1

AntimicrobialResistanceinMycobacteriumtuberculosis:theOddOneOut1

2

VegardEldholm1andFrançoisBalloux23

4

Correspondance: VegardEldholmv.eldholm@gmail.com5

FrançoisBallouxf.balloux@ucl.ac.uk6

7

1.DivisionofInfectiousDiseaseControl,NorwegianInstituteofPublicHealth,Lovisenberggata8

8,Oslo0456,Norway9

2.DepartmentofGenetics,EvolutionandEnvironment,UniversityCollegeLondon,Darwin10

Building,GowerStreet,LondonWC1E6BT,UK11

12

Keywords13

Tuberculosis,Antibiotics,Evolution,Mutation,Transmission,Latency,Biofilms14

15

2

Abstract16

Antimicrobialresistance(AMR)threatsaretypicallyrepresentedbybacteriacapableof17

extensivehorizontalgenetransfer(HGT).OneclearexceptionisMycobacteriumtuberculosis18

(Mtb).Itisanobligatehumanpathogenwithlimitedgeneticdiversityandalowmutationrate19

whichfurtherlacksanyevidenceforHGT.Suchfeaturesshouldinprinciplereduceitsabilityto20

rapidlyevolveAMR.Weidentifykeyfeaturesinitsbiologyandepidemiologythatallowsitto21

overcomeitslowadaptivepotential.Wefocusinparticularonitsinnateresistancetodrugs,22

unusuallifecycleincludinganoftenextensivelatentphaseanditsabilitytoshelterfrom23

exposuretoantimicrobialdrugswithincavitiesitinducesinthelungs. 24

3

SoSpecial25

Therapidincreaseofantimicrobialresistance(AMR;seeGlossary)inbacteriaisdrivenbythe26

widespreaduse,abuseandmisuseofantibiotics,andconstitutesoneofthemostchallenging27

healthcareproblemsglobally.WiththenotableexceptionofMycobacteriumtuberculosis(Mtb)28

theagentoftuberculosis(TB),allotherbacterialspecieslistedascurrentAMRthreatsbythe29

CentersforDiseaseControlhttp://www.cdc.gov/drugresistance/biggest_threats.htmltrend30

frequentlyexchangegeneticmaterialandfrequentlyacquirenovelmutationsthroughgainof31

newgenesbyhorizontalgenetransfer(HGT)ratherthandenovomutations(Figure1).Mtbhas32

avirtuallynon-existentaccessorygenome,meaningthatalmostallgenesarecommontoall33

strains.Evidencesuggeststhatthereislittleornorecombinationoccurringinthespecies.In34

additiontoitsstrictlyclonalreproduction,Mtbischaracterizedbyalowmutationrateand35

limitedgeneticdiversity,whichhasledtoitbeingconsideredasa‘monomorphicbacterium’36

[1].MtbalsostandsoutfrommostotherbacteriaconsideredasAMRthreatsbybeingan37

obligatepathogen.38

ThelackofHGTcombinedwithalowmutationratemakesMtbanaprioriunlikelyfoe39

outwittingconsiderableeffortsaimingatdefeatingit.Yet,multi-drugresistantTB[MDR-TB40

resistanttoisoniazid(INH)andrifampicin(RIF)]andextensively-drugresistantTB(XDR-TB;41

MDR-TBwithadditionalresistancetosecond-lineinjectabledrugsandfluoroquinolones)42

representasignificantandgrowingthreattoglobalhealthaccountingfornearlyhalfamillion43

newcasesandaround200,000deathsin2014alone44

http://www.who.int/tb/publications/global_report/en/.Resistancehasalsobeenshownto45

emergeessentiallyimmediatelyaftertheintroductionofnewdrugsandhighlyresistantstrains46

ofMtbhavebeenincirculationforatleastfourdecades[2,3].Inthisreviewweexplorewhy47

MtbissuchaseverepublichealthproblemdespitebeinganoutlieramongstotherAMR48

threats.Tosearchforclues,wereviewthekeyfeaturesofitsbiology,lifehistoryand49

epidemiologyinthewiderphylogeneticcontextofthebewilderingdiversityofmycobacteria.50

4

Weconcludethatcomparedtootherbacterialresistancethreats,thestrictrelianceofMtbon51

denovochromosomalmutationshasledtoarelativelyslowrateofemergenceofAMR.52

However,theproportionofresistantstrainsisincreasingandthistrendisprovingdifficultto53

containduetovariouspublichealthfailings,theintrinsicresistanceofmycobacteriatoarange54

ofantibioticsandlife-stylepropertiessuchastheabilityofMtbtohideinpulmonarycavities55

andlesionswithlimiteddrugpenetrance.56

RootsBloodyRoots57

ThegenusMycobacteriumcontainswellover100recognizedspeciesandprobablyanequally58

largenumberofspeciesyettobediscovered.Thegeneticdiversitywithindescribedspecies59

variessignificantly,andthisdiscrepancyfollowsasimplerule:themoreattentionagroupof60

mycobacteriahasreceived,themorelikelyithasbeensplitintomultiplespecies.Thegenus61

mainlycomprisesenvironmentalbacteria,butanumberofthesecancauseopportunistic62

infectionsinhumans(Figure2).Themorehost-specializedmembersexhibitaclonalmodeof63

inheritance,butrecombinationisfrequentinthegenusasawhole.64

Acomparativeanalysisincluding13mycobacterialspeciesfoundonlysporadicevidenceof65

recombinationincoregeneswithinMycobacteria,butgenomecontentanalysessuggestthat66

horizontalacquisitionofgenesisfrequentandplayedanimportantroleintheevolutionofthis67

group[4].Thepresenceofnumerousgenomicislandsdispersedacrossdifferentmycobacterial68

speciesisalsosuggestiveofextensivehorizontalgenetransferfromoutsidethegenus[5].A69

meiosis-likeconjugationalmechanismtermeddistributedconjugaltransfer(DCT),controlledby70

achromosomallyencodedmating-identitylocuswasrecentlydiscoveredinMycobacterium71

smegmatis[6].Thisintriguingmechanismenablesthetransferoflargeunlinkedstretchesof72

DNAacrossentirechromosomes.GenomicsignaturesindicativeofDCThavesubsequentlybeen73

identifiedinMycobacteriumcanettii,anenvironmentalmycobacteriumcloselyrelatedtoMtb,74

suggestingthatthismodeofhorizontalgenetransfermayplayanimportantroleinshapingthe75

evolutionofmycobacteria[7].76

5

Mycobacteriaareintrinsicallyresistanttoanumberofantimicrobialcompounds,an77

observationthatisoftenexplainedbythepresenceofanimpermeablemycolicacid-richcell78

envelope.Inaddition,mycobacteriaaremembersoftheorderActinomycetaleswhichalso79

includesStreptomycesspecies,wellknownfortheirabilitytoproduceawiderangeof80

antibiotics.Antibiotic-producingbacteriamusthavedefencemechanismsinplacetoguard81

themagainsttheirowntoxiccompounds.TheinduciblewhiB7multidrug-resistancesystem82

commontoallActinomycetaleshasbeenshowntoreducesusceptibilitytoawiderangeof83

antimicrobials,includingmacrolides,chloramphenicol,tetracyclineandaminoglycosides[8].In84

Mtb,theregulonincludesgenesinvolvedindrugefflux(tap),aputativemacrolideexporter85

(Rv1473),theribosomalmethyltransferaseermandtheaminoglycosideacetyltransferaseeis86

[8].87

Mtbandcloselyrelatedanimalstrains,togetherwiththeleprosybacilliareunusualinsofarthey88

constitutespecializedpathogensofhumansandothermammals(Figure2).Incontrasttomost89

othermycobacteria,thesebacillireadilytransmitbetweenmammalianhosts,ahallmarkoftrue90

pathogens[9].Despitethegeneralperceptionthatleprosyisadiseaseofthepast,nearlya91

quartermillioncasesarereportedyearly[10].Theruminant-infectingMycobacteriumavium92

subsp.paratuberculosisalsodeservestobementionedasahost-specializedpathogen.93

However,eventhoughthissubspeciescanonlygrowintracellularly,itcansurviveforlong94

periodsintheenvironmentandtransmitsviathefaecal-oralrouteandpossiblyalsovia95

nematodesandprotozoa[11].96

Up-andDown(sizing)97

Genome-levelcomparisonofMtbandM.marinumrevealedthatMtbhasalsoundergonea98

processofgenomedownsizingonitspathfromanenvironmentalancestortoaspecialized99

mainlyintracellularpathogen[12],butonafarmoremoderatescalethantheleprosybacilli.100

Thegeneraldownsizinghashoweveralsobeenaccompaniedbytheacquisitionofanumberof101

genes,includinggenesinvolvedinvirulence[12].Thereisstrongevidencethatrecombination102

hasbeenimportantinshapingtheearlyevolutionofMtbasitevolvedfromanancestorclosely103

relatedtopresent-daysmoothtuberclebacilli(STB)[13].Anumberofstrainsadaptedto104

6

variousmammalianhostshaveevolvedfromMtb.TogetherwithMtb,theseanimalstrains105

havebeengroupedtogetherintheso-calledMtbcomplex(MBTC).TheSTBM.canettii(notyet106

acceptedasavalidspeciesname)isalsogenerallyincludedintheMTBCbutevidencesuggests107

thatthisspeciesmightbemainlyenvironmental[14].ThehumanandanimaladaptedMTBC108

strainsrepresentaclonalexpansionrootedintheextensivelyrecombiningandgenetically109

diverseSTB.STBstrainscancauseTBinimmunocompetentindividuals,butaresignificantlyless110

virulentthanMtb,anddonotseemtotransmitbetweenhumans[9,15].Theyhaveonlybeen111

isolatedfromsporadichumancasesinEastAfrica,whichhasledtothesuggestionsthatthis112

regionoftheworldiswheretheMTBCoriginated.TheevolutionaryhistoryoftheMTBCseems113

tobeanalogoustotheclonalexpansionofanimal-adaptedM.aviumstrainsfromamore114

diverseenvironmentalgroup(Box1).115

NoconsensushasbeenreachedtodateonwhethermodernMtbhasretainedtheabilityto116

undergorecombination[16,17],butthesumofevidencesuggeststhatrecombinationis117

exceedinglyrare.Itisplausiblethatsomeanalysespointingtorelativelyhighratesof118

recombinationinMtbhadbeenmisledbyconvergentevolutionatarelativelyhighnumberof119

sites,manyofwhicharelikelyduetomultipleindependentemergenceofresistancemutations120

followingexposuretoantimicrobialcompounds[18].Indeed,bothconvergentparallel121

evolutionandrecombinationleadtoconflictsbetweenlocioverthebest-supportedtopologyof122

aphylogenetictree.123

DistanceEqualsRateTimesTime124

125

Comparingmutationratesacrossbacterialspeciesischallenging.Fluctuationassays[19],126

despiteoftenbeingregardedasthegoldstandard,onlyallowestimatingmutationratesifall127

possiblemutationsyieldingresistancetoagivenantimicrobialcompoundareknown,whichis128

generallynotthecase.Evenwhencomparisonsarerestrictedtoexperimentsusingthesame129

antimicrobialcompound,estimatedmutationratescanvaryduetovariationinthenumberof130

mutationsthatcanpotentiallybringaboutresistanceindifferentspecies.Additionally,the131

samemutationcouldyieldvariablelevelsofresistanceindifferentspecies132

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133

AnalternativeapproachtoestimatemutationratesreliesonBayesianphylogeneticanalysesof134

whole-genomesequencesfromclinicalisolatessampledoverseveralyearsandforwhich135

isolationdatesarepreciselyknown.SuchdataisavailableforallthespeciesillustratedinFigure136

1,andmutationestimatesfromindependentstudiesarehighlycongruentforMtb[20-22].In137

thiscomparisonMtbhasthelowestrateofall,withSalmonellaentericanextonthelist.This138

comparisonisbynomeansperfect,duetothevariationinsamplingandmethodologybetween139

studies.However,theapproachoffersthemajoradvantagethatsuchmutationrateestimates140

arescaledoverunittimeinnaturalconditions(ratherthanpergeneration).Assuch,these141

phylogeneticmutationrateestimatescapturethecapacityofdifferentbacteriatoadaptto142

antimicrobialsinthewild.ThegenerationtimeofMtbisindisputablyveryslowcomparedto143

thevastmajorityofclinicallyimportantbacteria.Asaresult,despitewhatseemstobea144

relativelyunremarkablemutationratepergenerationcomparedtoe.g.Escherichiacoli[23,24],145

MtbevolvesataveryslowpacecomparedtomostotherAMRthreats.146

147

Anextrapolationoftheseshortterm-termmutationratestomoreancienttimespointstoa148

fairlyrecentoriginofMtblessthan6,000yearsago[25,26],whichisincompatiblewith149

scenariosofancientoriginandajointcolonizationoftheglobeofMtbandanatomically150

modernhumanssome40,000-70,000yearsagothathavebeensuggested[27,28].Theseage151

estimatesalsofitsomewhatuncomfortablywithdirectevidenceforancientMTBCinfection152

fromPCRandthedetectionoflipidbiomarkerstargetingtheunusualandhighlystable153

componentsofthecellwall[29].TheseincludethedetectionofTBinhumanremainsfrom154

Syriasome11,000yearsago[30]andinabisonfromWyomingdatedto~17,000yearsago[31].155

AlsoofnoteisthedetectionofDNAharboringtheMtb-specificTbD1-deletioninawomenand156

childfromIsraelsome9,000yearsago[32].Atthistime,itseemsfairtoacceptthatthejuryis157

stilloutontheageofMtbandadditionalancientDNAgenomesequenceswillberequiredto158

obtainbetterlong-termcalibrationofmutationrates;theoldestMtbgenomesgeneratedto159

datebeingonlyabout250yearsold[26].160

161

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ComingBacktoLife162

Mtbhasaveryunusuallifecyclewithalonglatencyperiod.About90%ofinfectedpeople163

neverdevelopactivetransmissibleTBbutstayhealthyandasymptomatic[33],possiblybecause164

humanshaveadaptedtocontrolTBquiteefficientlyeventhoughimmuneactivationdoesnot165

leadtosterilization.AbouttwobillionpeopleareestimatedtobelatentlyinfectedwithTB[34],166

constitutingamassivechallengeforTBeradicationefforts.Aten-yearfollow-upstudyofTB167

contactsfoundthatthemajorityofpeoplethatdodevelopactiveTBdosowithinthefirstthree168

yearsofexposure[33].However,oldageisalsoknowntobeariskfactorforactiveTB[35].169

IfMtbhasco-evolvedwithhumanssincethedawnofourspecies,oratleastformillennia,a170

longlatencyperiodcouldberegardedasanadaptationtothesmallandisolatedpopulationsof171

ourancestors.ThisadaptationmighthaveallowedMtbtotransmitbetweenhostgenerations172

asactiveTBdevelopedintheelderlyandwastransmittedtothenextgenerations,without173

burningthroughandkillingoffsmallandisolatedbandsofhunter-gatherersandthus174

extinguishingitspopulationofhosts[36].AplausibleandworrisomescenarioisthatmodernTB175

strainsareevolvingtowardsshorterlatencyperiods.Therearenowmorethansevenbillion176

humansontheplanetandpotentialhumanTBhostsfrequentlytravelandmigratebetween177

countriesandcontinents.Assuch,acceleratedprogressiontoactivediseaseandhencepossible178

transmission,couldbeselectedfor.Inthiscontext,itisinterestingtonotethattheBeijingTB179

lineagewhichhasexpandedgloballyinrecentdecadesseemstobeassociatedwithaccelerated180

progressiontoactiveTBrelativetootherTBstrainsandMycobacteriumafricanum[37].181

SpaceOddity182

Globally,3.3%ofnewTBcaseswereestimatedtobeMDRin2014.Thisrelativelylowrate183

mightcomeasasurprisetomany,anddoesconcealsignificantvariationbetweenandwithin184

countriesandregions.InmanywesternEuropeancountriestheburdenislow,asexemplified185

bythesituationintheUK,where1.6%ofallcaseswereMDR-TBin2013186

https://www.gov.uk/government/publications/tuberculosis-in-england-annual-report.This187

figureinitselfdoesnotcapturethefullextentoftheproblemas7.1%ofstrainsinthecountry188

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areINHresistantinthe(Figure3),whichisunusuallyhighforawesternEuropeancountry.189

Again,thisfigureforINHresistancedoesonlyveryimperfectlycapturetherealityonthe190

ground.INHresistantstrainsintheUKareprimarilyfoundinLondonandarepatchily191

distributedevenatthatscale.192

TBdrugresistanceintheUKandinotherhighincomecountriesisaseriouspublichealthissue193

incurringasignificantfinancialburdenonpublichealthservices,eventhoughtherateof194

resistanceisrelativelylowcomparedtothosefoundinsomeotherresistancethreats(Figure3).195

Theextraordinarilyrapidpopulation-levelresponsetoantibioticsseenforexamplein196

StaphylococcusaureusandEnterococcusfaeciumisstrikingwhencomparedtoMtb,and197

probablypartlyreflectsitslowmutationrateandlackofrecombinationandofresistance-198

determinantsonmobilegeneticelements(Figure1).However,Mtbresistanceratesinhigh-199

incomecountriesarenotrepresentativeofthefrequencyofMDR/XDR-TBstrainsinotherparts200

oftheworld,andtheburdenduetoAMRinTBresistanceiscripplinginsomehotspots(Box2201

andFigure4).202

RoadtoResistance203

Althoughintrinsicallyresistanttomanydrugs,thereislittleevidencetosuggestthattheMtb204

genomeshouldbeespeciallypronetoevolvingadditionaldrugresistance(Figure1).Infact,205

ratesofRIFresistanceinM.leprae,whichisnotgenerallyregardedasamajorresistance206

threat,seemtomirrortheratesfoundinMtb.TheinabilitytocultureM.leprae,andthusto207

performphenotypicdrugsusceptibilitytesting(DST)complicatesanalysesofresistanceinthis208

bacterium.However,availabledatafromIndia,South-EastAsiaandColombiaallfoundabout209

3%ofallcasestobeRIFresistant[38-40],aratethatissimilartorecentestimatesofMtbRIF210

resistancegloballyat3.3%.211

ThefirstoutbreaksofMDR-TBwerelargelyrestrictedtoHIVco-infectedpatientsandoften212

confinedtohospitals[41-43].ThiscaneasilybeexplainedbyHIVinfectiontriggeringthe213

developmenttoactiveTB.Tomakemattersworse,HIVandTBmedicationshavebeenshown214

tonegativelyinterferewitheachother.However,strainswithextensiveresistanceprofileshave215

10

recentlybeenshowntohaveemergedandtransmittedwellbeforetheHIVepidemictookoff216

inthe1980s[2,3]andthereiscurrentlylittleevidencetosuggestacausalrelationshipbetween217

HIVco-infectionandincreasedemergenceorcirculationofMDR-TBstrains[44].218

InformerSovietEasternEuropestates,themassiveratesofdrugresistantTBhavebeen219

attributedtothecollapseofhealthsystemsfollowingthefalloftheSovietUnion[45].Similar220

forcesmightbeatplayinlower-middleincomecountriessuchasIndiatodaywhereanti-TB221

medicationisavailabletomost,butthehealthsysteminfrastructureisoftenweakand222

antimicrobialstewardshipislacking[46].Anotherpossibleexplanationfortheregional223

differencesinresistanceburdencouldbeduetophenotypicdifferencesbetweenstrains.Ifthe224

dominantlineageinaregionweremorepronetodevelopresistancethanotherlineages,this225

couldexacerbatetheresistanceburdenintheregion.226

TheBeijinglineage(Lineage2)isthedominatinglineageinlargepartsofAsiaandeastern227

Europeandisoftenassociatedwithdrug-resistance[47].Whethermembersofthelineageare228

actuallymorepronetodevelopresistance-conferringmutationsasrecentlysuggestedbyFord229

andcolleagues[48]remainsunclear.Intheirstudy,Fordetal.reliedheavilyonlabstrains[48]230

andanearliersimilarstudywithslightlylargersamplesizedidnotpointtoahigherrateof231

acquisitionofmutationsofLineage2strains[49].ItispossiblethattheBeijinglineagesimply232

happenedtobeattherightplaceattherighttimewiththecollapseoftheSovietUnion,233

leadingtotheemergenceofresistantstrainsstillcirculatinginlargenumberstoday.The234

observationthatBeijingisolatesareassociatedwithacceleratedprogressiontoactiveTB[37]235

couldbepartoftheexplanationfortherelativelyrecentandongoingglobalexpansionofthe236

lineage.237

Tosummarize,worldwidedistributionofMDR-TBisextremelyheterogeneousandhas238

undoubtedlybeenshapedbypastfailingsinpublichealthinfrastructureinvariouspartsofthe239

world.Thisheterogeneitymighthavebeenfurtherexacerbatedbyanintrinsicpropensityof240

certainlineagestoacquireresistancemorereadily.However,lineage-specificfactorsare241

difficulttoquantifybecauseMtblineagesarethemselvespatchilydistributed.242

11

243

MyBodyIsaCage244

ThefirstclinicalstageofTBinfectionistermedprimaryTBandtypicallyinvolvestheproduction245

andspreadofgranulomassystemicallyandtoregionallymphnodes[50].Withinafewweeks,246

immunitydevelopsandtheinfectionregresses,butisnotsterilized[50].TBisgenerallymore247

virulentinanimalsthaninhumans,andcommonanimalmodelssuchasmice,guineapigs,248

rabbitsandmonkeysalldevelopaggressiveprimaryTBthatisnottransmissibleandoften249

resultsindeath[51].HumansarespecialinthatprimaryTBisgenerallynotassociatedwith250

seriousillness.Inhumanshowever,Mtbentersalatentstagefollowingregressionofthe251

primaryinfection.Uponre-activationofthedormantorganismsorreinfectionwithnew252

organismsfromtheenvironment,softenedandfragmentedlungtissueiscoughedupleaving253

cavitiesthatharborsmallnumbersofbacilli.Thisearlystageofcavityformationcanerode254

arteriestoproduceheavybleeding,aclassicalsignofTB[50].Uponmaturation,cavities255

developathinfibrouswall.Theinnersurfaceiscoveredwithfluidcaseumwithnoviablecells.256

Mtbgrowsextracellularlyonthesurfaceofsuchcavitiesasapellicle(biofilm)[52].Mtbcan257

growinmassivenumbersonthesurfaceofcavitieswhereitcanbecoughedintothe258

environmentwhilethehostremainsinhealthexceptforthecoughing[50].Thisformofclinical259

TBisobviouslyextremelytransmissibleasbillionsofbacteriacanbeproducedeachday[53].260

InadditiontotheroleofcavitiesinthetransmissionofTB,theyconstituteasignificant261

complicationforsuccessfulantimicrobialtherapyasdifferentdrugspenetratecavitieswith262

varyingefficiency:Thefluoroquinolonemoxifloxacinseemtopenetratewell,whereasthefirst-263

linedrugsINH,RIFandpyrazinamide(PZA)arelessefficient[54].Themoreexperimentaldrug264

linezolidhasbeenshowntobeeffectiveagainstcavitaryTB,albeitoftenwithquiteseriousside-265

effects[55].Mathematicalmodellinghasrevealedthatusingdrugswithdifferentpenetration266

profilesleadstospatialmonotherapyandrapidevolutionofmultidrugresistance[56].Itisno267

surprisethen,thatcavitaryTBisassociatedwithtreatmentfailure[57]andisamajorriskfactor268

foracquiredresistancetosecond-linedrugs[58,59].Infact,arecentstudyfromGeorgiafound269

additionalresistancetoemergein58%ofcavitaryMDR-TBcasestreatedwithsecondline270

12

drugs,butin‘only’16%ofsuchpatientsnotpresentingwithcavities[58].Effortstooptimize271

regimensforprogressedcavitaryTBminimizingresistancedevelopmentarewarranted.272

TheemergenceofdrugresistantTBismostoftenattributedtopoorpatientadherencetodrug273

treatmentschemes,aproblemthatisamelioratedbydirectlyobservedtreatment(DOT).274

Patientsaretypicallyenrolledonanti-TBtherapyfor6to24months,dependingonresponse275

andtheresistancephenotypeoftheinfection.Inlightofthisonecannotexpecttheproblemof276

imperfectpatientcompliancetogoawayanytimesoon.Yet,basedonahollow-fibermodel,277

pharmacokineticvariabilityalonewasestimatedtoresultinacquiredmultidrug-resistancein278

about1%ofpatients,irrespectiveofadherence[60].Manybacteriaexhibitincreaseddrug279

tolerancewhengrowinginbiofilmsandthisphenomenonhasalsobeenobservedinMtb.Bacilli280

incavitiesarealsoseparatedfromthehost’simmunedefensesbythewallofthecavitythat281

preventspenetrationofviablecells.Whenallowedtoformbiofilmsinvitro,asmallbutpossibly282

clinicallyimportantsubpopulationemerges,whichisabletotolerateveryhighdosesof283

antimicrobials[61].Mtbbiofilmformationwasrecentlyshowntodependonketo-mycolicacids284

andwhenco-culturedwithawild-typestrain,evendrugsensitivebiofilm-defectivemutants285

werefoundtobecomedrugtolerant[62].Thebiologicalrelevanceofbiofilmformationwithin286

patientsremainstobedetermined,butthebiofilm-likegrowthofMtbwithinandonthe287

surfaceofcavities[52]suggestthatthisgrowthmodecouldbeclinicallyveryimportant.288

ChemicalWarfare289

Recentstudiesutilizingdeep-sequencingofpatientisolateshaverevealedasurprisingdegree290

ofMtbgeneticdiversitywithinpatients[63-67].Resistancemutationshavebeenfoundto291

emergemultipletimeswithinasinglepatient,generallyfollowedbyselectivesweepsresulting292

inoneclonereplacingthewholewithin-hostpopulation[63,66,67].LargeMtbpopulationsizes293

andsignificantgeneticdiversityupondiagnosissurelyplayimportantrolesintheemergenceof294

resistance,asadiversepopulationismorelikelytoencompassmutantswithdecreased295

susceptibilitytoanti-TBtherapeuticdrugs.Theimportanceofwithin-hostMtbpopulationsize296

13

andgeneticdiversityinresistancedevelopmentisaresearchavenuethatdeservesfurther297

attention.298

Evenmoreworrisomethanresistanceevolvinginindividualpatientsisthetransmissionof299

resistantstrainswithlittleornoapparentfitnesscosttothebacterium.Theoverallrobustness300

ofMtbwhenchallengedwithantimicrobialsledtostandardizeddrugtreatmentschemes301

includingacocktailoffourdrugs.Unfortunately,theseschemesarenotalwayspairedwith302

robustdrug-susceptibilitytesting.Ithasbeenarguedthatstandardizedtreatmentschemesfor303

susceptibleandMDR-TBintheabsenceofphenotypicresistancetestinghasbeenadirect304

driveroftheevolutionofXDR-TBinSouthAfrica[68].Itiswelldocumentedthatthemost305

commonlytransmittedRIF-resistancemutationrpoBS450Lincombinationwithsecondary306

compensatorymutationsinpolymerasesubunitsisassociatedwithlittleornofitnesscost[69,307

70]whereasthepictureislessclearforINH-resistance.Ithashoweverbeenshownthatthe308

mostcommonINH-resistancemutation,namelykatGS315Tretainsresidualcatalase-309

peroxidaseactivity,isvirulentinmice,andimportantly,transmitswellbetweenpeople[71,72].310

RecentstudieshavedocumentedthatMDR-TBstrainshavebeenincirculationfordecades[2,311

3].Thefour-druganti-TBregimencurrentlyinuseincludesdrugsthathaveallbeenused312

continuouslyagainstTBfor40-60years.Itmaythuscomeasnosurprisethatthishasselected313

forhighlytransmissibleMDR-TBstrains,andwemightperhapsconsiderourselvesluckythat314

theproblemisnotyetworsethanitis.315

ConcludingRemarks:KnowYourEnemy316

Essentiallyirrespectivelyofthefeatureunderscrutiny,Mtbstandsoutfromallotherbacteria317

consideredasAMRthreats.Someofthesepeculiaritiesshouldconstitutemajorchinksinits318

armormakingitatractabletargetforararesuccessinstemmingtheriseofAMRs.Inparticular,319

Mtbhasalowmutationrateandlimitedgeneticdiversity,lacksanymechanismforextensive320

HGTanddoesnotbenefitfromanyhidingplaceoutsideitshumanhost,suchasan321

environmentalorzoonoticreservoir.Itremainstobedefinedwhatexactformadetermined322

assaultagainstAMR-TB(andTBmoregenerally)shouldtake.However,itisclearthatany323

14

successfulpublichealthstrategywillhavetobeinformedbyrobustfundamentalscientific324

evidenceandbemultiprongedtobesuccessful.WehavelearnedalotaboutMtbandTB,in325

particularsincetheadventoffastandaffordablesequencingtechnologies.However,itwould326

befoolishtoassumethatthecurrentknowledgeissufficienttovanquishMtb(seeOutstanding327

Questions),asitremainsadeadlyandsurprisinglyadaptablefoedespiteitsapparentinherent328

weaknesses.329

330

Acknowledgements331

TheauthorsacknowledgesupportfromtheNorwegianResearchCouncil(grant221562), the332

ERC(grantERC260801–BIG_IDEA)andtheNationalInstituteforHealthResearchUniversity333

CollegeLondonHospitalsBiomedicalResearchCentre.Wearegratefulforthehelpand334

informationprovidedbyHelenDonoghueandRobertL.Hunter. 335

336

15

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87Turenne,C.Y.,etal.(2008)Mycobacteriumaviumsubsp.paratuberculosisandM.aviumsubsp.avium523AreIndependentlyEvolvedPathogenicClonesofaMuchBroaderGroupofM.aviumOrganisms.Journal524ofBacteriology190,2479-248752588Doig,K.D.,etal.(2012)OntheoriginofMycobacteriumulcerans,thecausativeagentofBuruliulcer.526BMCgenomics13,25852789Guenin-Macé,L.,etal.(2011)MycolactoneimpairsTcellhomingbysuppressingmicroRNAcontrolof528L-selectinexpression.ProceedingsoftheNationalAcademyofSciences108,12833-1283852990Fyfe,J.A.M.,etal.(2010)AMajorRoleforMammalsintheEcologyofMycobacteriumulcerans.Plos530Neglect.Trop.Dis.453191Dalal,A.,etal.(2015)ResistancePatternsamongMultidrug-ResistantTuberculosisPatientsin532GreaterMetropolitanMumbai:TrendsoverTime.PLoSONE10,e011679853392Isaakidis,P.,etal.(2014)AlarmingLevelsofDrug-ResistantTuberculosisinHIV-InfectedPatientsin534MetropolitanMumbai,India.PLoSONE9,e11046153593Skrahina,A.,etal.(2012)Alarminglevelsofdrug-resistanttuberculosisinBelarus:resultsofasurvey536inMinsk.EuropeanRespiratoryJournal39,1425-1431537

538

20

Figures539

540

541

Figure1.BasicGenomicFeaturesoftheMainBacterialResistanceThreats.Thecoregenome542proportionindicatestheproportionoftypicalindividualgenomesthatarecommontoall543membersofthespecies.Mutationratesarebasedonwhole-genomephylogeneticanalysesof544clinicalisolates.Thepresenceofplasmid-orphage-mediatedresistanceisindicatedinthe545figure.InthisfigureweconsideredM.tuberculosistohaveentirelylosttheabilitytorecombine546(seemaintextforabriefdiscussiononpossiblerecombinationinM.tuberculosis).547Acinetobacterbaumanniwasnotincludedinthefigureaswecouldnotfindrelevantestimates548formutationratesandrecombination/mutationratio. 549

21

550

Figure2.HistoryandHostRangeofImportantMycobacterialPathogens.Thefigureincludes551the most important pathogens in the genus Mycobacterium. SGM = slow-growing552mycobacteria; RGM = rapid-growing mycobacteria.M. tuberculosis and related species are553descendantsfromahypotheticalenvironmentalancestralspeciestermedM.prototuberculosis,554whereasM.ulceransevolvedfromaM.marinum-likeancestor.TheacquisitionofthepMUM555mega-plasmid containing genes required for the synthesis ofmycolactonewas central in the556evolution of this pathogen. Pictograms indicate host range, whereas the presence of a tree557specifically indicates that the species is environmental. M. avium and M. abscessus are558separatedintosubspecies. 559

22

560

561

Figure3.Population-levelResponsetoAntimicrobialsofSelectedPathogens,UnitedKingdom562Fromleft:PercentageofMtbclinicalisolatesresistanttoisoniazid(INH-resMtb)(From1960-5631983onlynewcases[73,74],from1988-2013allcases[75,76];InvasiveE.faeciumisolates564resistanttovancomycin(VAN-resEfa)[77];InvasiveS.aureusisolatesresistanttomethicillin565(MET-resSau). [78,79].ForE.faeciumandS.aureus,datafortheyears2001-2013was566retrievedfromhttp://www.bsacsurv.org/.Shadedareascorrespondto95%confidence567intervalsbasedonlocalregression.MtbdatafromEnglandandWales1960-1999;England,568WalesandNorthernIreland2000-2005;UK2005-2013.E.faeciumdatafromEnglandand569Wales1990-1998;UKandIreland2001-2013.S.aureusdatafromEnglandandWales1989-5701996;UKandIrelandfrom2001-2013.571

23

572

Figure4.HotspotsofMDR-TB.DataonMDR-TBincidencewascollectedfrom[80](Belarus),573[81](Mumbai)and[82](KwaZulu-Natal).NotethattheMDR-TBfrequenciesreportedherefor574Mumbaiaresignificantlyhigherthanthosereportednation-widefromtheWorldHealth575Organization.ThereportedMDR-TBincidenceinKwaZulu-NatalrepresentsRIF-resistant576isolatesasidentifiedbyTBXpert[83]. 577

24

TextBoxes578

Box1Youalllookthesametome 579

M.abscessusisarapidlygrowingenvironmentalspeciesbutalsoarelativelycommonsourceof580

softtissueinfections,disseminatedinfectionsinimmunocompromisedindividualsand581

pulmonaryinfectionincysticfibrosispatients.TreatingM.abscessusinfectionswith582

antimicrobialsischallenging,asthegroupisintrinsicallyresistanttomostavailabledrugs[84].583

Thespeciesharborsthreesubspecies,namelyabscessus,bolettiiandmasilliense.Themacrolide584

clarithromycinhasbeenusedfrequentlytotreatinfections,buttherelativelyrecentdiscovery585

thatnearlyallabscessusandbolettiistrains,butnotmasilliense,caninduceresistancetothe586

drugbyactivationoftheerm(41)gene[85],encodingaribosomemethylase[86],highlights587

criticaldifferenceswithinthisspeciescomplexandtheneedforimprovedtaxonomic588

assignmenttoolsforeffectivetreatment.589

M.aviumisaspeciesconsistingoffourmainsubspeciesrangingfromenvironmentaltomore590

specializedpathogens.M.aviumsubsp.homoinissuis,anenvironmentalspeciescausing591

opportunisticinfectioninimmune-compromisedpeople,isadiversegroupundergoing592

frequentrecombinationevents.Incontrast,thesubspeciessilvaticum,aviumand593

paratuberculosisrepresentclonallineagesradiatingoutofthehominisuisgroupthathave594

adaptedtovariousanimalhosts[87](Fig.2).595

M.ulceransfallssomewhereinthemiddleofthespectrumbetweenenvironmentalandhost-596

specializedmycobacteria.Thebacteriumcanbeconsideredasasemi-specializedpathogen597

mainlyduetotheacquisitionofthepMUMplasmidbyaM.marinum-likeancestor[88].The598

plasmidencodesthegenesnecessaryforthesynthesisofmycolactone,apolyketide-derived599

macrolidethatservesbothasatoxin,triggeringtissuedamage,andanimmunomodulatory600

compoundinhibitingthehostimmuneresponse.AnalogoustoMtbandtheleprosybacilli,M.601

ulceranshasundergonesignificantgenelossandcontains771pseudogenes,instarkcontrastto602

M.marinum,whereonly65inactivatedgeneshavebeenidentified[88].Thispseudogenization603

seemstohavebeenpartiallydrivenbytheexpansionofinsertionsequenceIS2404thatwas604

25

acquiredafterthesplitbetweenM.marinumandM.ulcerans.Insertionsequencesseemto605

haveplayedimportantrolesalsointhehost-adaptionofotherpathogenicmycobacteria.M.606

ulceranshasbeenidentifiedinawiderangeofenvironments,includingsoil,water,frogs,fish,607

mosquitos,waterbugsandmammals.However,thefindingthatmycolactonespecifically608

inhibitsT-cellcontrollingmammalianmicroRNAs[89]combinedwithaveryclosegenetic609

relationshipbetweenM.ulceransisolatesinhumansandopossumsinsouthwestAustralia[90]610

dosuggestthatthebacteriumhasevolvedtoaccommodateamammalianniche.611

612

26

Box2TerriblePlaces(forMDR-TB)613

InIndia,4.3%ofnotifiedTBcaseswereestimatedtobeMDR-TBin2013,butwithinthe614

countrytherearesignificantregionaldifferencesinresistanceburden.Asurveyoffour615

municipalwardsinMumbairevealedratesofMDR-TBcloseto30%intheyears2004-2007[81]616

(Figure4)andsubsequentstudieshaveconfirmedveryhighratesofdrugresistance[91,92].A617

significantproportionoftheMDR-isolatesareresistanttoadditionaldrugs,exemplifiedbya618

studyfrom2013thatfound10.6%ofallMDR-TBisolatestoqualifyforXDR-TBstatus[91].619

Resultsfromfirst-linediagnosticsreportedannuallyinSouthAfricafound6.6%ofallTBcases620

inSouthAfricatobeRIFresistantin2013/2014[83].However,intheprovinceofKwaZulu-Natal621

whichishometo30%ofallTBcasesinthecountry,andtheregionwiththehighestincidence622

ofMDR-TBintheworld,8.9%ofTBcaseswereRIFresistantinthesameperiod.RIF-resistance623

isoftenusedasaproxyforMDR,anassumptionthatiscorrectinmorethan90%ofthecasesin624

KwaZulu-Natal[82],butdoesnotholdforinstanceintheUK.Thesenumberssuggestthatthe625

WHOestimatesfrom2013ofratesofaround2.1%ofMDR-TBareoverlyoptimistic.626

AstudyconductedinMinsk(Belarus)in2009-2010revealingthatalmosthalfofallTBcases627

wereMDRraisedafeweyebrows[93].However,thesefigureswereconfirmedbyafollow-up628

country-widestudyoneyearlaterthatconfirmedthat45.5%ofallisolatesinBelarusare629

indeedMDR-TB.Possibly,evenmoreshockingwastheobservationthatamongMDR-TB630

isolates,11.9%wereXDR[80]. 631

632

27

GlossaryBox633

AncientDNA(aDNA):isDNAisolatedfromanyancientspecimen.Itisgenerallylooselyusedto634

describeanyDNArecoveredfrombiologicalmaterialthathasnotbeenpreservedspecifically635

forlaterDNAsequencing.ExamplesincludeDNArecoveredfromarchaeologicalandhistorical636

skeletalmaterial,mummifiedtissuesandarchivalcollectionsofnon-frozenspecimens.637

Antimicrobialresistance(AMR):isresistanceofamicroorganismtoanantimicrobial638

compoundtowhichitwasoriginallysensitive.Resistantorganisms(bacteria,fungi,virusesand639

someparasites)areabletowithstandexposuretoantimicrobialdrugs,sothatstandard640

treatmentsbecomeineffectiveandinfectionspersistincreasingtheriskoftransmissionto641

otherhosts.Theevolutionofresistantstrainsisanaturalphenomenongenerallyinducedby642

exposuretoantimicrobialdrugs.643

Directlyobservedtherapy(DOT):casemanagementthathelpstoensurethatpatientsadhere644

totreatment.DOTisconsideredthemosteffectivestrategyformakingsurepatientstaketheir645

medicines.646

Drugsusceptibilitytesting(DST):thevariousprocedurestofindoutwhichdrugsabacterial647

strainisresistantto.Thisrepresentsanessentialstepforrapididentificationofresistantstrains648

sothatpatientscarryingsuchstraincanbeputonadequatedrugtreatmentassoonas649

possible.650

Isoniazid(INH):anantibioticusedasafirst-lineagenttogetherwithrifampicin(RIF)inthe651

preventionandtreatmentofbothlatentandactiveTB.652

Multi-drug-resistanttuberculosis(MDR-TB):definedasaformofTBinfectioncausedby653

bacteriathatareresistanttotreatmentwithatleasttwoofthefirst-lineanti-TBdrugs,isoniazid654

(INH)andrifampicin(RIF).655

28

Mycobacteriumtuberculosiscomplex(MTBC):agroupofcloselyrelatedstrainsandspecies656

includingpathogensofhumansandanimalsaswellasthehighlydiverseprobably657

environmentalMycobacteriumcanettii.658

Rifampicin(RIF):anantibioticusedtotreatanumberofbacterialinfections.Itconstitutesone659

ofthetwofirst-lineagentstogetherwithisoniazid(INH).ItisontheWorldHealth660

Organization'sListofEssentialMedicines,themostimportantdrugsneededinafunctional661

basicpublichealthsystem.662

Smoothtuberclebacilli(STB):agroupofmycobacteriafoundinEasternSub-SaharanAfricaand663

areconsideredastheputativeancestorsofMtb.TheyincludeinparticularthespeciesM.664

canettiithatcancauseTBbutdoesnotseemtotransmitdirectlybetweenhumanhosts.665

Tuberculosis(TB):abacterialinfectioncausedbysomespeciesinthegenusMycobacterium,666

withthemainagentbeingM.tuberculosis.Theinfectiongenerallyresidesinthelungsbutcan667

spreadthroughthelymphnodesandbloodstreamtoanyorgan.Mostpeoplewhoareinfected668

byMtbremainhealthyandasymptomaticanddonottransmitthebacteriumtoothers.669

Extensivelydrug-resistantTB(XDR-TB):atypeofmultidrug-resistanttuberculosis(MDR-TB)670

thatisresistanttoisoniazidandrifampin,plusanyfluoroquinoloneandatleastoneofthethree671

injectablesecond-linedrugs(i.e.,amikacin,kanamycin,orcapreomycin). 672