesnotyield resultsofclinicalrelevanceifperformedaccordingtoastandard experimentalset-up.However,thedatafromthisstudysuggest that anECOFF MBCof >4mg/Lmay bea good indicatorof tri-closan‘resistance’.Weplantoundertakefurtherstudiestoassess this.

Summarising,anovelresistancemechanismwasidentiﬁedin clinicalisolatesbasedon‘heterodiploidy’duetoanadditionalcopy ofsh-fabIfromS.haemolyticus.Detectionofthesamesh-fabIislet instaphylococcalplasmidsindicatesthatthisnovelresistance ele-ment is being activelytransferred, most likely due to positive selectionbytriclosan.

Acknowledgments

TheauthorsaregratefulforhelpfuldiscussiontoUlkuYetis,

HansJoachimRoedger,TeresaCoque,AyseKalkanci,DiegoMora

and Stephen Leib who participated to the BIOHYPO research

project.

Funding: The workwas supported by EuropeanCommunity

FP7projectKBBE-227258(BIOHYPO),whichisaresearchproject aimedatevaluatingtheimpactofbiocideuseonthegenerationof antibioticresistance.Thefundershadnoroleinstudydesign,data collectionandanalysis,decisiontopublish,orpreparationofthe manuscript.

Competinginterests:MROhasreceivedfundingfromBASFfor

workonbiocides;however,thecompanydidnotinﬂuencethe

studydesignandtheworkcarriedoutforBASFisnotpartofthis study.Allotherauthorsdeclarenocompetinginterests.

Ethicalapproval:Notrequired.

References

[1]ClaytonEM,ToddM,DowdJB,AielloAE.TheimpactofbisphenolAandtriclosan onimmuneparametersintheU.S.population,NHANES2003–2006.Environ HealthPerspect2011;119:390–6.

[2]Scientiﬁc Committee on Consumer Safety (SCCS). Opinion on triclosan. Antimicrobial resistance. Brussels, Belgium: European Union; 2010.

http://ec.europa.eu/health/scientiﬁccommittees/consumersafety/docs/sccs o023.pdf[accessed08.02.12].

[3]Merlino J, Brown M. Biocides in the health industry. Microbiol Aust 2010;31:158.

[4]Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). Research strategy to address the knowledge gaps on the antimicrobial resistance effects of biocides. Brussels, Belgium: European Commission; 2010. http://ec.europa.eu/health/scientific committees/emerging/docs/scenihro028.pdf[accessed08.02.12].

[5]Scientiﬁc Committee on Emerging and Newly Identiﬁed Health Risks (SCENIHR). Assessment of the antibiotic resistance effects of biocides. Brussels, Belgium: European Commission; 2009.

http://ec.europa.eu/health/phrisk/committees/04scenihr/docs/scenihro021 .pdf[accessed08.02.12].

[6]RussellAD.Mechanismsofantimicrobialactionofantisepticsand disinfec-tants:anincreasinglyimportantareaofinvestigation.JAntimicrobChemother 2002;49:597–9.

[7]CooksonB.Clinicalsigniﬁcanceofemergenceofbacterialantimicrobial resis-tanceinthehospitalenvironment.JApplMicrobiol2005;99:989–96. [8]SchweizerHP.Triclosan:awidelyusedbiocideanditslinktoantibiotics.FEMS

MicrobiolLett2001;202:1–7.

[9]FanF,YanK,WallisNG,ReedS,MooreTD,RittenhouseSF,etal.Deﬁning andcombatingthemechanismsoftriclosanresistanceinclinicalisolatesof Staphylococcusaureus.AntimicrobAgentsChemother2002;46:3343–7. [10] BrenwaldNP,FraiseAP.Triclosanresistanceinmethicillin-resistant

Staphylo-coccusaureus(MRSA).JHospInfect2003;55:141–4.

[11] XuH,SullivanTJ,SekiguciJ,KirikaeT,OjimaI,StrattonCF,etal.Mechanism andinhibitionofsaFabI,theenoylreductasefromStaphylococcusaureus. Bio-chemistry2008;47:4228–36.

[12] CoiaJE,DuckworthGJ,EdwardsDI,FarringtonM,FryC,HumphreysH,etal. Guidelinesforthecontrolandpreventionofmeticillin-resistantStaphylococcus aureus(MRSA)inhealthcarefacilities.JHospInfect2006;63(Suppl.1):S1–44. [13]HeathRJ, Rubin JR, Holland DR, Zhang E, Snow ME, Rock CO.

Mecha-nismoftriclosaninhibitionofbacterialfatty acidsynthesis.JBiolChem 1999;274:11110–4.

[14]Slater-RadostiC,VanAllerG,GreenwoodR,NocholasR,KellerPM,DewolfJr WE,etal.Biochemicalandgeneticcharacterizationoftheactionoftriclosanon Staphylococcusaureus.JAntimicrobChemother2001;48:1–6.

[15]RawatR,WhittyA,TongeP.Theisoniazid–NADadductisaslow,tight-binding inhibitorofInhA,theMycobacteriumtuberculosisenoylreductase:adduct afﬁn-ityanddrugresistance.ProcNatlAcadSciUSA2003;100:13881–6. [16]GoodmanCD,McFaddenGI.Fattyacidbiosynthesisasadrugtargetin

apicom-plexanparasites.CurrDrugTargets2007;8:15–30.

[17]EscaichS,ProuvensierL,SaccomaniM,DurantL,OxobyM,GeruszV,etal.The MUT056399inhibitorofFabIisanewantistaphylococcalcompound. Antimi-crobAgentsChemother2011;55:4692–7.

[18]ClinicalandLaboratoryStandardsInstitute.Methodsfordilutionantimicrobial susceptibilitytestsforbacteriathatgrowaerobically;approvedstandard.8th ed.DocumentM7-A8.Wayne,PA:CLSI;2009.

[19]NationalCommitteeforClinicalLaboratoryStandards.Methodsfor determin-ingbactericidalactivityofantimicrobialagents;approvedguideline.Document M26-A.Wayne,PA:CLSI;1999.

[20] DecorosiF,SantopoloL,MoraD,VitiC,GiovannettiL.Theimprovementofa phenotypemicroarrayprotocolforthechemicalsensitivityanalysisof Strepto-coccusthermophilus.JMicrobiolMethods2011;86:258–61.

[21]CEN European Committee for Standardisation. EN 1276:2009/AC: 2010. Chemicaldisinfectantsandantiseptics.Quantitativesuspensiontestforthe evaluationofbactericidalactivityofchemicaldisinfectantsandantiseptics usedinfood,industrial,domesticandinstitutionalareas.Testmethodand requirements(phase2,step1).2010.

[22] KvamPH,VidakovicB.Nonparametricstatisticswithapplicationstoscience andengineering(Wileyseriesinprobabilityandstatistics).Hoboken,NJ:John Wiley&SonsInc.;2007.

[23]KahlmeterG,BrownDF,GoldsteinFW,MacGowanAP,MoutonJW,Osterlund A,etal.EuropeanharmonizationofMICbreakpointsforantimicrobial suscep-tibilitytestingofbacteria.JAntimicrobChemother2003;52:145–8. [24]HoltDC,HoldenMT,TongSY,Castillo-RamirezS,ClarkeL,QuailMA,etal.Avery

early-branchingStaphylococcusaureuslineagelackingthecarotenoidpigment staphyloxanthin.GenomeBiolEvol2011;3:881–95.

[25]ShearerJES,WiremanJ,HostetlerJ,ForbergerH,BormanJ,GillJ,etal.Major familiesofmultiresistantplasmidsfromgeographicallyandepidemiologically diversestaphylococci.G3(Bethesda)2011;1:581–91.

[26] LuH,TongePJ.InhibitorsofFabI,anenzymedrugtargetinthebacterialfatty acidbiosynthesispathway.AccChemRes2008;41:11–20.

[27]MartinezJL,FajardoA,GarmendiaL,HernandezA,LinaresJF,Martínez-Solano L,etal.Aglobalviewofantibioticresistance.FEMSMicrobiolRev2009;33: 44–65.

[28]Commission of the European Communities. Proposal for a regu-lation of the European parliament and of the council concerning the placing on the market and use of biocidal products. Brussels, Belgium: Commission of the European Communities; 2009. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=com:2009:0267:ﬁn:en:pdf

[accessed08.02.12].

[29]MaillardJY,DenverSP.Emergingbacterialresistancefollowingbiocide expo-sure:shouldwebeconcerned?ChimOggi2009;27:26–8.