hidden antibiotic resistance fitness costs revealed by ...dec 09, 2017 · 3 55 introduction 56...

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HiddenantibioticresistancefitnesscostsrevealedbyGWAS-basedepistasis1

analysis.2

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MahoYokoyama1,MaisemLaabei2,EmilyStevens3,LeannBacon3,KateHeesom4,4

SionBayliss1,NicolaOoi5,AlexJ.O’Neill5,EwanMurray6,PaulWilliams6,Anneke5

Lubben7,ShaunReeksting7,GuillaumeMeric1,BenPascoe1,SamuelK.Sheppard1,6

MarioRecker8,LaurenceD.Hurst1,andRuthC.Massey3*7

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1:MilnerCentreforEvolution,Dept.ofBiologyandBiochemistry,UniversityofBath,9 UK10

2:DivisionofMedicalProteinChemistry,DepartmentofTranslationalMedicine,11 LundUniversity,S20502,Malmö,Sweden12

3:SchoolofCellularandMolecularMedicine,UniversityofBristol,UK.13 4:UniversityofBristolProteomicsFacility,UniversityofBristol,UK.14 5:AntimicrobialResearchCentre,FacultyofBiologicalSciences,UniversityofLeeds,15

Leeds,LS29JT,UK16 6:CentreforBiomolecularSciences,SchoolofLifeSciences,Universityof17

Nottingham,NG72RD,UK.18 7:ChemicalCharacterisationandAnalysisFacility,FacultyofScience,Universityof19

Bath,BA27AY,UK20 8:CentreforMathematicsandtheEnvironment,UniversityofExeter,Penryn21

Campus,PenrynTR109FE,UK22 23 *forcorrespondence: [email protected] 25

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.CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted December 9, 2017. ; https://doi.org/10.1101/148825doi: bioRxiv preprint

https://doi.org/10.1101/148825http://creativecommons.org/licenses/by-nc-nd/4.0/

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ABSTRACT27

Understandinghowmulti-drugresistantpathogensevolveiskeytoidentifying28

meansofcurtailingtheirfurtheremergenceanddissemination.Fitnesscosts29

imposedonbacteriabyresistancemechanismsarebelievedtohampertheir30

disseminationinanantibioticfreeenvironment,however,somehavebeenreported31

tohavelittleornocost,whichsuggeststherearefewbarrierspreventingtheir32

globalspread.Onesuchapparentlycost-freeresistancemechanismacquiredbythe33

majorhumanpathogenStaphylococcusaureusistotheclinicallyimportantantibiotic34

mupirocin,whichismediatedbymutationofthehighly-conservedandessential35

isoleucyl-tRNAsynthethase(ileS)gene.InGenomeWideAssociationStudies(GWAS)36

ontwogeneticallyandgeographicallydistinctMRSAlineageswehavefoundthis37

mutationtobeassociatedwithchangesinbacterialvirulence,driventhrough38

epistaticinteractionswithotherloci.Giventhepotentialdualeffectofthismutation39

onbothantibioticresistanceandvirulenceweadoptedaproteomicapproachand40

observedpleiotropiceffects.Thisanalysisrevealedthattheactivityofthesecretory41

apparatusofthePSMfamilyofcytolytictoxins,thePmtsystem,isaffectedinthe42

mupirocinresistantmutant,whichexplainswhyitislesstoxic.Asanenergetically43

costlyactivity,thisreductionintoxicitymasksthefitnesscostsassociatedwiththis44

resistancemutation,acostthatbecomesapparentwhentoxinproductionis45

required.Giventhewidespreaduseofthisantibiotic,andthatthisresistanceoften46

resultsfromasinglenucleotidesubstitutionintheileSgene,thesehiddenfitness47

costsprovideanexplanationforwhythisresistancemechanismisnotmore48

prevalent.Thisworkalsodemonstrateshowpopulation-basedgenomicanalysisof49

virulenceandantibioticresistancecancontributetouncoveringhiddenfeaturesof50

thebiologyofmicrobialpathogens.51

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INTRODUCTION55

Antibioticresistancecanevolveinmanyways,andfrequentlyincurafitnesscostto56

theorganism1whichhastoeithermutatethetargetsiteoftheantibiotic,acquire57

andexpressageneencodinganalternativenon-susceptibleversionofthetarget58

protein,oracquireandproduceaneffluxpumpthatremovestheantibioticbeforeit59

canattackitstarget2.Asantibioticsaremostcommonlyusedforshortanddefined60

periodsoftime,resistantbacteriaareunderselectiontoreducethesecoststoavoid61

displacementoncetreatmenthasfinished.Inmanycasesthisisachievedthrough62

compensatorymutationsthatallowmanyresistancemechanismstobemaintained63

stablypopulationsforlongperiodsoftime3.However,someantibioticresistance64

mechanismshavebeenreportedtoincurnodetectablefitnesscosts4,5,6,which65

suggeststherearenobarrierstotheirwidespreaddissemination.66

Staphylococcusaureusisanexampleofamajorhumanpathogen7thathas67

becomemorechallengingtotreatduetotheemergenceofantibioticresistance,68

withMethicillin-ResistantS.aureus(MRSA)beingthemostnotableexample8.S.69

aureusresidesasymptomaticallyaspartofthenormalnasalfloraofupto50%of70

humans9,however,thisisasignificantriskfactorforinfection10,totheextentthat71

carriersareoftendecolonisedusingantibioticssuchasmupirocinpriortoinvasive72

proceduressuchassurgeryordialysis11.Mupirocinisapolyketideantibioticthatis73

appliedasanointmenttoeradicatenasalcarriageofMRSAinpatientsatriskof74

infection12.SuchdecolonisationhasbeenreportedtoreduceS.aureusinfectionsof75

post-surgicalwoundsby58%,ofhaemodialysispatientsby80%andofperitoneal76

dialysispatientsby63%13.77

Themoleculartargetformupirocinisthebacterialisoleucyl-tRNAsynthetase78

(IleRS),whichchargestRNAswiththeaminoacidisoleucine(Ile)12.Bybindingtothis79

enzymetheantibiotichaltsproteinsynthesis,soinhibitingbacterialgrowth14.Asa80

consequenceofthewidespreaduseofmupirocin,resistancehasemergedwherethe81

bacteriahavemutatedthegeneencodingIleRS,ileS,resultinginanaminoacid82

substitution(e.g.V588F,encodedbyaGtoTsinglenucleotidepolymorphism(SNP)83

atposition1,762intheileSgene(G1762T))whichaltersthestructureofthe84

protein’sactivesite,butretainsfunctionalityandrendersmupirocinlesseffective15.85

Thisconfersalowtointermediatelevelofresistancetotheantibiotic16.86




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Alternatively,thebacteriaacquireanalternativeIleRS,encodedbyamupAormupB87

gene,onaplasmid,whichconfersahigherlevelofresistance17,18.Theprevalenceof88

mupirocinresistancevarieswidely,withthehighestratesassociatedwithpatient89

groupsrepeatedlyexposedtotheantibiotic19,20.Ithasalsobeenshownthatin90

countriessuchasNewZealandandAustraliawhichhavepreviouslyreportedahigh91

prevalenceofmupirocinresistance,whenrestrictionswereputinplacelimitingthe92

useofthisantibiotic,theprevalenceofresistantstrainssignificantlydeclined20.This93

suggeststhatantibioticexposureisrequiredtomaintainselectionofthisresistance94

mechanismwithinapopulation,despiteitbeingreportedasincurringnofitness95

cost.96

TheileSgeneishighlyconservedacrossthethousandsofsequencedS.97

aureusisolates,andmanyfailedattemptstoinactivateitsuggestitsactivityis98

essentialtothebacteria.Itisthereforesurprisingthatthemutationthatconfers99

mupirocinresistance,byalteringthestructureoftheencodedprotein,doesnot100

appeartoaffectfitness.Especiallyconsideringthatthereplacementofthevaline101

588(V588)withphenylalanine,amuchbulkierresidue,islikelytofillanddistortthe102

Rossmanfoldoftheenzyme,whichisresponsibleforATPbindingactivityofthis103

enzyme4,10.However,inarecentgenomewideassociationstudy(GWAS)onthe104

majorhospitalacquiredMRSAclone,ST239,thismutationwassignificantly105

associatedwithdifferencesinthevirulenceofS.aureusisolates21.Itseffectontoxin106

secretion,amajoraspectofS.aureusvirulence,wasbelievedtoresultfromepistatic107

interactionsbetweenileSandotherpolymorphicloci.Uponanalysisofasecond108

geneticallyandgeographicallydistinctcollectionofclinicalisolatesoftheUSA300109

lineagewedetectedthisepistasissignalagain,andherewecharacterisetheeffect110

thismutationhasonbothtoxinproductionandbacterialfitness.111

112

RESULTSandDISCUSSION113

EpistasisbetweenthemupirocinresistanceconferringmutationintheileSgene114

andotherlociisassociatedwiththetoxicityoftheUSA300MRSAlineage.Having115

previouslyidentifiedtoxicity-associated,epistaticinteractionsoccurringbetweenthe116

mupirocinresistance(mupR)conferringmutationintheileSgene(G1762T)andother117

lociwithinacollectionofST239MRSAisolates21,wesoughttodeterminewhether118




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thiswaslineagespecific.Weanalysedtoxicityandsequencedataforacollectionof119

130USA300MRSAisolates22,wheretheSNPconferringmupRresistanceemergedfor120

theUSA300isolatesasthemostdominanttoxicity-affectingepistatically-interacting121

locus(fig.1),demonstratingthewidespreadnatureofthiseffectacrossdiverse122

clonallineages.SeeSupp.Table1forthelistoflociassociatedwiththemupR123

conferringSNPforboththeST239andUSA300collections.124

Toillustratetheepistaticeffectontoxicityweselectedatrandomoneofthe125

interactinglociandpresentthemeantoxicityofthefourcombinationsofeachallele126

ofthetwogenes(Table1).TheSNPweselectedwasatposition480640(relativeto127

theoriginofreplication)andconfersanon-synonymouschangeintheprotein128

encodedbytheopen-readingframewiththelocustagSAUSA300_0426,whichis129

describedasaconservedhypotheticalprotein.WhenincombinationwiththemupR130

encodingSNPinileS,strainswiththealleleofSAUSA300_0426containingaTat131

position480640aresignificantlymoretoxicthanthosewithaCatthissite.Whereas132

inthemupirocinsensitivestrains,thosewiththeCatposition480640aremoretoxic133

thanthosewithaTatthissite(Table1).Thisexampleillustratesthetoxicity134

affectingepistaticsignaldetectedbyouranalysis.135

136

MupirocinresistanceexertsapleiotropiceffectontheS.aureusproteome.137

AcrossboththeST239andUSA300collectionsofisolates,59lociassociatedwiththe138

toxicityofS.aureusthroughepistaticinteractionswiththemupRmutation(Supp.139

Table1).Tounderstandhowsuchpotentialinteractionscouldaffecttoxicitywe140

examinedthislistofloci,however,noknowntoxicityaffectinggeneswere141

identified,andnolociwerecommonbetweenthetwoclones.AsIleRSisinvolvedin142

thetranslationofproteins,andourGWASdatasuggeststhisantibioticresistance143

conferringmutationalsoaffectstheabilityofS.aureustosecretetoxins,we144

hypothesisedthatthismutationmayhavepleiotropiceffectsonproteinproduction,145

whichcouldexplaintheobservedepistasis.Toexaminethis,weisolatedamupR146

versionoftheS.aureuslaboratorystrainSH1000byplatingovernightcultureson147

agarcontaining4µg/mlmupirocin.MupirocinresistantcoloniesoftheSH1000strain148

wererecovered,andtoconfirmthattheV588FconferringSNPwaspresentthe149

coloniesweresequencedandcomparedtoSH1000.Weselectedacolonywhichwe150




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havedesignatedMY40,wheretheonlynon-synonymousSNPfoundinthisstrain151

wasthatconferringtheV588FchangeinIleRS,althoughasmallnumberofnon-152

synonomousSNPdifferencesweredetected(Supp.Table2).153

154

WeperformedTandem-Mass-Tagging(TMT)proteinmassspectroscopy23onwhole155

celllysatesofthewildtypeSH1000andmupRstrainMY40.Theproteinswere156

extractedfromtriplicate18hrculturesinTryptoneSoyBroth(TSB),andofthe3026157

openreadingframespredictedfortheNCTC8325chromosome(whichistheclosest158

referencegenometoSH1000),thisproteomicapproachwasabletodetectand159

quantify1284proteins,whereweusedacut-offofaminimumofatwo-fold160

differenceinproteinabundancetoidentifydifferentiallyproducedproteins.When161

wecomparedtheproteomeofSH1000andMY40,therewere140proteinsthat162

weredifferentiallyproduced(Supp.Table3),whichverifiedourhypothesisthatthis163

resistancemutationhaspleiotropiceffects.However,whenwecomparedthislistof164

differentiallyproducedproteinswithourlistoflociassociatedwithtoxicitythrough165

epistasiswiththemupRmutation(Supp.Table1)therewasnooverlap,suggesting166

anytoxicityaffectinginteractionsbetweentheselocimustbeindirect.167

168

Exposuretomupirocinandmupirocinresistancehavecommoneffectsonprotein169

production.ToexaminewhetherthemupRmutationhasasimilareffectonIleRS170

activityasthepresenceofmupirocinhas,alongsideourproteomicanalysisofthe171

themupRmutantwealsoanalysedlysateofSH1000exposedtothehighest172

concentrationsofmupirocinforwhichwefoundnoinhibitionofgrowth(10ng/ml).173

Thisconcentrationwasselectedtoavoidanyconfoundingeffectsdifferencesin174

growthratesmighthaveontheproteome.ExposureofthewildtypeSH1000strain175

tomupirocinresultedindifferentialproductionof67proteinswhencomparedto176

theuntreatedSH1000(Supp.Table4),18ofwhichwerealsodifferentinthemupR177

mutant,suggestingmanycommondown-steameffectsofinterferingwiththe178

activityofIleRS.Theseincludeanincreaseintheproductionofseveralofthe179

proteinsinvolvedinIlebiosynthesis(i.e.LeuA,LeuCandIlvA),whichsuggeststhat180

thebacteriaarecompensatingfortheinterferenceinIleRSactivityduetothe181

mutationandexposuretotheantibiotic.Anothercommonexpressiondifference182




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wasthatseveraloftheiron-regulatedsurfacedeterminant(Isd)proteinswere183

expressedatlowerlevelsinboththemutantandthemupirocin-exposedwildtype184

whencomparedwiththeuntreatedwildtype.Asyetwehavenoexplanationforthis185

observation.186

187

Effectofmupirocinresistanceonvirulenceregulatingproteins.Thefirstvirulence188

relatedproteinabundancedifferencebetweentheSH1000anditsmupRmutantwe189

notedwastheAgrAprotein24.ThisisthecytoplasmicresponseregulatoroftheAgr190

toxicityregulatingsystemwhichwasproducedatsignificantlyhigherlevelsinthe191

mutant(7-fold,Supp.Table3).TheagrAandagrCgenesareco-transcribed,192

however,therewasnodifferenceinAgrCproteinabundanceacrosstheprotein193

preparations,whichsuggeststhattheeffectonAgrAabundancemustbepost-194

transcriptional.TheAgrAproteinneedstobephosphorylatedtobecome195

transcriptionallyactive,assuch,wereitactivewewouldexpecttoseeincreased196

transcriptionoftheRNAIIIeffectormoleculeoftheAgrsystem.Totestthiswe197

performedqRT-PCRonmRNAextractedfromboththeSH1000andmupRmutant,198

wherewefoundnodifferenceinRNAIIIexpression(n=6,two-tailedt-test,p=0.49),199

whichsuggeststhatalthoughAgrAmaybemoreabundantinthemutantitdoesnot200

seemtobetranscriptionallyactive.201

202

Otherknownvirulenceregulatorswerealsoidentifiedasbeingdifferentially203

producedinthemupirocinresistantstrain.BoththeSrrBandSarRproteins24were204

expressedathigherlevelsinthemutant,whereastheRotprotein24wasexpressedat205

lowerlevelscomparedtothewildtypestrains.Threeotherknownregulatory206

proteinswerealsodifferentiallyexpressed(i.e.aGntRfamilytranscriptional207

regulator,thelyticregulatoryproteinSAOUHSC_02390,andthePuroperon208

regulator,PurR),whichwhenconsideredalongsidetheeffectonthethreevirulence209

regulatorssuggeststhatmupirocinresistanceresultsinasignificantre-wiringofS.210

aureusregulatoryprocesses.211

212

Effectofmupirocinresistanceontoxinproduction.Ofthetoxinsencodedonthe213

SH1000genome,therewassignificantlymoreofbothdeltatoxinandPSMa1inthe214




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lysateofmupirocinresistantmutant(32-and21-foldrespectively,Supp.Table3).215

ThesearetwoofthemostabundantlyproducedmembersofthePhenolSoluble216

Modulin(PSM)familyofcytolytictoxins25.AsouroriginalGWASanalysiswas217

focusedontoxicity,wehypothesisedthatthesedifferencesmayexplainthe218

associationweobserved,anditsuggeststhatthemutantshouldbemoretoxicthan219

thewildtypestrain.Totestthiswequantifiedthecytolyticactivityofthewildtype220

andmutantbyincubatingbacterialsupernatantwithculturedTHP-1cells,whichare221

sensitivetothemajorityofcytolytictoxinsproducedbyS.aureus,includingthe222

PSMs22.Whilewefoundthatmupirocinresistancedidinfluencedtoxicity,theeffect223

wastheoppositetowhatourproteomicanalysissuggesteditshouldbe,inthatthe224

mupirocinresistantMY40strainkilledonly71%ofthecellscomparedtothewild225

typemupirocinsensitiveSH1000strainwhichkilled87%ofthecells(n=6,twotailed226

t-test;p=0.023).Asourtoxicityassaysusesbacterialsupernatant,whereasour227

proteomicanalysiswasonwholecelllysates,itispossiblethatthedifferencein228

abundanceofthetoxinsisnotequivalentbetweentheintra-andextra-cellular229

environments.ToexaminethiswequantifiedtheabundanceofPSMsinthebacterial230

supernatantswherewefoundthePSMstobe3.4-foldlessabundantinthe231

supernatantofthemupRmutant(n=6,twotailedt-test;p=0.001,arepresentative232

imageofispresentedinfig.2a),whichexplainsourcytolyticresults.233

234

MupirocinresistanceaffectstheactivityofthePSMsecretoryapparatus,Pmt.As235

thePSMsaremoreabundantintheintracellularenvironmentofthemupRmutant,236

butlessabundantintheextracellularenvironment,wehypothesisedthatPSM237

secretionmaybeaffectedinthemutant.ThisisfacilitatedbytheactivityofthePSM238

exportsystem,Pmt26,andalthoughthepmtgeneshavenotbeenannotatedonthe239

NCTC8325genome,theyarepresent(locustagsSAOUHSC_02155,SAOUHSC_02154,240

SAOUHSC_02153,SAOUHSC_02152,SAOUHSC_02151).Thelocusencodesa241

regulatoryprotein,twomembraneboundABCtransporterproteinsandtwoATP242

bindingproteinsthatfueltheexportsystem,wherethefourstructuralgenesare243

transcribedfromasinglepromoter.EachATPbindingproteininteractswithits244

pairedtransporterproteinandthemaintainedselectionofthe1:1:1:1stoichiometry245

suggestsitiscriticaltotheexportersactivity.InourmupRmutantwefoundthat246




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therewasmorethantwiceasmuchofoneofthetwoATPbindingproteins247

(SAOUHSC_02152,genenamepmtC)comparedwiththewildtypestrain.We248

hypothesisedthatinterferencewiththestoichiometryoftheproteinsinthisexport249

systemmayexplainwhywehavemorePSMinsidethecellbutlessoutside.Totest250

thisweclonedandexpressedthepmtCgene,fromaninduciblepromoterinthe251

SH1000wildtypebackground,andfoundthattherewas2.8-foldlessPSMsinthe252

extracellularenvironmentwhenthisATPasewasoverexpressed(n=6,twotailedt-253

test;p=0.003,arepresentativeimageofispresentedinfig.2b).Thissuggeststhat254

theeffectmupirocinresistancehasonS.aureustoxicityismediatedbyinterfering255

withtheactivityofthePSMsecretoryapparatus.Itisinterestingtoconsiderthatthe256

completeinactivationofthePmtsystemhasbeenshowntobelethaltothebacteria,257

presumablyasaresultofthedamagethePSMscancausetointernalmembrane258

structures26.WhilewedemonstrateapartialblockingofthePmtsystemby259

mupirocinresistance,andsomeaccumulationofPSMsinternally,itmustbeasub-260

toxiclevel,asweseenoinvitrogrowthdefectsassociatedwiththis.261

262

ReducingtheproductionoftoxinsalleviatesthefitnesscostofmupR.Withone263

consequenceofthesignificantchangesmupRcausestotheproteomeofS.aureus264

beingareductionintoxinproduction,andwithtoxinproductionandsecretionbeing265

anenergeticallycostlyactivity,wehypothesizedthatthedownregulatoryeffectof266

mupirocinresistanceontoxinproductionmaymaskoralleviatetheresistance267

relatedfitnesscoststhatareincurred.Totestthiswequantifiedtherelativefitness27268

ofthemupSandmupRstrainsbycompetitionintwogeneticbackgrounds;oneinthe269

SH1000wild-typebackground(i.e.SH1000andMY40)wherethebacteriacan270

expresstoxins.Fortheother,weinactivatedtheAgrquorumsensingsysteminboth271

SH1000andMY40bytransducingintheinactivatedAgrsystem(anerythromycin272

resistancehasbeeninsertedintoit)fromthestrainROJ4828,resultinginstrains273

MY18andMY41.Asreportedpreviously,inthewildtypebackgroundwitha274

functionalAgrsystem,therewasnodifferenceinfitnessbetweenthemupSSH1000275

andthemupRMY40strains(fig.3A;n=10;two-tailedt-testp=0.6).However,when276

wequantifiedtherelativefitnessintheAgrdefectivebackgroundwhereneither277

straincouldproducetoxins,suchthatanyalleviationoffitnesscoststhatresultfrom278




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therelativereductionintoxicityofthemupRmutantwasnullified,wefoundthe279

fitnessofthemupRMY41straintobesignificantlylowerthanthemupSMY18strain280

(fig.3A;n=10;two-tailedt-testp=0.04).Thisdemonstratesthatthismupirocin281

resistanceconferringmutationdoesincurafitnesscost,butthiscostcanbemasked282

byreducingthecostlyproductionoftoxins.283

284

Astheabilitytoproducetoxinsisselectedforinenvironmentssuchasthenose29,285

wehypothesisedthatinsuchanenvironment,thecostsassociatedwiththis286

resistancemechanismshouldbecomeapparent,withouthavingtogenetically287

manipulatetheAgrsystem.Totestthiswedevelopedagrowthmediawithverylow288

levelsofnutrients(0.1XTSB)butadded5%horsebloodsuchthatstrainsthatcan289

producetoxinscanreleaseandutilisefurthernutrientsfromthesebloodcellsfor290

growth.Toavoidthelesstoxiccellsbenefitingfromnutrientsthemoretoxiccells291

release,wequantifiedthegrowthrateofSH1000anditsmupRmutantseparately,292

quantifyingtheirMalthusiansparametersat3,6and24hoursinthismedium.After293

3hrofincubationinthemediumtherewasnodetectablegrowthofeitherstrain,294

however,by6hrsbothbacterialstrainshadgrown,althoughthemupRstrainwas295

significantlymorefitthantheSH1000strain(fig.3B;n=6;two-tailedt-testp=0.007).296

Thissuggeststhebacteriaenteralonglagphaseinthismedium,butonceadapted297

(after3hrs)therewassufficientnutrientsavailabletosustainsomegrowth,withthe298

SH1000strainatanapparentdisadvantage,presumablybyexpendingenergyon299

producingmoretoxinsthanitsmupRmutant.Afterthe6hrtime-pointtherewasa300

distinctshiftintherelativefitnessofthestrains,withtheSH1000strainbecoming301

relativelymorefitthanthemupRstrain(fig.3B;n=6;two-tailedt-testp=0.04).The302

increasedrelativegrowthrateofthewildtypeSH1000strainispresumablyasa303

resultofitsincreasedcapabilitytolysecellsandreleasenutrientsnecessaryforits304

growth.Whiletherelativefitnessofanyorganismisdependentuponits305

environment,herewedemonstratehowreadilythiscanfluctuatewithinan306

environment.Thatthefitnessconsequencesofmupirocinresistancebecame307

apparentwhencelllysisbecamenecessarymayprovideanexplanationforwhy,308

despitetheeaseatwhichthismutationcanoccur,itisnotmoreprevalentandis309

readilylostwithinhealthycommunities20.310




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311

ThemupRmutationhasdifferingeffectsontoxicityindifferentS.aureus312

backgrounds.Ourproteomicanalysisdidnotprovideanyevidenceinsupportofa313

directinteractionoccurringbetweenthemupRileSgeneandthelociourGWAS314

analysishasidentifiedasinteractingwiththislocustoaffecttoxicity(fig.1andSupp.315

Table1).AsGWASinbacteriacanbeconfoundedbypopulationstructuresand316

linkagedisequilibrium,itisthereforepossiblethatthesepolymorphiclociareinstead317

reflectiveofthegeneticbackgroundsofthebacteriathataresensitivetotheeffect318

ofthemupRmutationtovaryingdegrees.Iftrue,thentheintroductionofthemupR319

mutationintodifferentS.aureusstrainsshouldhavedifferingeffectsontoxicity.To320

testthisweisolatedmupRcoloniesoftwotemporallyandgeographicallydiverse321

isolatesfromthesamecloneasSH1000(ST8asdeterminedbyMLST),inthe322

laboratorystrainRN6390BandintheUSA300clinicalisolateUSFL34.Thepresence323

oftheV588Fmutationwasconfirmedbysequencingandtheeffectofthemutation324

ontoxicityquantified.DespitereducingthetoxicityoftheSH1000backgroundstrain,325

themupRmutationhadnoeffectontoxicityineitherRN6390BorUSFL34(n=6;two-326

tailedt-testp=0.59and0.12respectively),supportingourhypothesisthatstrains327

responddifferentlytothisantibioticresistantmechanism,andprovidingan328

explanationforourepistasisfindings.329

330

CONCLUSION331

Understandingtheevolutionofantibioticresistance,bothintermsofhowit332

emergesandhowitbecomesstablymaintained,iscriticalifidentifyingmeansof333

curtailingitsfurtheremergencearetobedeveloped.Withsomeantibioticresistance334

mechanismsbeingreportedasincurringnofitnesscosts4-6,andotherswhere335

physiologicalandregulatorycompensationofcostshavebeendemonstrated30-32,it336

isperhapssurprisingthattheyhavenotbecomemoreprevalent.Here,byadoptinga337

population-basedfunctionalgenomicsapproach,weuncoverhiddenfitnesscosts338

associatedwithanapparentlycost-free,antibioticresistancemechanism.We339

demonstratethatthemupirocin-resistanceconferringmutationofthegene340

encodingtheIleRSenzymehaspleiotropiceffectsonbacteria,whichinhindsightis341

perhapsunsurprisinggiventhehighlyconservedandessentialnatureofthisgene.In342




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thisinstance,whilereducingtheenergeticexpenseassociatedwithtoxinproduction343

providesareliefoftheresistance-associatedfitnesscosts,thisappearstobe344

unsustainablewhentheabilitytoproducetoxinsisrequired.Giventhatthenoseof345

healthycarriershasbeenshowntobesuchatoxicity-dependentenvironmentforS.346

aureus,thistoxicityrelatedfitnessoff-settingmayexplainwhythisresistance347

mechanismisnotmoreprevalent,andisreadilydisplaced,oncethisantibiotichas348

beenremovedfromtheirenvironment.Aneffectwewouldhavebeenunableto349

explainhadwenotadoptedapopulation-basedapproachtostudyingthismajor350

bacterialpathogen.351

352 353




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MATERIALSANDMETHODS354

Strainsandgrowthconditions355

AllstrainsusedinthisstudyarelistedinSupp.Table5.S.aureusstrainsweregrown356

at37oC,ineithertrypticsoyagarorbroth(TSA/TSB)withtheappropriateantibiotic357

wherenecessary.TheE.coliTOP10straincontainingthepAgrC(his)Aplasmidwas358

growninLuria-Bertani(LB)mediawith100µg/mlampicillin.THP-1cellsweregrown359

inRPMI1640supplementedwithfoetalbovineserum(10%),L-glutamine(2mM),360

penicillin(100units/ml)andstreptomycin(100µg/ml)andincubatedat37oCwith5%361

CO2.362

363

Selectionofthemupirocinresistantstrain.364

AsanessentialgeneileScannotbeinactivated,andasaconsequence,ahomologous365

recombinationbasedmutationalapproachwereunsuccessful.Therefore,to366

generateisogenicmupirocinresistantandsensitivestrainsweutilisedaselection367

basedmethodwhereanovernightcultureofamupirocinsensitivestrain(e.g368

SH1000wasplatedontoagarplateswith4µg/mlmupirocin.Thiswasincubatedat369

37oCfor48hrandcoloniesthatgrewwerefurtherisolatedbystreakingontofresh370

mupirocinplates.371

372

GenomesequencingofthemupRstrain.373

S. aureus strainMY40was sequenced in this study; DNAwas extracted using the374

QIAampDNAMiniKit(QIAGEN,Crawley,UK),usingmanufacturer’sinstructionswith375

1.5μg/μL lysostaphin(AmbiProductsLLC,NY,USA)tofacilitatecell lysis.DNAwas376

quantifiedusingaNanodropspectrophotometer,aswellastheQuant-iTDNAAssay377

Kit (Life Technologies, Paisley, UK) before sequencing. High-throughput genome378

sequencingwas performed using aMiSeqmachine (Illumina, San Diego, CA, USA)379

and the short read, paired-end data was assembled using the de novo assembly380

algorithmSPAdes(Bankevichetal(SPADES).SequencedataarearchivedintheNCBI381

repositories: GenBank Accession: SUB2754769, Short Read Archive (SRA):382

SRR5651527, associated with BioProject: PRJNA384009. Assembled genomes are383

alsoavailableonFigShare(doi.org/10.6084/m9.figshare.5089939.v1).384

385




14

Toxicityassays386

THP-1cells33weregrownasdescribedaboveandharvestedbycentrifugationand387

washedinPBSanddilutedtoafinaldensity(determinedbyhaemocytometer)of388

2x106cellspermlofPBS.Bacterialsupernatantwasharvestedafter18hrsofgrowth389

inTSBat37oC.20µlofthebacterialsupernatantwasmixedwith20µlofTHP-1cells,390

andincubatedfor12minsat37oC.260µlofGuavaViaCount(Milipore)wasaddedto391

thesample,andincubatedatroomtemperaturefor5minsbeforeanalysingthe392

viabilityontheGuavaflowcytometer(Milipore).393

394

Fitnessassays.395

Thestrainswereculturedindividuallyovernightanddilutedto104cfu/ml.Forthe396

directcompetitions25µlofeachdilutedculturewasaddedinto5mlfreshTSB,and397

grownat37°Cwithshakingfor24h.Themixedculturewasdilutedandplatedonto398

agarplateswithandwithout4µg/mlmupirocinandincubatedat37°C.Theresulting399

colonieswerecounted,andthenumberofcoloniesfromthemupirocinplatewas400

subtractedfromthecountfromnoantibioticplate.TheMalthusianparameterwas401

calculatedusingthefollowingformula:402

Ln(finaldensity(colonyformingunits(CFU)/ml)/startingdensity(CFU/ml))403

TheMalthusianparametersofthemupSandmupRstrainswerecomparedusinga404

twotailedt-test.Fortheindividualfitnessassaysthestrainswerecultured405

individuallyovernightanddilutedandaddedto0.1XTSBmadewithphosphate406

bufferedsalineratherthanwatertomaintaintheintegrityofthehorsebloodcellsto407

whichand5%horseblood(Oxoid)wasadded.Bacterialgrowthwasdeterminedby408

platingtheculturesonTSAplatesafter3,6and24hrsofincubationat37oCinair.409

410

DeletionoftheagrlocusfromSH1000andMY40.411

PhagetransductionwasusedtoconstructthemupRandmupSAgrmutants.IntheS.412

aureusstrainROJ48,theentireAgrlocushasbeenreplacedwithanerythromycin413

resistancecassette28.ThiswasmovedfromROJ48intoSH1000byphage414

transductionasfollows:ROJ48ϕ11lysateswerepreparedfrom200µlofovernight415

ROJ48cultureinLK(1%Tryptone,0.5%yeastextract,1.6%KCl)whichwasaddedto416

3mloffreshLKand3mlofphagebuffer(10mMMgSO4,4mMCaCl2,50mMTris-HCl417




15

pH7.8,100mMNaCland0.1%gelatinepowderinmolecular/MiliQwater),andto418

this500µlϕ11-RN6390Blysatewasadded.Thiswasincubatedat30oCshakinguntil419

themediabecameclear,whichindicatedbacteriallysis.Thelysateswerethenfilter420

sterilised,andasecondroundoflysiswascarriedoutonROJ48withthisfirstround421

lysate.Afterthesetwolysissteps,transductionintoSH1000andMY40strainswas422

performedbyadding200µlofovernightcultureto1.8mlLKwith10µl1MCaCl2,and423

500µltheϕ11-ROJ48lysate.Thiswasincubatedat37oCwithshakingfor45min,424

then1mlicecold20mMtrisodiumcitratewasaddedandthetransducingmixture425

wasplacedonicefor5min.Thebacteriawereharvestedbycentrifugationandre-426

suspendedwith1mlicecold20mMtrisodiumcitrate.Thiswasincubatedonicefor427

2.5h,andplatedontoTSAplateswith20mMtrisodiumcitrate,erythromycinand428

lincomycin(25µg/ml)whichwasincubatedovernightat37oC.429

430

qRT-PCR431

Overnightculturesofwerediluted1:500into3mlfreshTSB-chloramphenicol.After432

18hrofgrowth2mlofthisculturewasmixedwith4mlRNAProtectBacteria433

(Qiagen),andtheRNeasyMiniKit(Qiagen)wasusedtoextractRNAfollowingthe434

manufacturer’sprotocol.Lysostaphin(200µg/ml)wasaddedtoTris-EDTAbuffer435

(Ambion),andthiswasaddedtothesampleaftertheRNAProtectstepbefore436

continuingwiththeprotocol.WhentheRNAwasextracted,TurboDNA-freekit437

(Thermo)wasusedtoremovegenomicDNAfromtheRNAsamples;3µlTurboDNase438

wasaddedtothesampleandincubatedfor1.5hat37oC,theafurther4µlTurbo439

DNasewasaddedandincubatedfor1.5h.35µlDNaseinactivationreagentwas440

addedtothesamplestoinactivatetheDNaseaccordingtotheprotocol.The441

concentrationofRNAinthesamplesweremeasuredusingQubitRNABroadRange442

kit(Thermo)andnormalisedbeforeusingQuantiTectReverseTranscriptionKit443

(Qiagen)toconverttheRNAsamplesintocDNAaccordingtothemanufacturer’s444

protocol.Afteraddingthereversetranscriptase,thesampleswereincubatedat42oC445

for20minbeforeraisingthetemperatureto95oCfor3minstoinactivatethe446

reversetranscriptase.PrimersforgyrB,ahousekeepinggene,wasusedalongside447

thoseforRNAIIItostandardisetranscriptlevels(gyrBforward:448

CCAGGTAAATTAGCCGATTGC,gyrBreverse:AAATCGCCTGCGTTCTAGAG.RNAIII449




16

forward;AGCATGTAAGCTATCGTAAACAAC,RNAIIIreverse;450

TTCAATCTATTTTTGGGGATG).ssoAdvancedSYBRGreenSupermix(Bio-Rad)was451

used,usingastandardcurveofknowngenomicDNAconcentrationsforeachprimer452

set.5µlofsamples,standardsandwaterwerepipettedintothewellsofa96-well453

PCRplate.Thesupermixwasaddedtowaterandprimersaccordingtothe454

manufacturer’sprotocol,and15µlofthismixwaspipettedovertheDNAsamples.455

ThiswasthenplacedintoaqPCRmachine,andrunusingthemanufacturer’s456

recommendation.ThequantityofRNAIIIcDNAwasdividedbythequantityofgyrB457

cDNAtogetaratioofRNAIIItranscriptionlevels.458

459

PSMquantificationinsupernatants.460

AnovernightcultureofSH1000andMY40wasdiluted1:1000into50mlfreshTSB,461

andgrownfor18h.Thecultureswerecentrifugedat18,000rpmfor10min,and462

35mlofthesupernatantwasmixedwith10mlbutanol.Thesampleswereincubated463

at37°Cshakingfor3h,andwerethencentrifugedat3,000rpmfor3minand1mlof464

theupperorganiclayerwastakenoff.Thesampleswerethenfreeze-driedovernight465

andthenre-suspendedin160µl8Murea,separatedonanSDS-PAGEgelandPSMs466

quantifiedbydensitometryanalysisusingtheImageJsoftware.467

468

TMTLabellingandHighpHreversed-phasechromatography469

Aliquotsof100µgofuptotensamplesperexperimentweredigestedwithtrypsin470

(2.5µgtrypsinper100µgprotein;37°C,overnight),labelledwithTandemMassTag471

(TMT)tenplexreagentsaccordingtothemanufacturer’sprotocol(ThermoFisher472

Scientific,)andthelabelledsamplespooled.Analiquotofthepooledsamplewas473

evaporatedtodrynessandresuspendedinbufferA(20mMammoniumhydroxide,474

pH10)priortofractionationbyhighpHreversed-phasechromatographyusingan475

Ultimate3000liquidchromatographysystem(ThermoFisherScientific).Inbrief,the476

samplewasloadedontoanXBridgeBEHC18Column(130Å,3.5µm,2.1mmX150477

mm,Waters,UK)inbufferAandpeptideselutedwithanincreasinggradientof478

bufferB(20mMAmmoniumHydroxideinacetonitrile,pH10)from0-95%over60479

minutes.Theresultingfractionswereevaporatedtodrynessandresuspendedin1%480




17

formicacidpriortoanalysisbynano-LCMSMSusinganOrbitrapFusionTribridmass481

spectrometer(ThermoScientific).482

483

Nano-LCMassSpectrometry484

HighpHRPfractionswerefurtherfractionatedusinganUltimate3000nanoHPLC485

systeminlinewithanOrbitrapFusionTribridmassspectrometer(ThermoScientific).486

Inbrief,peptidesin1%(vol/vol)formicacidwereinjectedontoanAcclaimPepMap487

C18nano-trapcolumn(ThermoScientific).Afterwashingwith0.5%(vol/vol)488

acetonitrile0.1%(vol/vol)formicacidpeptideswereresolvedona250mm×75μm489

AcclaimPepMapC18reversephaseanalyticalcolumn(ThermoScientific)overa150490

minorganicgradient,using7gradientsegments(1-6%solventBover1min.,6-15%491

Bover58min.,15-32%Bover58min.,32-40%Bover5min.,40-90%Bover1min.,held492

at90%Bfor6minandthenreducedto1%Bover1min.)withaflowrateof300nl493

min−1.SolventAwas0.1%formicacidandSolventBwasaqueous80%acetonitrile494

in0.1%formicacid.Peptideswereionizedbynano-electrosprayionizationat2.0kV495

usingastainlesssteelemitterwithaninternaldiameterof30μm(ThermoScientific)496

andacapillarytemperatureof275°C.497

AllspectrawereacquiredusinganOrbitrapFusionTribridmassspectrometer498

controlledbyXcalibur2.0software(ThermoScientific)andoperatedindata-499

dependentacquisitionmodeusinganSPS-MS3workflow.FTMS1spectrawere500

collectedataresolutionof120000,withanautomaticgaincontrol(AGC)targetof501

200000andamaxinjectiontimeof50ms.Precursorswerefilteredwithanintensity502

thresholdof5000,accordingtochargestate(toincludechargestates2-7)andwith503

monoisotopicprecursorselection.Previouslyinterrogatedprecursorswereexcluded504

usingadynamicwindow(60s+/-10ppm).TheMS2precursorswereisolatedwitha505

quadrupolemassfiltersettoawidthof1.2m/z.ITMS2spectrawerecollectedwith506

anAGCtargetof10000,maxinjectiontimeof70msandCIDcollisionenergyof35%.507

ForFTMS3analysis,theOrbitrapwasoperatedat50000resolutionwithanAGC508

targetof50000andamaxinjectiontimeof105ms.Precursorswerefragmentedby509

highenergycollisiondissociation(HCD)atanormalisedcollisionenergyof60%to510

ensuremaximalTMTreporterionyield.SynchronousPrecursorSelection(SPS)was511

enabledtoincludeupto5MS2fragmentionsintheFTMS3scan.512




18

513

Proteomicdataanalysis514

TherawdatafileswereprocessedandquantifiedusingProteomeDiscoverer515

softwarev2.1(ThermoScientific)andsearchedagainsttheUniProtStaphylococcus516

aureusstrainNCTC8325databaseusingtheSEQUESTalgorithm.Peptideprecursor517

masstolerancewassetat10ppm,andMS/MStolerancewassetat0.6Da.Search518

criteriaincludedoxidationofmethionine(+15.9949)asavariablemodificationand519

carbamidomethylationofcysteine(+57.0214)andtheadditionoftheTMTmasstag520

(+229.163)topeptideN-terminiandlysineasfixedmodifications.Searcheswere521

performedwithfulltrypticdigestionandamaximumof2missedcleavageswere522

allowed.Thereversedatabasesearchoptionwasenabledandallpeptidedatawas523

filteredtosatisfyfalsediscoveryrate(FDR)of5%.524

525 526 REFERENCES527 1. MouradeSousa,J.,Balbontín,R.,Durão,P.&Gordo,I.Multidrug-resistant528 bacteriacompensatefortheepistasisbetweenresistances.PLOSBiol.15,e2001741529 (2017).530 2. Tenover,F.C.MechanismsofAntimicrobialResistanceinBacteria.Am.J.531 Med.119,S3–S10(2006).532 3. Levin,B.R.,Perrot,V.&Walker,N.Compensatorymutations,antibiotic533 resistanceandthepopulationgeneticsofadaptiveevolutioninbacteria.Genetics534 154,985–997(2000).535 4. Hurdle,J.G.,O’Neil,A.J.&Chopra,I.Theisoleucyl-tRNAsynthetasemutation536 V588Fconferringmupirocinresistanceinglycopeptide-intermediateStaphylococcus537 aureusisnotassociatedwithasignificantfitnessburden.J.Antimicrob.Chemother.538 53,102–104(2003).539 5. Stanczak-Mrozek,K.I.etal.Within-hostdiversityofMRSAantimicrobial540 resistances.J.Antimicrob.Chemother.70,2191–2198(2015).541 6. Knight,G.M.,Budd,E.L.&Lindsay,J.A.Largemobilegeneticelements542 carryingresistancegenesthatdonotconferafitnessburdeninhealthcare-543 associatedmeticillin-resistantStaphylococcusaureus.Microbiol.(UnitedKingdom)544 159,1661–1672(2013).545 7. Rasigade,J.-P.&Vandenesch,F.Staphylococcusaureus:apathogenwithstill546 unresolvedissues.Infect.Genet.Evol.21,510–4(2014).547 8. Lowy,F.Antimicrobialresistance:theexampleofStaphylococcusaureus.J.548 Clin.Invest.111,1265–1273(2003).549 9. Wertheim,H.F.etal.TheroleofnasalcarriageinStaphylococcusaureus550 infections.LancetInfect.Dis.5,751–762(2005).551 10. Gordon,R.J.&Lowy,F.D.PathogenesisofMethicillin‐Resistant552 StaphylococcusaureusInfection.Clin.Infect.Dis.46,S350–S359(2008).553




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11. Abad,C.L.,Pulia,M.S.&Safdar,N.DoestheNoseKnow?AnUpdateon554 MRSADecolonizationStrategies.Curr.Infect.Dis.Rep.15,455–464(2013).555 12. Thomas,C.M.,Hothersall,J.,Willis,C.L.&Simpson,T.J.Resistancetoand556 synthesisoftheantibioticmupirocin.Nat.Rev.Microbiol.8,281–289(2010).557 13. Simor,A.E.Staphylococcaldecolonisation:Aneffectivestrategyfor558 preventionofinfection?LancetInfect.Dis.11,952–962(2011).559 14. BertinoJr,J.S.Intranasalmupirocinforoutbreaksofmethicillin-resistant560 Staphylococcusaureus.AmJHeal.Pharm54,2185–2191(1997).561 15. Antonio,M.,McFerran,N.&Pallen,M.J.MutationsaffectingtheRossman562 foldofisoleucyl-tRNAsynthetasearecorrelatedwithlow-levelmupirocinresistance563 inStaphylococcusaureus.Antimicrob.AgentsChemother.46,438–442(2002).564 16. Patel,J.B.,Gorwitz,R.J.&Jernigan,J.A.MupirocinResistance.Clin.Infect.565 Dis.49,935–941(2009).566 17. Udo,E.E.&Sarkhoo,E.Geneticanalysisofhigh-levelmupirocinresistancein567 theST80cloneofcommunity-associatedmeticillin-resistantStaphylococcusaureus.568 J.Med.Microbiol.59,193–199(2010).569 18. Seah,C.etal.MupB,aNewHigh-LevelMupirocinResistanceMechanismin570 Staphylococcusaureus.Antimicrob.AgentsChemother.56,1916–1920(2012).571 19. Hetem,D.J.&Bonten,M.J.M.Clinicalrelevanceofmupirocinresistancein572 Staphylococcusaureus.J.Hosp.Infect.85,249–256(2013).573 20. WilliamsonD.A.,CarterG.P.&HowdenB.P.CurrentandEmergingTopical574 AntibacterialsandAntiseptics:Agents,Action,andResistancePatterns.Clin.575 Microbiol.Rev.30,827-860(2017).576 21. Laabei,M.etal.PredictingthevirulenceofMRSAfromitsgenomesequence.577 GenomeRes.24,839–849(2014).578 22. Laabeietal.EvolutionaryTrade-OffsUnderlietheMulti-facetedVirulenceof579 Staphylococcusaureus.PLoSBiol.13(9):e1002229(2015).580 23. DayonL&JCSanchez.RelativeproteinquantificationbyMS/MSusingthe581 tandemmasstagtechnology.MethodsMolBiol.893:115-27(2012).582 24. Bronner,S.,Monteil,H.&Prévost,G.Regulationofvirulencedeterminantsin583 Staphylococcusaureus :complexityandapplications.FEMSMicrobiol.Rev.28,183–584 200(2004).585 25. Qi,R.etal.IncreasedInVitroPhenol-SolubleModulinProductionis586 AssociatedwithSoftTissueInfectionSourceinClinicalIsolatesofMethicillin-587 SusceptibleStaphylococcusaureus.J.Infect.72,302–308(2017).588 26. Chatterjee,S.S.etal.EssentialStaphylococcusaureustoxinexportsystem.589 Nat.Med.19,364–367(2013).590 27. Lenski,R.E.,Rose,M.R.,Simpson,S.C.&Tadler,S.C.Long-Term591 ExperimentalEvolutioninEscherichiacoli.I.AdaptationandDivergenceDuring592 2,000Generations.Am.Nat.138,1315–1341(1991).593 28. Jensen,R.O.,Winzer,K.,Clarke,S.R.,Chan,W.C.&Williams,P.Differential594 RecognitionofStaphylococcusaureusQuorum-SensingSignalsDependsonBoth595 ExtracellularLoops1and2oftheTransmembraneSensorAgrC.J.Mol.Biol.381,596 300–309(2008). 597 29.ReckerM,etal.ClonaldifferencesinStaphylococcusaureusbacteraemia-598 associatedmortality.NatMicrobiol.2,1381-1388(2017).599




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30. Pacheco,JO,Alvarez-Ortega,C,Rico,MA&Martínez,JL.Metabolic600 CompensationofFitnessCostsIsaGeneralOutcomeforAntibiotic-Resistant601 PseudomonasaeruginosaMutantsOverexpressingEffluxPumps.mBio.8,e00500-17602 (2017).603 31. FreihoferP,etal.Nonmutationalcompensationofthefitnesscostof604 antibioticresistanceinmycobacteriabyoverexpressionoftlyArRNAmethylase. 605 RNA.22,1836-1843(2016).606 32. Collins,Jetal.OffsettingvirulenceandantibioticresistancecostsbyMRSA.607 ISMEJ.4,577-84(2010).608 33. Tsuchiya,S.etal.Establishmentandcharacterizationofahumanacute609 monocyticleukemiacellline(THP‐1).Int.J.Cancer26,171–176(1980).610 611 612 613 614




21

615 FIGURES616

617

618 Fig.1:EpistasisbetweenthemupirocinresistanceencodingmutationintheileSgene619

andmanyotherlociisassociatedwiththetoxicityoftheUSA300lineageofMRSA.620

Thisheatmapillustrateswherespecificcombinationsofthepolymorphicsiteinthe621

ileSgeneandpolymorphicsiteselsewhereonthechromosomeareassociatedwith622

thetoxicityofindividualisolates.ThemupRconferringsiteisindicatedontheXandY623

axisbytheredarrow.624

625

626

627

0

500000

1e+006

1.5e+006

2e+006

2.5e+006

3e+006

0 500000 1e+006 1.5e+006 2e+006 2.5e+006 3e+006

2

3

4

5

6

7

-log 1

0(p)

SNP1 (position)

SNP2

(pos

ition

)

0.5Mbp 1Mbp 1.5Mbp 2.5Mbp2Mbp 3MbpSNPposition

(relativetotheoriginofreplication)

0.5Mbp

1Mbp

1.5Mbp

2.5Mbp

2Mbp

3Mbp

SNPposition

(relativ

etotheoriginofreplication)




22

628 629 Fig.2:PSMabundanceintheS.aureussupernatant.Thebacterialstrainswere630 growninTSBfor18hrs.ThePSMswereharvestedfromthebacterialsupernatantby631 butanolextractionandrunonanSDS-PAGEgel.a:ThereissignificantlylessPSMin632 thesupernatantofthemupirocinresistantS.aureusstraincomparedtothewild633 typemupirocinsensitivestrain.b:Over-expressionofthepmtCgene,whichencodes634 oneoftheATPbindingproteinsofthePSMsecretorysystem,Pmt,inthewildtype635 SH1000straincausesareductionintheabundanceofthePSMintheS.aureus636 supernatant.AnAgrmutanthasbeenprovideasacontrol.AfulllengthSDS-PAGE637 gelhasbeenprovidedinSupplementarymaterial(Supp.Fig.1),toillustratewhywe638 onlyprovidea‘letter-box’snap-shotofthegelshere.639 640 641 642

mupS mupR

23

643 644 Fig.3:MupirocinresistanceaffecttherelativefitnessofS.aureus.a:theeffectof645 mupirocinresistanceontherelativefitnessofS.aureuswasdeterminedinstrains646 withandwithoutafunctioningAgrsystembydirectcompetitioninTSB.Therewas647 nodifferenceinfitnessintheAgr+vebackground,butintheabsenceofAgr,the648 mupirocinresistantstrainwaslessfitthatthemupirocinsensitivestrain.b:The649 effectofmupirocinresistanceonrelativefitnesswasdeterminedbyindividual650 cultureinanutrientpoorenvironment(0.1XTSB)supplementedwith5%horse651 blood.Attheearlystagesofgrowth(0-6hrs)themupirocinsensitivestrainwasmore652 fit,whereasbetween6and24hrwhenthenutrientsintheTSBweredepletedand653 celllysisbecamenecessary,themupirocinsensitivestrainswasrelativelymorefit.654 655

TSB(1X) TSB(0.1X)+5%horseblood

MupSagr +ve

MupSagr -ve

MupRagr +ve

MupRagr -ve

MupS0-6hrs

MupR0-6hrs

MupS6-24hrs

MupR6-24hrs

P=0.6

P=0.04

P=0.007

P=0.04

a b




24

656 657 TABLES658

Table1:Toxicityaffectingepistaticinteractions.AnexampleofaSNPassociatedby659

ourepistasisanalysistobeinteractingwiththemupirocinresistanceconferringSNP660

toaffecttoxicity.Themeantoxicityofeachofthefourcombinationsofthefour661

allelesispresented+/-the95%confidenceintervals.662

PercentageCelldeath(+/-95%confidence)

SAUSA300_0426(T) SAUSA300_0426(C)

Mupirocinresistant 45.6%(+/-5.7) 4.6%(+/-2.8)Mupirocinsensitive 20.65%(+/-4) 53%(+/-9.2)

663

664



hidden antibiotic resistance fitness costs revealed by ...dec 09, 2017 · 3 55 introduction 56...

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