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Use of collateral sensitivity networks to design drug cycling protocols that avoid
resistance development.
Imamovic, Lejla; Sommer, Morten
Published in:
Science Translational Medicine
Link to article, DOI:
10.1126/scitranslmed.3006609
Publication date:
2013
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Citation (APA):
Imamovic, L., & Sommer, M. (2013). Use of collateral sensitivity networks to design drug cycling protocols that
avoid resistance development. Science Translational Medicine, 5(204).
https://doi.org/10.1126/scitranslmed.3006609

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Use of Collateral Sensitivity Networks to Design Drug Cycling Protocols
That Avoid Resistance Development
Lejla Imamovic and Morten O. A. Sommer
Sci Transl Med 5, 204ra132 (2013);
DOI: 10.1126/scitranslmed.3006609
Editor's Summary

Resistance is Futile
In an emergency situation, people are often instructed to remain calm and proceed in an orderly fashion. The
same advice may apply to the current antibiotic-resistance crisis. Imamovic and Sommer now show how collateral
sensitivity profiles-- deciphered by treating bacteria with multiple antibiotics-- can help to order drug deployment in
sequences that thwart the development of antibiotic resistance.
Cells or organisms that have developed resistance to one drug sometimes display a greater sensitivity to a
second drug often from a distinct structural class, a concept called collateral sensitivity. The authors tested whether
application of this concept can aid in the management of bacterial infections by evolving resistance in Escherichia coli
33
to 23 known antibiotics and then mapping the resulting collateral sensitivity and resistance profiles. On the basis of 11
00
their derived collateral sensitivity network, the authors designed a new treatment framework-- collateral sensitivity 22
cycling-- in which sequential treatment of E. coli cultures with antibiotics that display compatible collateral sensitivity 4, 4,
11
profiles is predicted to select against drug-resistance development. The authors chronicled hundreds of such drug r r
sets and validated their predictions E. coli and the antibiotics gentamicin and cefuroxime by showing that cyclical ee
bb
deployment of the drugs selected against resistance to either antibiotic. This proof of principle for collateral sensitivity mm
cycling as a sustainable treatment regimen was then validated with two related bacterial pathogens. It remains to be ee
vv
seen whether deployment of cancer therapeutics in an orderly fashion also curbs drug resistance. oo
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RESEARCH ARTICLE
ANTIBIOTICS
Use of Collateral Sensitivity Networks to Design Drug
Cycling Protocols That Avoid Resistance Development
Lejla Imamovic1 and Morten O. A. Sommer1,2*
New drug deployment strategies are imperative to address the problem of drug resistance, which is limiting
themanagementofinfectiousdiseasesandcancers.WeevolvedresistanceinEscherichiacolitoward23drugs
used clinically for treating bacterial infections and mapped the resulting collateral sensitivity and resistance
profiles,revealingacomplexcollateralsensitivitynetwork.Onthebasisofthesedata,weproposeanewtreatment
framework—collateral sensitivity cycling—in which drugs with compatible collateral sensitivity profiles are used
sequentiallytotreatinfectionandselectagainstdrugresistancedevelopment.Weidentifiedhundredsofsuchdrug
sets and demonstrated that the antibiotics gentamicin and cefuroxime can be deployed cyclically such that the
treatmentregimenselectedagainstresistancetoeitherdrug.Wethenvalidatedourfindingswithrelatedbacterial
pathogens. These results provide proof of principle for collateral sensitivity cycling as a sustainable treatment
3
paradigmthatmaybegenerallyapplicabletoinfectiousdiseasesandcancer. 1
0
2
4,
1
INTRODUCTION r
thatevolutionofdrugresistanceisrelatedtodrugexposure;thus,by e
b
Therapiddevelopmentofdrugresistanceisamajorthreattothesuc- withdrawingadrugfromtheclinicforagivenperiod,theselective m
e
cessfulmanagementofinfectiousdiseasesandcancer,whichcollectively benefitofresistanceisremoved,resultinginadecreaseinresistance v
areresponsibleformorethanhalfofdisease-relateddeathsglobally frequencies(19–21).Thesepredictedadvantagesofconventionalanti- No
(1,2).Theevolutionandspreadofresistancereducesorentirelyelim- bioticcyclingrestontheassumptionthatresistanceisaccompaniedby n
o
inatestheutilityofmostdrugsusedforthetreatmentofbacterial,fun- abiologicalfitnesscost.Thelargerthefitnesscost,themorerapidly g
gal,parasitic,andviralinfections(3–6).Inparticular,treatmentfailureis theresistancefrequencyofapopulationwilldecreaseintheabsenceof or
rapidlyincreasingasaresultofthealarmingdevelopmentofmultidrug theselectivepressureimposedbydrugtreatment(22).Thus,ifdrug g.
a
resistanceinGram-negativebacterialpathogens,suchasEscherichia useisreduced,theresistantmutantswiththehighestfitnesscostsare m
coliandKlebsiella,whichcontributetomostofthedeathscausedby expectedtobeoutcompetedbytheirnonresistantcounterpartsand ce
hospital-acquiredinfections(7).Theburdenofresistanceisalsoevident disappearfromthebacterialpopulation. en
withcancertherapy,inwhichdrugresistanceremainsamajorcauseof However,thespreadofresistantbacterialstrainsstilloccurseven ci
s
treatmentfailureanddeathamongcancerpatients(8,9). thoughresistancecanbecostlyintermsofbacterialfitness(23–25). m.
Despitethedesperateneedfornewinfectiousdiseasetherapies, Inaddition,notallresistancecarriesfitnesscost(26,27),andthefit- st
limitedprogresshasbeenmadeinthediscoveryoffirst-in-classantibac- nesscostcanbeamelioratedbycompensatorymutations(22,28). m
o
terialdrugstobalancetherelentlessresistancedevelopment(10,11). Finally,ithasbeenshownthatresistantsubpopulationsofbacteria r
Anotherpromisingapproachforaddressingdrugresistanceisthrough canpersistfordecadesafterdrugremoval(29). d f
e
rationaltreatment(deployment)strategies.Themostprominentstrate- Current antibiotic cycling strategies implement cycles of drug d
a
giesaredrugcombinationtherapiesanddrugstewardshipprograms. restriction and prioritization on an ad hoc basis, andthe benefits o
Combinationtherapieshavebeendeployedclinicallybothtoprevent ofthisstrategyremaincontroversial(30–33).Here,weproposeafun- wnl
o
theriseofresistanceandtoimprovetreatmentefficacy.Theabilityofdrug damentallydifferentapproachtodrugcyclingthatbuildsonthefind- D
combinationstoimprovetreatmentoutcomeshasbeendemonstratedina ingthatthedevelopmentofresistancetoonedrugcanperturbdrug
varietyofdiseaseareas,includingHIVinfection(4,12–15).Ithasalsobeen susceptibilityprofilesinthebacterialcell;thisadaptationresultsincol-
demonstratedinbacteriathatantagonisticdrugcombinations,inwhich lateralresistanceandcollateralsensitivitytowardotherdrugsthrough
theconstituentdrugscounteracteachother’seffects,canbeusedtoselect perturbationofgeneexpressionnetworksthataffectacell’svulnera-
againstresistancethatresultsfromsuppressivedruginteractions(16). bilitytochemicals(34–36)(Fig.1,AtoC).Thecollateraleffectsof
Antimicrobialstewardshipprogramsaimtodecreasedrugresist- resistanceevolutionprovidearationaleforanewtreatmentstrategy:
ancebyavoidingunnecessaryuseandchoosinganadequatedosewith collateralsensitivitycycling(Fig.1D).
the shortest effective duration of treatment (17, 18). Drug cycling Consideratargetbacterialpopulationundergoingtreatmentwithdrug
programsareaformofantimicrobialstewardshipthatinvolvesthe A(Fig.1D).WhenresistancetodrugAevolveswithinthetargetpopula-
scheduledrotationofdistinctantibioticclassesthatexhibitcompara- tion,treatmentcanbeswitchedbydeploymentofdrugB,towhichthe
bleantimicrobialactivityspectra.Therotationcanincludeanynum- drugA–resistantpopulationdisplayscollateralsensitivity(Fig.1D).
berofdrugs,butmustbeconcludedwithreapplicationoftheoriginal With this treatment regimen, the drug A–resistant bacterial sub-
drug.Thecurrentrationalebehindantibioticcyclingreliesonthefact populationwouldbeeradicatedmoreeffectivelythanthesubpopula-
tionthathasnotdevelopedresistancetodrugA.Ifthedrugsarechosen
1DepartmentofSystemsBiology,TechnicalUniversityofDenmark,DK-2800Lyngby, carefully tohavereciprocalcollateralsensitivity profiles, collateral
Denmark.2NovoNordiskFoundationCenterforBiosustainability,TechnicalUniversity
sensitivitycyclingshouldlimitevolutionofdrugresistanceinthetarget
ofDenmark,DK-2970Hørsholm,Denmark.
*Correspondingauthor.E-mail:[email protected] bacterialpopulation.Becausecollateralsensitivityderivesfromchanges
www.ScienceTranslationalMedicine.org 25September2013 Vol5Issue204204ra132 1
RESEARCH ARTICLE
incellularnetworksthatdrivetheevolutionofdrugresistance,this chemicalclassesandmechanismsofaction(Table1andtableS2).
phenomenonisasystems-levelcharacteristicandhasbeenobserved Foreachresistantisolate,weperformeddose-responseexperiments
indiversebacteria(34,37),viruses(38),andcancers(9,39).Thus,if todeterminethesusceptibilityprofiles(Fig.1,AtoC)forthe23anti-
collateralsensitivitycanbeexploitedtocounterresistanceandmain- biotics(fig.S1).Theresistancelevelsacquiredduringadaptationfor
taindrugeffectivenessinonebiologicalsystem,itshouldbegenerally 23E.colistrainsasmeasuredbyminimuminhibitoryconcentrations
deployablewithinmanydifferentdiseaseareasinwhichevolutionof (MICs)inculturewereequaltoorabovethecurrentclinicalbreak-
drugresistanceoccurs.Wehypothesizedthatitwouldbepossibleto pointsaccordingtoTheEuropeanCommitteeonAntimicrobialSus-
identifysetsofdrugswithreciprocalcollateralsensitivityprofilesthat ceptibilityTesting(EUCAST)(tableS1).Seventeenofthe23resistant
enabletheirapplicationinacyclicalmannerthatkillsbacteriawhile E.coliisolatesexhibitedcollateralsensitivitytowardatleastoneother
sustainablyselectingagainstdrugresistance(Fig.1D).BecauseGram- drug(Fig.2).Withtheexceptionofthecefuroxime-resistantstrain,which
negativebacteriaareassociatedwithprolongedhospitalization,treat- showednocollateralresistancetowardtheb-lactamamoxicillin,allresist-
mentfailure,andincreasedmortalityratesworldwide(7,40–42),we antstrainswerecollaterallyresistanttowarddrugsfromthesamechemical
testedtheconceptofcollateralsensitivitycyclingintheGram-negative classandtoatleastonedrugfromadifferentclass(Fig.2andfig.S1).
bacteriumE.coli. Excludingdrugswiththesamemechanismofaction,weobservedcollat-
eralresistanceuptoa>32-foldincreaseintheMICcomparedtothewild-
typeE.coli(WT).Ahighlevelofcollateralresistancewasobserved
RESULTS betweenamphenicolandtetracyclinedrugclassesaswellasbetweenam- 13
phenicol,b-lactam,andquinolonedrugclasses(Fig.2Aandfig.S1).The 0
2
Collateral sensitivity profiles b-lactamandquinolonedrugclassesarecommonlyusedinclinicalcy- 4,
WeevolvedparallellineagesofE.coli(MaterialsandMethods)tobe- clingprograms(20,33),andthecollateralresistanceobservedcould 1
r
comeresistantto23clinicallyrelevantantibioticsspanning11distinct contributetothefailureofsuchdrugcyclingprograms. e
b
Althoughweobservedcollateralsen- m
e
sitivitywithmagnitudesofdowntoan v
A B C o
eightfold reduction of MIC, the most N
frequentlyobservedcollateralsensitivity n
o
wasatwofoldreductioninMICrelative g
htw htw htw ttooWtheT.amE.incoolgiltyhcaotsaidcqeucihreedmriceasilstcalanscse, g.or
orG orG orG whichisfrequentlyusedfortreatment ma
e
of hospital-acquired infections with c
n
Drug 1 Drug 2 Drug 3 mgeunlstid(4ru2g)-r(edseisttaainlstGonradmru-ngegcaotmivmepoanthuos-ecie
s
MICR > MICWT MICR = MICWT MICR < MICWT arelistedintableS2),exhibitedincreased m.
sensitivityto14unrelateddrugsfromsev- st
Fig.1. Collateralsensitivity eralchemicallydiversedrugclasses,includ- m
tfaciiylonecndsliiosnitfbgsda.asr(uepAgdpotsloiuncsaCtchte)eipoDgtneirbotiiewlnirttmydhpironurfoaga-- D Drug A t1 Drug itanentgrdabcny-ilctarlicontfeausmr,acsnh,tlopoironalmy(mFpighy.xen2in)ic.so,Tlm,hfeoasccfrooomlllaiytdecerinasl,, aded fro
WT(blackline)andresistant B sensitivityofisolatesresistanttodiffer- o
nl
strain(greenline)asafunction entdrugswithinaparticularchemicalclass w
ofvaryingdrugconcentrations. t0 t2 was not alwaysuniform. For instance, Do
(A)Collateralresistance.Asthe
WT collateralsensitivitytowardpolymyxins,
concentrationofdrug1increases, B
Dr g Drug A-resistant strain fosfomycin,andazithromycinwas ob-
aresreisstiasntacnettsotrtahiniswdrituhgcwolillaltoeurat-l ug A Dru Drug B-resistant strain servedforE.colistrainsresistanttothe
aminoglycosidesamikacin,gentamicin,
competetheWT(orangeshade).
(B)Nochangeinsusceptibility.As t3 andkanamycin,butnotforE.colistrains
theconcentrationofdrug2increases,theresistantstrainwillperformthesameastheWT.(C)Collateral resistanttotheaminoglycosidestrepto-
sensitivity.Whentheconcentrationofdrug3increases,theWTwilloutcompetetheresistantstrain(blue mycin.Ontheotherhand,anisolatere-
shade).(D)Ageneralmodelillustratingtheprincipleofcollateralsensitivitycyclingshowingeradicationof sistanttotheamphenicolchemicalclass
resistantstrainswhendrugswithreciprocalcollateralsensitivityprofiles(AandB)arerotated.Considerthat (chloramphenicol)increasedsensitivity
treatmentofaWTdisease-causingcellpopulation(blackcircles)startswithdrugA(violetarrow)attimet0. towardstreptomycinbutnottowardany
OvertimeinthepresenceofdrugA,resistancetodrugAdevelops(violetcircles),andeventually,drugA
other aminoglycosides tested (Fig. 2).
becomesineffective(t).Then,treatmentisswitchedtodrugB(greenarrow),towhichdrugA–resistantcells
1 These findings indicate that drugs be-
hadbecomecollaterallysensitive(t ).ThistreatmentwillleadtoaneradicationofthedrugA–resistant
2 longingtoaspecificclassdonotalways
cellsandselectionforcellswithWTresistancelevels(MIC ).Eventually,resistancetodrugB(green
circles)develops(t )andtreatmentisswitchedbacktodWruTgA,towhichdrugB–resistantcellshad inducethesamecollateralsensitivity.In-
3
becomecollaterallysensitive,resultinginaneliminationofdrugB–resistantcells(t ).Thus,through stead,drug-specificresistanceevolution
0
therationalcyclingbetweendrugsAandB,drugresistance—andpossiblytreatmentfailure—canbe playsaroleindeterminingthecollateral
selectedagainst. sensitivityprofiles.
www.ScienceTranslationalMedicine.org 25September2013 Vol5Issue204204ra132 2
RESEARCH ARTICLE
Table1.Listofantibioticsusedinthestudy.
Antibiotic Abbreviation Class Target
Amikacin AMI Aminoglycoside Proteinsynthesis,30S
Gentamicin GEN Aminoglycoside Proteinsynthesis,30S
Kanamycin KAN Aminoglycoside Proteinsynthesis,30S
Streptomycin STR Aminoglycoside Proteinsynthesis,30S
Ampicillin AMP b-Lactam Cellwall
Amoxicillin AMX b-Lactam Cellwall
Piperacillin PIP b-Lactam Cellwall
Cefuroxime CFX b-Lactam Cellwall
Cefepime CFP b-Lactam Cellwall
Nalidixicacid NAL Quinolone DNAgyrase
Ciprofloxacin CIP Quinolone DNAgyrase 3
1
0
Levofloxacin LEV Quinolone DNAgyrase 2
Tetracycline TET Tetracycline Proteinsynthesis,30S 4,
1
Minocycline MIN Tetracycline Proteinsynthesis,30S r
e
b
Tigecycline TGC Tetracycline Proteinsynthesis,30S m
e
Chloramphenicol CHL Amphenicol Proteinsynthesis,50S v
o
Azithromycin AZY Macrolide Proteinsynthesis,50S N
n
Colistin COL Polymyxin Lipopolysaccharide o
g
PolymyxinB POL Polymyxin Lipopolysaccharide r
o
Fosfomycin FOS Fosfomycin Cellwallbiogenesis g.
a
Rifampicin RIF Rifamycin RNApolymerase m
e
Nitrofurantoin NIT Nitrofuran Multiple nc
e
Trimethoprim TRI Dihydrofolatereductaseinhibitor Folicacidbiosynthesis ci
s
m.
st
Identification of collateral sensitivity cycles profileobserved betweenthosedrug groups(Figs.2Aand 3Cand m
o
Giventhattheevolutionofresistancetoward23clinicallyrelevant tableS3). r
antibioticsledtosubstantialcollateralsensitivityacrossninediverse However,notalldrugscanbeusedinsimpletwo-drugcycles.For d f
e
chemicalclasses,weidentifiedallpossiblecollateralsensitivitycycles instance,sequentialdeploymentofdrugswithcollateralresistancewill d
a
withinourdataset.First,wecreatedacollateralsensitivitynetwork provideaselectiveadvantagetotheresistantstrainovertheWTandlead o
forE.coliwhereinthenodesofthenetworkarethedrugsandthe toamplificationofresistance.Yet,drugswithcollateralresistanceprofiles wnl
o
directionaledgesofthenetworkrepresentcollateralsensitivitiesre- canbeincludedinthesamecollateralsensitivitycyclewheninterspersed D
sultingfromresistanceevolutiontowardeachofthesedrugs(Fig.3A). byadrugtowardwhichthedrugshaveacollateralsensitivity.Insuch
Forexample,whenastraindevelopsresistancetogentamicin,several cases,collateralsensitivitycyclingwithtwodifferentaminoglycosidedrugs
drugscanbechosentowhichgentamicin-resistantstrainswillbecol- withcollateralresistancecanbeachievedifquinoloneandb-lactamdrugs
laterallysensitive(forexample,colistin,nalidixicacid,orcefuroxime) areincludedinthecycle(forexample,cefuroxime→gentamicin→nalidixic
(Fig.3A).Again,whenresistanceevolvestothenextdrug(forexample, acid→amikacin)(Fig.3AandtableS3).Sequentialdeploymentofsuch
colistin),thetreatmentcanbeswitchedtothenextpossibleoptionto drugsetsbasedontheircollateralsensitivityprofilesshouldcontinueto
whichthecolistin-resistantstrainwillbecollaterallysensitiveandsoon. selectagainstresistancedespitecollateralresistancebetweensomeofthe
Weanalyzedall possiblesequentialdrugdeploymentstrategies cyclecomponentdrugs.
composedoftwotofourdrugs(Fig.3A)thatformedclosedloops
(cycles)withinourcollateralsensitivitynetwork.Weidentified207 Selection against drug resistance through collateral
possiblecycles(Fig.3BandtableS3),suggestingthatseveraloptions sensitivity cycling
existfordeploymentoftailoredcollateralsensitivitycyclesinresponse Totestwhethercollateralsensitivitycyclingofdrugscansuppress
to specific resistance patterns. Certain drugs were includedmore WTresistancedevelopmentovertime,wechosetwoclinicallyrelevant
frequentlyintheidentifiedcollateralsensitivitycycles.Forexam- drugsfromtheaminoglycosideandb-lactamclasses(gentamicinand
ple,ofthe22two-drugcyclesidentified(Fig.3C),59%involveddrugs cefuroxime) that exhibited reciprocal collateral sensitivity profiles
belongingtotheaminoglycoside,polymyxin,ortetracyclinechemi- (Fig.2A).First,wecreatedisogenicindicatorE.colistrainsthatexpress
cal classes as aresultof the strong reciprocal collateral sensitivity eithercyanfluorescentprotein(CFP)oryellowfluorescentprotein
www.ScienceTranslationalMedicine.org 25September2013 Vol5Issue204204ra132 3
RESEARCH ARTICLE
A B
Drug
Strain Sensitivity Resistance
AMI GEN KAN STR AMP AMX PIP CFX CFP NAL CIP LEV TET MIN TGC CHL COL POL AZY FOS RIF INT TRI sensitivity
AMI 14 AMI
GEN 12 GEN
KAN 12 KAN
STR 1 STR
AMP 1 AMP
AMX 0 AMX
PIP 1 PIP
CFX 3 CFX
e
c CFP 0 CFP
n
ats NAL 4 NAL
ise CIP 0 CIP
r n LEV 0 LEV
iartS TMEITN 57 TMEITN
3
TGC 8 TGC 1
CHL 2 CHL 20
COL 9 COL 4,
POL 9 POL 1
AZY 2 AZY er
FOS 0 FOS mb
RIF 3 RIF e
NIT 3 NIT ov
TRI 0 TRI N
n
15 10 5 0 5 10 15 o
senDsriutigvity 2 6 5 5 2 3 4 3 0 4 0 0 8 6 5 5 7 7 5 7 1 9 2 Number of drugs org
g.
a
m
32x 16x 8x 4x 2x 0 2x 4x 8x 16x 32x
e
c
Sensitivity MICWT Resistance n
e
Fig.2. Collateralsensitivityprofilesofdrug-resistantE.colistrains.(A)A resistancefor23drug-resistantE.colistrains(drugsfromthesameclassareex- sci
heatmapshowingdrugsusceptibilityprofilesofdrug-resistantE.colistrainsrel- cluded).Throughoutthefigure,bluecoloringreferstocollateralsensitivity,or- m.
ativetotheWT.SusceptibilityprofilesweredefinedonthebasisofMICinhibition angecoloringreferstocollateralresistance,andwhitecoloringreferstono st
curves(fig.S1).(B)Distributionofdrugcollateralsensitivityandcollateral changeinsusceptibilityrelativetoWT.DrugabbreviationsareshowninTable1. m
o
r
d f
e
(YFP)(MaterialsandMethods).Then,weusedtheCFP-labeledE.coli mixedwithCFP-labeledWTcellsandexposedtogentamicin(1mg/ml), d
a
straintoselectresistantmutantsthatgrewingentamicinat32times resultinginthepreferentialandcompletekillingoftheYFP-labeled o
nl
theMICoftheWTstrain(32×MIC )byrepeatedexposurestoin- cefuroxime-resistantcellsandsurvivaloftheCFP-labeledWTcells(Fig. w
WT
o
creasingconcentrationsofgentamicin.Next,wemixedtheYFP-labeled 4C).Thesedatademonstratedhowcollateralsensitivitycyclingcanbeap- D
WTstrainandtheCFP-labeledgentamicin-resistantstrainandexposed pliedtoselectagainstresistanceandmaintainWTresistancelevelsto
thismixedpopulationtoawiderangeofconcentrationsofcefuroxime. clinicallyrelevantdrugs.Notethatcollateralsensitivityandnotthefitness
Fromtheseexperiments,wediscoveredthatgentamicinresistancecan costofresistanceisthebasisoftheselectionagainstresistance,becausewe
beselectedagainstatcefuroximeconcentrationsof4mg/ml(Fig.4A). readilyrecoveredresistantmutantsfromthemixedpopulationintheab-
Throughasimilarapproach,wefoundthatgentamicinconcentrations senceofdrugselection(Fig.4,AandB).
of1mg/mlselectagainstcefuroximeresistance(Fig.4B). Toassessthereproducibilityofcollateralsensitivityphenotypes
Wethenconductedanexperimenttoprovideproofofprinciple resultingfromresistancedevelopment,werepeatedthecompetition
forcollateralsensitivitycycling.WegraduallyevolvedaCFP-labeled assaybychangingthedrugtowardwhicheachofthelabeledstrains
E.coliWTpopulationtobecomeresistanttowardgentamicinduring wasadapted.Again,weobtainedcomparableresults,whichisconsistent
8daysofrepeatedexposure.Once32×MIC resistancehadbeen withtheassumptionthatevolutionarypathwaysleadingtocollateral
WT
achieved,theCFP-labeledgentamicin-resistantcellsweremixed1:1with sensitivityhavearepeatableadaptivebasis(fig.S2).
YFP-labeledWTcells,andthemixedpopulationwasexposedtocefurox-
ime(4mg/ml)overnight.Thistreatmentswitchledtothepreferentialand Effect of collateral sensitivity on killing kinetics
completekillingoftheCFP-labeledgentamicin-resistantcells(Fig.4C). and mutant selection
Then,weevolvedthesurvivingYFP-labeledcellstobecomeresistantto Wehaveshownthatsequentialdosingofgentamicinandcefuroxime
cefuroximeduring8daysofrepeatedexposure.Once128×MIC resist- constitutesacollateralsensitivitycyclethatselectsagainstresistance.
WT
ancehadbeenachieved,theYFP-labeledcefuroxime-resistantcellswere However,inclinicalsettings,allinfectingpathogensmustbeerad-
www.ScienceTranslationalMedicine.org 25September2013 Vol5Issue204204ra132 4
RESEARCH ARTICLE
A B
PIP 160
AZY
CFX cles 120
y
c
KAN NIT AMP mber of 80
Nu 40
GEN POL
CHL
0
2 3 4
TET AMI Number of drugs
FOS
COL TGC AMX
MIN
3
1
0
NAL 2
C 4,
RIF 1
STR CHL er
b
TRI m
TGC KAN e
v
o
POL CFX N
n
AZY o
g
COL MIN or
AMI g.
0 15 a
Number of drugs m
with collateral sensitivity TET NAL e
c
n
NIT GEN e
ci
s
STR m.
st
Fig. 3. Collateralsensitivitycycles.(A)Collateralsensitivitynetwork berofedgesproceedingfromaparticularnodeandrepresentsthe m
ofE.coli.Nodesofthenetworkarethedrugs,andtheedgesofthe numberofdrugswithcollateralsensitivity.(B)Distributionofthenum- o
networkrepresentcollateralsensitivitiesresultingfromresistanceac- berofpotentialcollateralsensitivitycyclesasafunctionofthenumber d fr
quiredtothosedrugs(Table1).Forexample,ifE.colihasevolvedresist- ofdrugsincludedinthecycle.(C)Drugpairswithreciprocalcollateral e
ancetorifampicin(RIF),itdisplayscollateralsensitivitytotetracycline sensitivity.Thecolorcodingofthenodes(orangetoblue)isproportion- d
a
(TET),minocycline(MIN),tigecycline(TGC),andtrimethoprim(TRI).The altothenumberofcollateralsensitivitypairsofwhichagivendrugisa o
colorcodingofthenodes(orangetoblue)isproportionaltothenum- part(Table1). wnl
o
D
icated;hence,drugconcentrations aboveMIC mustbeapplied. concentrationfallsbelowthemutantpreventionconcentration(MPC).
WT
Wetestedwhethercollateralsensitivityaffectedthekillkineticsof TheMPCisthelowestconcentrationofadrugthatpreventsresistant
theresistantbacteriawhendrugswereappliedatconcentrationsthat mutantsfromappearinginapopulationofaspecifiedsize(43,44).To
exceededtheMIC .Wedeterminedtime-killcurvesforWTE.coli prevent resistance development within thetargetpopulation, drug
WT
andstrainsresistanttocefuroximeorgentamicininthepresenceof concentrationsatthesiteofinfectionshouldbeabovetheMPC.We
16×MIC concentrationsofeitherdrug.Inbothcases,resistantstrains speculatedthattheMPCwouldbereducedproportionallytothere-
WT
wereeradicatedmorequicklythantheWTwhenexposedtothedrugto ductioninMICwhencollateralsensitivityisobserved.Thiswould
whichtheywerecollaterallysensitive(Fig.5,AandB).Thisfindinghas providefurtheradvantageofcollateralsensitivitycyclinginaclinical
importantimplicationsforfutureclinicalapplication,becauseitsug- setting.Totestthishypothesis,wedeterminedtheMPCofgentamicin
geststhattreatmentwithinthecollateralsensitivitycyclingparadigm andcefuroximeforWTE.coliandforstrainsresistanttogentamicin
couldoffermoreeffectiveeradicationofresistantinfectionscompared andcefuroxime.Wefoundthatbothgentamicin-andcefuroxime-
tocyclingofdrugswithoutreciprocalcollateralsensitivities. resistantstrainshadlowerMPCvaluesrelativetoWTforthedrugsto
Wehavecharacterizedcollateralsensitivityintermsofthedrug whichtheywerecollaterallysensitive(Fig.5C).Hence,theadministra-
concentration,whichinhibitsmeasurablegrowthofWTandresistant tionofdrugsbasedoncollateralsensitivitycanpreventtheappearance
strainsdefinedbytheirMIC.However,drugresistancecanbeselected ofresistantmutantsatalowerconcentrationforstrainswithresistance
foratconcentrationsexceedingtheMICduringtherapyifthedrug comparedtotheWT.Thisfurthersupportsthehypothesisthatcollat-
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RESEARCH ARTICLE
A WT C
Gentamicin R
10 Adaptation Adaptation
to gentamicin to cefuroxime
lm 8
1024 Resistant
/UFC goL01 64 µ)lm/g( CIMemixorufec 848 WSeTnsitive
2 Time
0 0.5 1 2 4 8 16
Cefuroxime (µg/ml)
32 Resistant
µC (g/ml)ntamicin 1 WT
MIge 0.5 Sensitive 13
B WT 0
Time 2
10 Cefuroxime R 4,
1
WTCFP WTYFP WTCFP er
lm 8 b
/ m
U e
FC 6 Population v
01 No
go 4 n
L o
g
2 r
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 o
0 0.12 0.25 0.5 1 2 4 g.
a
Gentamicin (µg/ml) Time (days) m
e
Fig. 4. SelectiveeliminationofresistantE.colistrainsbycollateral pleteeradicationoftheCFP-labeledpopulation.ThesurvivingYFP-labeled nc
sensitivitycycling.(AandB)SurvivalofWTE.colistrainsandstrainsre- populationwasthenexposedtocefuroximeoveran8-dayperioduntil cie
sistanttogentamicinorcefuroximewithinamixedpopulationatvarious a 128-fold increase in MIC was achieved. At this point, the YFP- s
concentrationsof(A)cefuroximeor(B)gentamicin.Colony-formingunits labeledcefuroxime-resistanWtpTopulationwasmixedwithaCFP-labeled m.
(CFU)indicatetheproportionofthemutantandWTcellsinthemixed WTpopulationandexposedtogentamicin(1mg/ml),resultinginthe st
m
population.Resultsaremeansofthreereplicates±SD.(C)Collateralsen- eradicationoftheYFP-labeledcefuroxime-resistantpopulation.The
o
sitivitycyclingofgentamicinandcefuroxime.ACFP-labeledWTpopula- datawereconsistentacrossthreereplicates.Greencolorreferstotreat- r
tionwasexposedtogentamicinoveraperiodof8daysuntila32-fold mentwithgentamicin,andvioletreferstotreatmentwithcefuroxime.In d f
e
increaseinMICWTwasachieved.Atthispoint,theCFP-labeledgentamicin- thebottompanel,theproportionofthepopulationthatisCFP-labeled ad
resistantpopulationwasmixedwithaYFP-labeledWTpopulation,and (green)orYFP-labeled(violet)isrepresentedduringthe17-daydrugex- o
treatmentwasswitchedtocefuroxime(4mg/ml),resultinginthecom- posureperiod. nl
w
o
D
eralsensitivitycyclingcanreducethelikelihoodofmultidrugresistance sitivitywithmagnitudesofdownto32-foldcomparedtoMIC (figs.S4
WT
evolution,becauselowerdrugconcentrationsareneededtopreventthe andS5).ThesusceptibilityprofilesoftheevolvedclinicalE.coliisolates
selectionofmultidrug-resistantstrainscomparedtocyclingofdrugs agreedwell,ingeneral,withthoseofthelaboratorystrain(Fig.6).Indeed,
withoutreciprocalcollateralsensitivityprofiles. 94%oftheconditionstestedshowedeitherthesameornochangeintheir
responsetodrugexposureafterresistanceevolution.Sixpercent(fourcon-
Collateral sensitivity in clinical isolates of E. coli ditions)oftheresistance-inducedcollateralsusceptibilitychangesdiffered
To exploretheclinicalrelevanceofcollateralsensitivitynetworks,we betweenthelaboratorystrainandthetwoclinicalisolates.Inthreeof
determinedthecollateralsensitivityprofilesoftwopathogenicE.coliiso- fourcases,thedifferencesobservedincollateralsensitivityandcollateral
lates(MaterialsandMethods).Thepathogenicisolateswereadaptedto resistancebetweentheisolatesinvolvedstrainsresistanttonitrofurantoin
eightantibioticsthatspannedvariouschemicalclasses.Theeightanti- orchangesinthesusceptibilitytowardnitrofurantoin.Nitrofurantoinis
bioticswerechosenasarepresentativesubsetoftheoriginal23antibiotics usedinthetreatmentofcomplicatedurinarytractinfection(45)(tableS2),
(Table1),andthedistributionsofcollateralresistanceandsensitivityob- anditaffectsseveraltargetsinthebacterialcell(46),whichmay,atleast
servedforthestrainswithresistancetowardbothdrugsetsweresimilar(fig. partially,explainthevariationobservedwithrespecttocollateralsensitivity
S3).Wedeterminedthecollateraleffectsofdrugresistancedevelopmentfor andcollateralresistanceforthestrainstested.Thissimilaritysuggeststhat
eachisolateandfoundthatallresistantisolatesexhibitedcollateralsensitiv- collateralsensitivityprofilesarelargelypreservedacrossmultipleisolates.
ity(Fig.6).Foroneoftheclinicalisolates,weevenobservedcollateralsen- Overall,therobustnessofthecollateralsensitivityprofilesfortheevolved
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RESEARCH ARTICLE
A WT Gentamicin R Drug
7
GEN AMP CFX NAL MIN COL AZY NIT
16x Cefuroxime
MG1655 GEN
6 EC3770 GEN
lm EC3856 GEN
/ 5
U
F MG1655 AMP
C
go01 4 EECC33787506 AAMMPP
L
3
MG1655 CFX
e
2 0 0.16 0.3 0.5 1 2 4 5 6 24 cnatsis EECC33787506 CCFFXX
e
Time (hours) r g MG1655NAL
urd EC3770 NAL
B WT Cefuroxime R de EC3856 NAL
v
7 lo 3
6 16x Gentamicin ve dna e MEECCG331786755065MMMIIINNN 4, 201
lm/UFC 5 talosI MECG31767505CCOOLL mber 1
g01 4 EC3856 COL ve
o o
L MG1655AZY N
3 EC3770 AZY n
o
EC3856 AZY g
2 r
o
0 0.16 0.3 0.5 1 2 4 5 6 24 MG1655NIT g.
a
Time (hours) EC3770 NIT m
EC3856 NIT e
c
C n
WT e
ci
Gentamicin R s
32x 16x 8x 4x 2x 0 2x 4x 8x 16x 32x m.
2 4 8 16 st
Cefuroxime (µg/ml) Sensitivity MICWT Resistance m
o
Fig. 6. CollateralsensitivityprofilesforE.coliclinicalisolates.Aheat d fr
WT mapshowingdrugsusceptibilityprofilesoftworesistantE.colipathogenic e
isolates(EC3770 andEC3856)andtheMG1655 strain, which evolved d
a
Cefuroxime R resistancetoeightdrugs.Susceptibilityprofilesweredefinedonthebasis o
nl
0.5 1 2 4 ofMICinhibitioncurvesfortheresistantstrainsandWT(figs.S1,S4,and w
S5).Bluecoloringreferstocollateralsensitivity,orangecoloringreferstocol- o
Gentamicin (µg/ml) D
lateralresistance,andwhitecoloringreferstonochangeinsusceptibility
Fig.5. Alterationofthekillingdynamicandmutantselectionwindow relativetoWT.DrugabbreviationsarelistedinTable1.
ofresistantE.colistrainsbycollateralsensitivity.(AandB)Inthepres-
enceofadrugtowhichresistantstrainsarecollaterallysensitive,both
resistancewhenselecteddrugsarecycledoptimally.Incollateralsen-
gentamicin-andcefuroxime-resistantstrainswereeradicatedfasterthan
sitivitycycling,selectionagainstresistancewasbasedprimarilyonan
theWT.Resultsaremeansofthreereplicates±SD.(C)Strainsresistant
togentamicinorcefuroximealsohadalowerMPCrelativetoWTwhen increaseindrugsensitivityimposedbyrationallyselecteddrugswith
exposedtodrugstowhichtheywerecollaterallysensitive. reciprocal collateral sensitivities,whichis fundamentally different
fromtheprincipleofcurrentdrugcycling,whichreliesonthedilution
ofresistantstrainsintheabsenceofselectionforresistanceandresulting
clinicalisolatessuggeststhatgeneralcollateralsensitivitycyclescouldbe fromfitnesscostsofresistance.
effectivefortreatingawiderangeofE.colipathogens. Theaimofanydrugtreatmentisthecompleteeradicationofthe
diseasebyexposingdisease-causingcellstotherapeuticconcentra-
tions of drug within the host. In principle, drug treatment based
DISCUSSION oncollateralsensitivitycyclingshouldexhibitsuperiorefficacyinpa-
tientsinfected withboth the fullysusceptible bacterialpopulation
Thisstudyprovidesproofofprinciplethatanunderappreciatedside andresistantmutantscomparedtocyclingofdrugswithoutcompatible
effectofresistance,collateralsensitivity,canbeusedtotargetdrug collateralsensitivityprofiles.
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RESEARCH ARTICLE
Ourfindingssuggestthatcollateralsensitivitycyclingshouldbeap- MIC and IC determination
90
plicablefortheclinicalmanagementofGram-negativebacterialinfec- Strainsweretakenfrom−80°C,streakedontoLBagarsupplemented
tions using already approved antibiotics. It is, perhaps, even more withantibiotic(Table1),andincubatedovernightat37°C.ForMIC
importanttoapplythisprinciplefortreatmentofchronicinfections determination,overnightcultureswerepreparedfromculturesfrom
andinfectioninwhichmultiplepathogensareinvolved,suchascystic LB agar plates. About 1 × 104cells were prepared form overnight
fibrosis(47).Insuchcases,theapplicationofthree-orfour-drugcycles culturesandgrownat37°Cwithshakingin96-wellmicrotiterplates
couldenhancetheefficacyoftreatmentwhendesignedoptimallyon containing200mlofmediumperwell.MICsweredeterminedinlog-
thebasisofcommonpathogensusceptibilityprofiles.However,fur- arithmicdrugconcentrationgradientwithtwofolddilutions.Endpoint
therdrugresistanceandsensitivityprogramsareneededtoexplore absorbancemeasurements(A )weretakenafter18hoursofincuba-
600
theconceptwithinthesetherapeuticareas,andclinicalstudiesare tionwithaplatereader(EpochBiotech)andbackground-subtracted.
needed totestwhetherourexpectationsareconfirmedinhospital Thepercentageofinhibitionwascalculatedaccordingtothefollowing
settings. formula:1−[A drug/A control].Theinhibitoryconcentrationwas
600 600
Here,wedemonstratedtheprincipleofcollateralsensitivitycycling definedasthelowestconcentrationofthedrugthatinhibited90%ofthe
forresistanceacquiredthroughadaptiveevolution,whichisthemain growthofthestraintested(IC ).Forstrainsinwhichinhibitionwas
90
pathwayleadingtoresistanceininfectiousdiseases,suchastuberculosis notuniformacrossthethreesetsofexperimentsperformed,twoaddi-
andmalaria,andincancer.Indeed,drugswitchingbasedoncollateral tionalsetsofexperimentswereperformedtodeterminedrugsuscepti-
sensitivityobservedincancertreatmenthasshownsuccessfultherapeu- bility.TheresultsobtainedaresummarizedinFig.2. 3
1
ticresults(48,49).Weanticipatethatcollateralsensitivitycyclingwill 0
2
contributetothesustainableuseofdrugsintheclinicforthemanage- Susceptibility profiles 4,
mentofdiseasesinwhichevolutionarydrugresistanceisaconcern. StrainsusceptibilityprofilewasdeterminedrelativetotheWT(Fig.2 1
r
andfig.S1).Collateralresistancewasdefinedwhenthegrowthofa e
b
resistantstrainwasinhibitedatahigherdrugconcentrationrelative m
MATERIALS AND METHODS totheWT(MICResistantstrain>MICWT;Fig.1A).Nochangeinsen- ove
sitivitywasdefinedwhenthegrowthofresistantstrainandWTwas N
Media, bacterial strains, and drugs inhibitedatthesamedrugconcentration(MIC =MIC ; n
ResistantstrainswerederivedfromE.coliMG1655andtwopathogenic Fig.1B).Collateralsensitivitywas defined whReensisttahntestgrarionwth ofWthTe og
E.colistrainsEC3770andEC3856providedbythelaboratoryofclin- resistantstrainwasinhibitedatlowerconcentrationsthantheWT or
icalmicrobiologyatHvidovreHospital(Denmark).Allstrainsusedin (MIC <MIC ;Fig.1C).Collateralsensitivityprofiles g.
Resistantstrain WT a
thestudyarederivedfromE.coliMG1655.Strainswereadaptedtothe wereusedtovisualizecollateralsensitivitynetworkswithCytoscape m
e
23antibioticslistedinTable1.Allexperimentswereconductedinmod- 3.0.1.software. c
n
ifiedLBmedium[peptone(10 g/liter),yeastextract(5g/liter),and e
NaCl(10g/liter)].Alldrugsolutionswerepreparedfrompowderstock Isogenic CFP- and YFP-labeled strains ci
s
andfilter-sterilized.Thedrugstockswerestoredat−20°Cformaximum pZE21 MCS1 (50) plasmid containing kanamycin resistance marker m.
of90days. withfluorescentlylabeledCFPorYFPwaselectroporatedintoE.coli st
MG1655.Theselabeledisogenicstrainswereusedforthecompetition m
o
Strain adaptation assaydescribedbelow. r
Strainsevolvedresistancetoselecteddrugsbygradientplatemethod(34) d f
e
onLBagar.Theselectionforresistancewasrepeatedduringaperiodof10 Competition assay d
a
days.OvernightculturesofE.coliwerestreakedontothewholegra- Westartedthisexperimentbyselectingtheresistantmutanttoward o
nl
dientplate.After18hoursofincubationat37°C,theedgeofgrowth gentamicin.WTlabeledwithCFPwasgraduallyadaptedto32×MIC w
o
wassweptwithaloopandappliedtoafreshgradientplate.Thepro- gentamicinaccordingtotheprotocoldescribedabove.Then,theCFP- D
cesswasrepeateduntilgrowthwasobtainedfromthewholeplate.Af- labeledgentamicin-resistantstrainwasmixedinequalproportions
terthisprocedure,strainsevolvedresistanceto21drugs(Table1andtable withtheWTlabeledwithYFP.Eachstrainatabout5×104CFU/ml
S1).Strainsadaptedtopolymyxindrugclass(colistinandpolymyxinB) wasincubatedin5mlofLBbrothsupplementedwithvariouscon-
withgradientplatesdidnotcorrelatebetweentheresistanceonsolid centrationsofcefuroxime(0,0.5,1,2,4,8,and16mg/ml).After18hours
andliquidmedium(strainresistanceinliquidmediumwasfourfold ofincubationat37°Cwithshaking,serialdilutionswereplatedonto
lowerthanthelevelofadaptationongradientplate).Therefore,the LBagarplatessupplementedwithkanamycin(50mg/ml)andwith
resistancetocolistinandpolymyxinBwasevolvedbyserialpassagein kanamycin and gentamicin (8 mg/ml). Plate supplemented with
liquidmediumaccordingtothefollowingprotocol.About106cells kanamycin allowedgrowthofWTandresistantstrain,whereasthe
fromovernightcultureswereaddedtotwofoldserialdilutionofco- growthofWTwasinhibitedonagarplatessupplementedwithboth
listinorpolymyxinBin10mlofLBbroth.Aftertheovernightin- kanamycin and gentamicin. Viable cell growth was evaluated at
cubationat37°C,absorbancemeasurementsat600nm(A )were 18hoursafterincubationat37°C.Onthebasisofthisexperiment,
600
takenwithaplatereader(EpochBioTek).Theculturesgrownatthe weselectedtheconcentrationofcefuroxime(4mg/ml)atwhichonly
highestantibioticconcentrationforwhichA valuewasabove0.4 WT strain survived,whereas gentamicin-resistant strain was erad-
600
wereusedforfurtheradaptation.Again,about106cellsfromovernight icated. After this selection step, we continued the exposure of the
cultureswereaddedtotwofoldserialdilutionofcolistinorpolymyxinB YFP-labeledWTstraintotheincreasingconcentrationsofcefuroxime,
andincubatedovernight.Thisstepwasrepeatedfor10days.Allstrains asdescribedabove.After7daysofcefuroximeexposure,thestrain
werestoredin15%glycerolat−80°C. evolved 128× MIC was selected (YFP-labeled cefuroxime-resistant
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