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International Journal of Antimicrobial Agents
jou rn a l h o m e pa g e:h tt p : / / w w w . e l s e v i e r . c o m / l o c a t e / i j a n t i m i c a g
Challenging the concept of bacteria subsisting on antibiotics
Fiona Walsh
a,∗, Sebastian G.B. Amyes
b, Brion Duffy
aaBacteriology,Agroscope–ResearchStationChangins-WädenswilACW,FederalDepartmentofEconomicAffairs,EducationandResearch(EAER), Wädenswil,Switzerland
bMolecularChemotherapy,MedicalMicrobiology,MedicalSchool,UniversityofEdinburgh,Edinburgh,UK
a r t i c l e i n f o
Articlehistory:
Received28January2013 Accepted28January2013
Keywords:
Catabolism Antibiotic Subsistence
-Lactam Streptomycin Trimethoprim
a b s t r a c t
Antibioticresistanceconcernshavebeencompoundedbyareportthatsoilbacteriacancataboliseantibi- otics,i.e.breakdownand usethemasasolecarbon source.Todatethishasnotbeenverifiedor reproduced,thereforeinthisstudysoilbacteriawerescreenedtoverifyandreproducethishypothe- sis.Survivalinhighconcentrationsofantibioticswasinitiallyobserved;however,onfurtheranalysis thesebacteriaeitherdidnotdegradetheantibioticsortheyusedanintrinsicresistancemechanism(- lactamases)todegradethe-lactams,asdemonstratedbyhigh-performanceliquidchromatography.
Theseresultsdidnotverifyorreproducethehypothesisthatbacteriasubsistonantibioticsorcatabolise antibioticsaspreviouslyreported.Thisstudyidentifiedthatbacteriawithacatabolisingphenotypedid notdegradestreptomycinortrimethoprimandthereforecouldnotutilisetheantibioticsasanutrient source.Therefore,weconcludethatsoilbacteriadonotcataboliseantibiotics.
© 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
Antibioticresistanceis emergingasoneofthegreatestchal- lengestohumanhealth[1].Withinthepast20years, antibiotic resistancehasdevelopedfromresistancetosingleclassesofantibi- oticstomultidrugresistance(MDR)andextensivedrugresistance (XDR)[2].Catabolism,i.e.thecapacityofbacteriatonotonlyresist buttosubsistonantibiotics,hasbeenpresentedasapotentially crucialstepintheevolutionofantibioticresistancefromMDRand XDRtountreatable infections. In 2008,a novelhypothesiswas introduced,namelyantibioticcatabolism,definedbytheidenti- ficationofsoilbacteriafromdifferentphylathatwerecapableof degradingandutilisingdifferentclassesofantibioticsasasolecar- bonsource[3].Bacterialcatabolismofantibioticsisaconceptthat surpassesantibioticresistanceandMDRintermsofcomplexityin treatingandmanagingbacterialinfections.Therefore,itisimpor- tant forthefuture successful managementof antibiotic-treated bacterialinfectionsthatweareabletodistinguishbetweenconven- tionalresistancemechanismsandtheabilityofbacteriatosubsist onantibiotics.
Researchonantibioticdegradationwasreportedinthe1970s, butthenovelstudyonmultipleantibioticclassescatabolisedby soilbacteriaexposedthefullextentanddistributionofantibiotic- degradinggenesintheenvironment[4].Thisstudyhasspecifically
∗Correspondingauthor.Tel.:+41447836329.
E-mailaddresses:fiona.walsh@agroscope.admin.ch,fiona1walsh@gmail.com (F.Walsh).
led tothe scientific belief that soil bacteria contain previously uncharacterisedantibiotic-degradingresistanceenzymesandthat soil bacteriacan subsistonantibiotics, withrespect toat least 18differentantibiotics[3].Theonlyresistance-degradingmecha- nismdescribedpriortothisstudywasthedegradationof-lactam antibioticsbythe-lactamaseresistancemechanisms.Thecharac- terisedmechanismsofresistancetoallotherclassesofantibiotics donotincludedegradation.Thisstudyrevolutionisedthecurrent thinkingonhowbacteriacanresistantibioticswhilstalsoidentify- ingsoilbacteriaasapotentiallylargereservoirofnovelresistance mechanisms.Antibioticcatabolismwouldprotecttheentirebac- terial community from the inhibitoryeffects of antibiotics and concurrentlyincreasethepopulationsizeastheyusetheantibiotics asanutrientsource.
Althoughthehypothesisofantibioticcatabolismbysoilbacteria waspublished5yearsago,thishypothesishasneitherbeenrepro- ducednorverified.Theaimsofthecurrentstudywerethereforeto verifythehypothesisofsoilbacteriasubsistingonantibiotics.
2. Materialsandmethods 2.1. Samplingandsitedescriptions
Thesoilsamplingsites,locationsandelevationsaredescribed inTable1.SoilpHwasdeterminedbysuspending1gofsoilin 2.5mLof0.01MCaCl2andmeasuringthepHusingaglasselec- trode[5].ThepHofeachsoilwasdeterminedthreetimesandthe mean±standarddeviationisgiveninTable1.
0924-8579/$–seefrontmatter© 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
http://dx.doi.org/10.1016/j.ijantimicag.2013.01.021
F.Walshetal./InternationalJournalofAntimicrobialAgents41 (2013) 558–563 559
Table1
Geographicanddescriptivecharacteristicsoftheanalysedurban,agriculturalandpristinesoilsamples.
SampleID Description Environmentalmatter Elevation(m) Latitude Longitude pH(n=3)
FWS1 WädenswilAppleOrchard Orchardsoil 407 47.2333 8.6667 7.0±0.1
FWS2 BenedictineAbbey Farmsoil 880 47.1167 8.75 7.1±0.1
FWS3 Hospitalgarden Lawnsoil 667 46.7167 9.4333 7.3±0
FWS4 LindauAppleOrchard Orchardsoil 485 47.4833 8.2 4.1±0.2
FWS5 GüttingenAppleOrchard Orchardsoil 503 47.6 9.2833 5.3±0.1
FWS6 Rütlimeadow Meadowsoil 835 46.9667 8.6 7.1±0.2
FWS7 AreabesideLakeZürich Lawnsoil 408 47.3667 8.55 7.0±0.1
FWS8 Matterhornmountaintrail Alpinesoil 1936 46.0167 7.75 7.2±0.1
FWS9 Farmlandtreatedwithpigmanure Manuredsoil 592 47.1833 8.3167 5.7±0.2
FWS10 MountainnearZürich Mountainforestsoil 700 47.3496 8.492 7.0±0
2.2. Samplingstrategy
Foreachsamplesite,eightsoilcoresampleswerecombined.
Soilsamplesconsistedofeightsoilcores(10cmdepth)perrepli- catetakenusingastainlesssteelcorerwithaninternaldiameterof 2.5cm.Soilcoreswerepooledforeachreplicateinthefield.Pooling ofsoilcoresisstandardlyappliedinordertoobtainmorerepre- sentativesamplesforacertainfieldplotoraspecificexperimental treatment[6–8].
2.3. Isolationofantibiotic-catabolisingbacteriafromSwisssoil
Soilbacteria withanantibiotic-catabolising phenotypewere isolatedand cultured usingmethods described previouslyfrom tensoilsunderavarietyofanthropogenicinfluencesfromurban, pristineandfarmlandsoils(Table1)[3].Minimalmedium[single carbonsource(SCS)]brothculturesofputativecatabolisingbacteria were serially diluted and spread-plated onto SCS agar supple- mentedwiththeappropriatecorrespondingantibiotic[3].Negative controlscomprisedSCSagarwithoutantibiotic,inoculatedinthe samemannerasdescribedbyDantasetal.[3].Theantibioticstested comprisedpenicillin,dicloxacillin,amikacin,cefalexin,kanamycin, gentamicin, sisomicin, streptomycin, vancomycin, levofloxacin, ciprofloxacin,sulfamethizole,nalidixicacid,chloramphenicol,d- cycloserine,cefotaxime,novobiocin,trimethoprim,sulfisoxazole, tetracycline,erythromycin,colistinandrifampicin.Allantibiotics and chemicals, except kanamycin and vancomycin (Carl Roth GmbHandCo.KG,Karlsruhe,Germany),wereobtainedfromSigma- AldrichChemieGmbH(Buchs,Switzerland).
Twobacteriadescribedashavingthecapacitytocatabolisecar- benicillin and penicillinwere obtainedfromthe Dantasgroup:
CA-S3F-1,Burkholderiasp.;andPE-S2R-1,Burkholderiasp.Dueto unforeseencircumstancesintheDantaslaboratory,furtherisolates catabolisingnon--lactamantibioticscouldnotbeprovided.
Theabilitiesoftheantibiotic-catabolisingbacteriatogrowor surviveinsterile-filteredSCSmediumwithoutaddedantibiotics wereinvestigated.Assimilableorganiccarbon-freeglasswarewas prepared as previously described [9]. Bacteria were inoculated intoSCSmediumandgrownat22◦Cfor3days.Then,10Lwas removedandaddedtoSCSmediumwithandwithoutantibioticata finalconcentrationof1g/L.Totalbacterialcountswereperformed onDays0,5,7,14,21and28byplatecountsonLuria–Bertani(LB) agar.Thetotalorganiccarbonanddissolvedorganiccarbon(DOC) concentrationsweredeterminedaspreviouslydescribed[10].
2.4. Resistanceprofilingofantibiotic-catabolisingbacteria
Theresistanceprofileofeachisolatewasdeterminedasprevi- ouslydescribedusing1mg/mLofeachantibiotic[11].Antibiotic susceptibility tests were performed in duplicate. Where there wasadiscrepancybetweenthetworesults,susceptibilitytesting
wasperformedathirdtime[12,13].Twoconsistentsusceptibility resultsweretakenasthefinalresult.
2.5. Bacterialphylogeneticprofiling
Thephylogeneticprofilesofthebacteriaweredeterminedas previouslydescribed[3].Briefly,thevariableregions(nucleotides 63–1389) of the 16S rRNA genes were amplified by PCR and sequenced.Thephylogenyofthebacteriabasedonsequencevari- ationofthe16SrRNAgeneswasidentifiedusingBLASTnandthe Greengenes 2011database.Aphylogenetic treeofthebacterial speciesbasedonthe16SrRNAsequenceswasconstructedusing theneighbour-joiningalgorithminARB[14].
2.6. High-performanceliquidchromatography(HPLC) investigationofbacterialantibioticdegradation
Degradationof carbenicillin (bacteriaCA-S3F-1, Burkholderia sp.), penicillin (bacteria PE-C-1, Pseudomonas sp. and bacteria PE-S2R-1,Burkholderiasp.),streptomycin(bacteriaST-C-1,Achro- mobactersp.)andtrimethoprim(bacteriaTR-C-1,Pseudomonassp.) wasinvestigatedbyHPLC[1]performedusingaDionexPDA-100 photodiodearraydetector,Chromeleonv.6.80software(Dionex, Idstein,Germany)andtheconditionsdescribedinTable2.Aliquots (80L)ofculturesgrowninSCSmediumcontaining1mg/mLof antibioticwereinoculatedinto20mLofSCSmediumcontaining 1mg/mLof appropriateantibioticandincubatedat22◦Cfor 28 days.SCS–antibioticmediumcontainingnobacteriawasalsoincu- batedat22◦C.Then,1mLwasremovedatDays0,2,4,7,14,21 and28andwasanalysedinduplicateusingHPLC.Followingthe initialresultsoftheHPLCexperiments,allbacterialisolateswere alsoinoculatedintoSCSmediumcontainingnoantibioticasneg- ativecontrolsatafinalconcentrationof103 or104CFU/mLand incubatedat22◦Cfor28days.
2.7. Biologicalactivityoftheantibioticsolutionsfollowing incubationwiththecatabolisingbacteria
Thebiologicalactivitiesofthetrimethoprimandstreptomycin HPLCsolutionscontainingtherespectivecatabolisingbacteriafrom Days0,14 and28wereinvestigatedusinganadaptationofthe antibioticdisk diffusionassay [15]. Staphylococcus aureus ATCC
Table2
Descriptionofthehigh-performanceliquidchromatography(HPLC)experimental conditionsandcolumns.
Antibiotic Wavelength (nm)
Flowrate (mL/min)
Column
Penicillin 220 0.7 Nucleosil100-5,C18
Carbenicillin 220 0.7 Nucleosil100-5,C18
Streptomycin 195 0.7 Nucleosil100-5,C18
Trimethoprim 250 0.7 Nucleosil100-5,C18
Fig.1.Antibioticresistanceprofilesof140isolatesfromtensoils,showingpercentageofisolatesresistanttoantibioticsataconcentrationof1mg/mL.
25923(0.5McFarland)wasspreadonLBagarplates.TheHPLCsolu- tions(Days0,14and28)weresterile-filteredand15Lwasadded toasterilefilterpaperdiskontheS.aureusagarplates.Theplates wereincubatedat37◦Candthezonediametersofthediskdiffusion assaysweremeasuredafter16h.
2.8. Extractionofˇ-lactamaseenzymesandHPLCinvestigation ofˇ-lactamdegradationbytheˇ-lactamaseenzymes
CulturesofCA-S3F-1,PE-C-1andPE-S2R-1grownintherespec- tiveantibiotic(1mg/mL)-containingLBbrothat26◦Cwithshaking overnightwerediluted1in10inantibiotic(1mg/mL)-containing LBbrothandincubatedforafurther4h.Thefiltered(0.2mfilters), washedsupernatantswereusedinthecrude-lactamaseenzyme assays using nitrocefin at 100M at a wavelength of 486nm [16].EscherichiacolitransformedwithpUC19plasmid(AmpC- lactamase-producing)wasusedasapositivecontrol.Degradation ofcarbenicillinandpenicillinwasinvestigatedusingHPLCinthe samemannerastheHPLCantibioticdegradationexperiments.
3. Results
Bacteria presenting a catabolism phenotype (growth in SCS medium containing 1mg/mL of antibiotic) were isolated from all ten soil sources consisting of farmland, pristine soil and urban environments (Table 1). The antibiotics on which the bacteria presented a catabolism phenotype comprised peni- cillin, dicloxacillin, amikacin, kanamycin,gentamicin, sisomicin, streptomycin, cefalexin, vancomycin, trimethoprim, cefotaxime, sulfamethizole and erythromycin, representing many different classes ofantibiotics, including natural,semisynthetic and syn- thetic. All tensoils contained bacteria presenting a catabolism phenotype both to the -lactam antibiotic penicillin and the macrolideerythromycin.Intotal,140bacteriawithanantibiotic- catabolisingphenotypewereidentified.
Antibioticresistanceprofiling oftheisolatedbacteriaidenti- fiedthat>80%werecapableofgrowthat1mg/mLof almostall antibiotics(Fig.1).Thepatternanddistributionofhigh-levelresis- tancedidnotinallcasescorrespondtothelevelsofcatabolism
against that antibiotic. However, for the fluoroquinolones and tetracycline, whichhad lowlevelsofresistance, nocatabolising isolateswereidentified.16SrRNAgeneticphylotypingidentified thatthemajorityofbacteriawerePseudomonadales(n=67)fol- lowedbyBurkholderiales(n=22)(Fig.2),whichweresimilarto thephylotypespreviouslyidentified[3].TheActinomycetaleswere theonly Gram-positive bacteriaidentified. Allbacterialisolates werecapableofgrowthinSCSmediumwithoutantibiotic,asthis mediumwasnotcarbon-freeasdescribedbyDantasetal.butcon- tained15mgofethylenediaminetetra-aceticacid(EDTA)perlitre ofSCSmedium[3].DOC testsidentifieda DOCconcentrationof 8.53mg/Lin theSCSmedium [9]. Thus, themedium contained sufficientcarbonwithoutantibioticstosupportbacterialgrowth.
Thisconstituentwasoverlookedbytheseminalstudy[3].Growth ofselectedstrainsofbacteriainSCSmediumwithoutantibiotics increasedbyaminimumof2loggrowthover28days(Fig.3).
TheHPLCexperimentswereperformedtoinvestigatewhether theantibioticsstreptomycin(bacteriaST-C-1,Achromobactersp.), trimethoprim(bacteriaTR-C-1,Pseudomonassp.),penicillin(bacte- riaPE-C-1,Pseudomonassp.andPE-S2R-1,Burkholderiasp.)and carbenicillin(bacteriaCA-S3F-1,Burkholderiasp.)weredegraded inthepresenceofthecatabolisingbacteria(Fig.4a).Thebacte- ria used in the penicillin and carbenicillin HPLC experiments includedisolates(bacteriaPE-S2R-1andbacteriaCA-S3F-1)previ- ouslyreportedasantibioticcatabolisers[3].Threedifferentclasses of antibiotic, which have different target sites and resistance mechanisms, were chosen. Streptomycin is a protein synthesis pathwayinhibitor,trimethoprimisadihydrofolatesynthesispath- wayinhibitor,andthe-lactamscarbenicillinandpenicillinare cellwallsynthesis inhibitors.Therewerenostatisticallysignifi- cantdifferencesbetweentheconcentrationsofstreptomycinand trimethoprimantibioticswithandwithoutthebacteria,asdeter- mined by Student’s t-test. Bacteria were viable after 28 days incubationastestedbygrowthonLBagar.Theseresultsindicated thatstreptomycinandtrimethoprimwerenotdegradedover28 days andthebacteria werestillviable at Day28. Theseantibi- oticswerenotcatabolisedeventhoughthebacteriapresentedwith thesamephenotypeaspreviouslydescribedantibioticcatabolis- ers.Thebiologicalactivitiesofstreptomycinandtrimethoprimat
F.Walshetal./InternationalJournalofAntimicrobialAgents41 (2013) 558–563 561
Fig.2. Phylogeneticdistributionof140bacterialisolatesfromtensoils.
Day28 wereinvestigated usingantibioticdiskdiffusionassays.
The diameters of the zones of inhibition did not decrease for streptomycinortrimethoprimsolutionsbetween0and28days.
StreptomycinandtrimethoprimwereasbiologicallyactiveatDay 28asatDay0,confirmingthattheydidnotlosetheirbiological activitiesaswouldbeexpectediftheyweredegraded.
The-lactamantibioticspenicillinandcarbenicillindegraded in thepresence and absence ofthe bacteria. Penicillinand the
-lactamantibiotics arenotstableand degradeeasily,which is whypenicillinwassodifficulttoproduceinlargequantitiesini- tiallyafteritsdiscovery.Therateandextentofdegradationwere higherwhenthebacteriawerepresentthaninthemediumlack- ingbacteria(Fig.4a).The-lactamantibioticsaretheonlyclass ofantibioticsknowntobedegradedduetoaresistancemecha- nism,namelythe-lactamases.PseudomonasandBurkholderiaspp.
bothcontainchromosomallymediatedAmpCenzymescapableof degrading-lactamantibiotics[17,18].
The -lactam-degrading bacteria (PE-C-1, PE-S2R-1 and CA- S3F-1)wereinvestigatedfortheproductionof-lactam-degrading
-lactamases.Productionof-lactamaseswasconfirmedbydiges- tionofnitrocefinasdetectedbyultravioletspectrophotometryover time.Therateof-lactamdegradationwasdeterminedbyplotting theabsorbanceagainsttimeforeachbacteria.Usingtheseplots,the slopesofthelinesforCA-S3F-1(carbenicillin),PE-S2R-1(penicillin) andPE-C-1(penicillin)were0.0642x+0.1202,0.0097x+0.0977and 0.0041x+0.1278,respectively,indicatingthatdegradationofthe
-lactamringwasfastestbythe-lactamaseextractedfromCA- S3F-1, followed by PE-S2R-1 and then PE-C-1. Therefore, each bacteriacontained-lactamasescapableofdegradingthe-lactam ringofthe-lactamantibiotics.Theconcentrationofproteininthe assayswasthesameandthusthedifferencesinratesofdegradation werenotduetovariationsinprotein(enzyme)concentrations.
Degradation of carbenicillin and penicillin by the extracted
-lactamaseswasfurtherinvestigated byHPLC (Fig. 4b).These resultsidentifiedthatthe-lactamasesextractedfromPE-S2R-1 (describedinapreviousstudyasa-lactamcataboliser)digested
Fig.3.Growthofthefivebacteriapresentingcatabolismphenotypesinsinglecarbonsource(SCS)mediumwithoutantibioticover28days.Thefivebacteriaarethoseused inthehigh-performanceliquidchromatography(HPLC)experiments:PE-S2R-1,Burkholderiasp.;PE-C-1,Pseudomonassp.;CA-S3F-1,Burkholderiasp.;ST-C-1,Achromobacter sp.;TR-C-1,Pseudomonassp.
Fig.4.High-performanceliquidchromatography(HPLC)analysisofantibioticdegradationover28daysinthepresenceof(a)bacteriaandthecorrespondingantibiotics and(b)-lactamasesandthecorrespondingantibioticsincomparisonwithdegradationbythebacteria.PE-S2R-1,Burkholderiasp.;PE-C-1,Pseudomonassp.;CA-S3F-1, Burkholderiasp.;ST-C-1,Achromobactersp.;TR-C-1,Pseudomonassp.
penicillintoalmostnothingbyDay4.Therateofdegradationby theextracted-lactamaseisalmostidenticaltothatofthebacteria (PE-S2R-1).The-lactamasesfromCA-S3F-1andPE-C-1showed asimilarrateofdegradation.Therefore,noadditionalmechanism otherthantheantibioticresistancemechanism(-lactamase)was requiredbyanyofthesebacteriatodegradethe-lactamantibi- oticspenicillinandcarbenicillin.
Asstreptomycinandtrimethoprimwerenotdegradedafter28 daysof incubation withthebacteria,concentrations ofthefive bacteria usedin the HPLCexperiments weremonitored in SCS mediumwithoutantibioticover28days.Thehypothesiswasthat thebacteria may beviable at the end of the 28 days but that theyhave not grown over the 28 days and thus couldsurvive
ina non-growingstate.Theinitialbacterialcountsrangedfrom 1.27×103CFU/mLto1.55×104CFU/mL(Fig.3).
4. Discussion
Reproducibilityandverificationofresultsarevitalcomponents ofthebasis ofscientific discovery.We aimedtoreproduceand verifythediscoveryofsoilbacteriacapableofcatabolisingantibi- otics[3].Withinthe4yearssincethishypothesiswasreported,the paperdescribingtheseresultshasbeencited145times(Google Scholar,28January2013).However,thehypothesishasnotbeen verifiedorreproduced.Wepropose,basedontheresultsofthe current studyanda lackofverifiedresultsconcurringwiththe
F.Walshetal./InternationalJournalofAntimicrobialAgents41 (2013) 558–563 563
hypothesisofsoilbacteriacatabolisingantibiotics,thatsoilbacte- ria do not catabolise theantibiotics and do not use anynovel mechanismofdegradation.Usingbacteriafromthepreviousstudy andfromthisstudy,wehaveidentifiedthatthe-lactamswere degradedbythewell-characterised-lactamaseresistancemech- anism.Thesoilbacteriadonotthereforerepresentasourceofnovel antibiotic-degradingenzymes.Thisstudyhasalsoidentifiedthat bacteriawiththecatabolisingphenotypedidnotdegradestrep- tomycinortrimethoprim.Weproposethatnoevidencehasbeen showntoindicatethatsoilbacteriacandegradeanyotherclasses ofantibiotics.The‘carbon-free’medium(SCS)contained15mg/mL EDTAthat,accordingtoDantasetal.,wouldbesufficienttosupport bacterialgrowth[3].Thiswasoverlookedbytheseminalstudy.
Usingthesamemethodologiesaspreviouslyreported,thisstudy hasidentifiedsoilbacteriawithhigh-levelresistanceto14different antibioticsfromdifferentclasses[3].Bacteriawiththesamepheno- typehavepreviouslybeendescribedassubsistingontheantibiotics asasolecarbonsource[3].However,thecurrentstudyhasiden- tifiedthatbacteriawithacatabolismphenotypedidnotdegrade streptomycinortrimethoprim.Resistancetotheseantibioticshas todatebeenrestrictedtotargetsitemutation,modification,efflux, productionofanadditionaltarget,orhyperproductionofthetarget site [19]. High-level resistance in Pseudomonas and Burkholde- ria spp. has been associated with intrinsic resistance to these antibiotics[20,21].The-lactamantibioticsweredestroyedinthe presenceofthesoilbacteria,howeverdestructionoftheantibi- oticswasdue totheproductionof -lactamases. Productionof
-lactamases wasfirst reported in 1940 [22]. Therehas been, however,sincethennoevidencetosuggestthatproductionof- lactamasesislinkedtotheutilisationandsubsistenceofbacteria onantibioticsasasolecarbonsource.
Using the same methods and some of the bacteria as the previousstudydescribingsubsistenceonantibioticsasasolecar- bonsource,wecometoacontradictoryconclusion.Weconclude that thebacteria previously described as using antibiotics as a solecarbonsourcehaveeithernotdegradedtheantibiotics,and thuscannotutilisewholeantibioticsasacarbonsource,orhave degradedtheantibioticsusingwell-characterisedresistancemech- anisms,whichhavenotpreviouslybeenlinkedtoutilisationasa solecarbonsource.
Acknowledgments
ThisworkwasconductedwithintheEuropeanresearchnetwork COSTTD0803Detectingevolutionaryhotspotsofantibioticresis- tancesinEurope(DARE).TheauthorsthankA.Baechli,T.Poigerand H.-U.Weilenmannfortechnicalassistanceanddiscussion.
Funding:ThisprojectwasfundedbytheSwissFederalOfficefor Agriculture,theSwissFederalOfficefortheEnvironmentandthe SwissExpertCommitteeforBiosafety(SECB).
Competinginterest:Nonedeclared.
Ethicalapproval:Notrequired.
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