Antimicrobial resistance acquisition via natural transformation: context is everything

Antimicrobial resistance acquisition via natural transformation: context is everything

Macaulay Winter

, Angus Buckling

, Klaus Harms

, Pa˚l Jarle Johnsen

and Michiel Vos

Naturaltransformationisaprocesswherebacterialcells activelytakeupfreeDNAfromtheenvironmentandrecombine itintotheirgenomeorreconvertitintoextra-chromosomal geneticelements.Althoughthismechanismisknownto mediatetheuptakeofantibioticresistancedeterminantsina rangeofhumanpathogens,itsimportanceinthespreadof antimicrobialresistanceisnotalwaysappreciated.Thisreview highlightsthecontextinwhichtransformationtakesplace:in diversemicrobiomes,ininteractionwithotherformsof horizontalgenetransferandinincreasinglypolluted

environments.Thisexaminationoftheabioticandbioticdrivers oftransformationrevealsthatitcouldbemoreimportantinthe disseminationofresistancegenesthanisoftenrecognised.

Addresses

1EuropeanCentreforEnvironmentandHumanHealth,Universityof ExeterMedicalSchool,EnvironmentandSustainabilityInstitute,Penryn Campus,TR109FE,UnitedKingdom

2DepartmentofBiosciences,UniversityofExeter,PenrynCampus, TR109FE,UnitedKingdom

3MicrobialPharmacologyandPopulationBiologyResearchGroup, DepartmentofPharmacy,FacultyofHealthSciences,UiTTheArctic UniversityofNorway,Tromsø,Norway

Correspondingauthor:Vos,Michiel(m.vos@exeter.ac.uk)

CurrentOpinioninMicrobiology2021,62:133–138 ThisreviewcomesfromathemedissueonEnvironmental microbiology

EditedbyMarie-CecilePloyandThomasBerendonk

https://doi.org/10.1016/j.mib.2021.09.009

1369-5274/ã2021TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.

org/licenses/by/4.0/).

Introduction

Thewidespreadselectionfor antibioticresistancegenes (ARGs) through over and misuse of antimicrobials and exposure to pollutants that co-select for antimicrobial resistance (AMR) is one of the most pressing concerns inhealthcareglobally[1].TomitigatethespreadofARGs withinandbetweenreservoirsofenvironmentalbacteria andhumanpathogens,itisvitaltogainadetailedunder- standingofthemechanismsthatdrivetheirtransmission.

One mechanism of horizontal gene transfer (HGT) is natural transformation, a process wherein cells take up

DNAfromtheextracellularenvironmentandincorporate it intotheirchromosomeor reassembleitas partof the self-replicatingepisome[2].Transformationisevolution- aryconserved,phylogeneticallywidespread[3]andcapa- ble of mediatingtheacquisition of largetracts of DNA (7 50kb) [4].

Importantly, transformationhasbeenshowntoresultin the transfer of clinically relevant ARGs in a variety of human pathogens (Table 1). These examples notwith- standing,transformationisgenerallyconsideredtonotbe as importantinthetransferofARGscompared toHGT mechanisms based on Mobile Genetic Elements (MGEs), particularly conjugation (e.g. Refs. [5,6]).

Although this may be true, it is likely that the role of transformation in ARG dissemination is underappreci- ated. This could be due to two main reasons. First, transformation-mediated gene transfer is more difficult to quantify than mostother HGT mechanisms. Unlike MGEs suchas prophagesorintegrativeconjugativeele- ments,transformationdoesnotleavedistincttracesinthe DNAoftherecipient.Transformationcanalsoresultin thedeletion,rather thantheadditionof DNA,which is undistinguishablefrom mutationalloss.In addition,the presenceofplasmidsmightoftenautomaticallybeattrib- utedtoconjugation,whereasitispossiblethatsomewere taken up via transformation. Second, only a relatively small number of bacteria have been shown to take up DNA under laboratory conditions, likely because the specific physiological requirements to initiate the com- petence staterequired for transformation werenot met [2,3]. Furthermore, in those species amenable to lab experiments, transformation rates could frequently be underestimated using standard antibiotic marker-based assays[7].

In thisshort review,we will focusonahost of exciting new studies that indicate that transformation could be moreimportantthanoftenisassumed,particularlyinthe contextofAMRspread.Specifically,wewillfocusonthe role of inter-strain and inter-species interactions, the interaction oftransformation withMGEs andtheeffect ofpollutiononratesoftransformation,specificallyinthe lightof ARGspread.

Transformationand interactionsbetween strains andspecies

Recent years have seen somewhat of a paradigm shift whereitisnolongerautomaticallyassumedthattransfor- mation-mediated recombination is atthe mercy of ran- dom death of donor bacteria that happen to be in the vicinityofrecipientcells.Instead,ithasbecomeclearthat recipientsoftentakeanactiveroleinacquiringDNA[8].

Forinstance,Vibriocholerae[9]andAcinetobacterbaylyi[10]

employ Type-VIsecretion system (T6SS)to lyse other strainsand species,freeingDNAwhichsubsequentlyis takenupbytransformation.Transformationefficiencyis predictedtodecreasewithincreasinggeneticdivergence becauseof theconstraints imposed byhomology-based recombination[11].However,whenstrainsobtainDNA forrecombinationbylysingotherstrains,increaseddiver- gence in toxin gene content facilitates lysis, making transformation-mediated recombination with more divergeddonorstrainsmoreefficientthanthatwithmore closelyrelatedstrains[12].AroundhalfofallV.cholerae strains harbour temperate kappa phage K139 which is ableto killneighbouringnon-lysogenicstrains;genomic DNAthusreleasedcouldbeshowntobeincorporatedby thelysogenichost [13].Inthis case, thelysogenactsat leastpartlyin theinterestof itshost and thuscouldbe hypothesised to function akin to T6SS. Both genetic mechanisms provide the host strain with an advantage becausecompetitorsarelysed(interferencecompetition, a form of natural selection) but also because they may facilitate thetargeted uptakeand recombination of for- eignDNA(akintomatechoice,aformofsexualselection [14]).

Bothphylogeneticdistanceand ecologicalspecialisation generallyformstrongbarrierstoHGT[15–17]andtrans- formationismosteffectivebetweenrelatedstrainsinha- bitingthesame(micro)niche.However,thevastnumber of bacterialcells meansthat HGTisa ‘numbersgame’

and ARGs occasionally crossdeep ecological and taxo- nomic divides [15,18]. Proteobacteria are prevalent in soils but also represent an important group of human pathogens and are thought to be an important conduit throughwhichARGsmovebetweenlineagesandniches [16].ArecentgenomicstudyonsoilActinomycetesand Proteobacteriabuilds aconvincing caseof genetransfer fromProteobacteriatoActinomycetesandbackagain,in whattheauthorstermthe‘carrybackmodel’[19].Here, aProteobacteriumtransfersaplasmidtoanActinomycete viaconjugation,afterwhichanactinobacterialtransposon insertsintotheproteobacterialplasmid.Aftercelldeath oftheactinobacterialhost,thisDNAisreleasedintothe environmentwhereitcanbetakenupbyProteobacteria throughnaturaltransformation,withtheflankingproteo- bacterialsequencemediatingefficient recombination of actinomyceteARGsintothechromosome.Goodinsilico evidenceexistsforallstepsinthisprocessandthisstudy furthermoreexperimentallydemonstratedtheuptakeof recombinant proteobacterial-actinomycete DNA by A.

baylyivianaturaltransformation [19].

Transformationandmobile genetic elements TextbookexplanationsofHGTcustomarilylisttransfor- mation, transduction and conjugation as distinct pro- cesses, but it is clear from the example above that HGT can progress via combinations of transformation

Table1

Examplesofinter-speciesARGtransferinhumanpathogensmediatedbynaturaltransformation.

Recipientspecies DonorSpecies ARGs Antibiotics Description Reference

A.baumannii K.pneumoniaeCRKp,Providencia rettgeriM15758(bla_NDM-1)and methicillin-resistantStaphylococcus aureus‘Cordobes’clone(SAC) (mecA)

blaTEM-1,blaKPC-2, bla_SHV-11,bla_SHV-

12,bla_KPC,bla_OXA-

23,bla_NDM-1,mecA

Meropenem, cefotaxime, ampicillin

Interspeciescell-freeDNAwas addedtoA.baumanniiculturesand successfullytransformed.Resulting transformantswereresistantto multipleantibiotics

[46]

Enterococcus faecalis

Escherichiacoli,Pseudomonas aeruginosaandSalmonella aberdeen

Notspecified Kanamycin, ampicillin, tetracycline

TransferofRP4fromchlorine-killed antibiotic-resistantbacteriato chlorine-injuredbacteriavianatural transformation.

[41]

Haemophilus influenzae,H.

suis,H.

parainfluenzae

H.influenzae,H.suis,H.

parainfluenzae

Notspecified Streptomycin DNAconferringstreptomycin resistancefromHaemophilus specieswasusedtotransform differentspeciesofHaemophilus

[49]

S.pneumoniae, S.oralis,S.mitis

S.oralis,S.mitis,S.sanguisandS.

constellatus,S.pneumoniae

parCandgyrA Fluoroquinolones (Pefloxacinand Sparfloxacin)

Cell-freeDNAconferring fluoroquinoloneresistancefrom Streptococcusspecieswasusedto transformmultipleother

Streptococcusspecies

[50]

Neisseria meningitidis

N.cinereaandN.flavescens penA Penicillin Cell-freeDNAconferringpenicillin resistancefromNeisseriaspecies wasusedtotransformN.

meningitidis

[51]

andMGE-basedmechanismsaswell.Agoodexampleof the blurred boundariesbetween HGT mechanisms are plasmids, which canbe transferredvia conjugation,but also via transformation, transduction [20] and vesicle transfer [21] (thelatter ofwhich is rarely listed among HGTmechanisms).Asisthecaseforconjugation,trans- formation can facilitate plasmid spread to phylogeneti- cally unrelated recipients [22], but the relative impor- tance of conjugation versus transformation in plasmid spread hasnotreceivedmuchattention.Distinctadvan- tages anddisadvantagescouldbelistedforeachmecha- nism.Disadvantagesforconjugationinclude:alivedonor cell and closecell–cell proximityare required,resident plasmidscouldsuppresstransferofcompatibleplasmids, plasmidtransferratescanbeslow[23],andsome(smaller) plasmidsrequiremobilization byco-residentconjugable elements.Amaindisadvantageoftransformationrelative to conjugation is that it is sensitive to the action of DNases and extracellular DNA degradation (although it has been argued that transformation is less sensitive to disinfection thanconjugation in thatitis notdepen- dent onlive cells[4]).Sincetransformation isbased on the uptakeof single strandsof DNA,atleasttwo com- plementary strands need to be taken up for successful plasmidreassemblyinthenewhost.Therefore,plasmid transformation efficiency is expected to drop with the squareoftheplasmidsize[24].Indeed,plasmidtransfor- mationhasbeenshowntobelesseffectivethanchromo- somal DNA (e.g. Ref. [25]). When comparing transfor- mationefficiencyofgenomicDNAversusplasmidDNA, plasmidshavetheadvantagethattherearenobarriersof sequencehomology(sincenorecombinationisinvolved) and relative uptake efficiencycouldbe increasedwhen plasmidcopynumbersindonor cellsarehigh.

Thetransferofgeneticelementsviatransformationisnot limited to plasmids. Recent experimental studies have shown thatgeneticelements harbouringARGs that are notcapableoftheirownmobilisationcanbetransferred via transformation as well. Experiments using A. baylyi havedemonstratedthatintegronsontransposonsoronIS elements [26], or ARGs located in fully heterologous DNA [27] can insertchromosomally. Efficientincorpo- ration of the DNA requires sections of DNA with sequence identity or similarity and therefore likely depends on the cellular homologous recombination machinery. Genomicinsertionefficiency decreaseswith increasing sequenceheterology due to DNA mismatch repair [28] or other recombination obstacles such as disruption of synteny[29].One-sided sequencehomol- ogy may be sufficient in some prokaryotes to allow integration, by using a second recombination event betweenheterologousDNAmolecules(homology-facili- tatedillegitimaterecombination)[30,31].Intheabsence of any homology, genomic integration of genes occurs rarely by mechanisms still poorly understood (double illegitimate recombination) [32]. As an interesting

exception, transposable elements taken-up by a cell caninsertthemselvesintothechromosomeevenwithout homologyrequirements[26],indicating thattransforma- tionofARGsassociatedwithtransposonscouldbeespe- cially efficient.Arecentstudyin A.baylyidemonstrated thatthegenesconferringthetranspositionareexpressed whentheforeignDNAcontainingthesegenesistempo- rarilyprotectedfromdegradationinthecytoplasm[33].

Finally,diverseconjugativeelementshavebeenshownto disable transformation in a range of species through a variety of pathways, including insertion directly into competence-related genes [34],sRNA-mediated silenc- ing[35]ornucleaseproduction[36].Ithasbeenproposed that transformation could be selectively advantageous because of its tendency to remove MGEs from the chromosome via recombination of flanking sequences of donor DNA that lack the same MGEs [34]. Any MGEabletodisruptprocessesthatcouldresultintheir removalthusisexpectedtobeselectivelyfavoured.Such interactions between transformation and MGE-based mechanismsofHGTseempervasiveacrossmanybacte- rial speciesand arelikely important driversofbacterial genomeevolution[4,34].

Transformationand anthropogenic pollution Unprecedented, human-driven global changes increas- ingly affect the distribution, activity and diversity of microbialcommunities,andcouldevenchangethemode and rate of bacterial evolution itself [37]. It has been known forsome timethatstress imposedbyantibiotics canupregulatecompetence[38,39].Severalrecentstud- ies haveprovided evidencethatevendisinfectantssuch as chlorine[40]and triclosan[41] foundin low concen- trations in contaminated environments can upregulate transformation,therebyenhancingthepotentialforARGs tospread.Onestudyfoundthataby-productofdrinking water disinfection,bromoacetic acid,increasedtransfor- mationfrequencyinA.baylyiinadose-dependentmanner [42].Thiswasfoundtobemediatedviaoxidativestress leadingto upregulationofRecA.Mixtures ofwaterdis- infection by-products at environmentally relevant con- centrations resulted in alow but significant increasein transformation frequency. Several non-antimicrobial drugs,includingthecommonlyprescribedanti-inflamma- torydrugDiclofenac,werefoundtoelevatethetransfor- mationrateofaplasmidcontainingampicillinandtetra- cycline resistance genes in A. baylyi to asimilar extent (1 3-fold) asintheafore-mentionedstudy[43].Again, thiseffectwasmediatedthroughanincreasein reactive oxygen species, but in addition genes involved in the transformation process were found to be upregulated [43].Asanytypeofcelldamagecanresultin thestress responseslinkedtotheupregulationoftransformation,it islikelythatotheranthropogenicstressors,forexample, metal pollutionor increasedsolar irradiation,couldalso be important [42]. Finally, stressors can co-occur and

additively or synergistically interact and apart from increasing transformation frequencies could simulta- neously increase ARG transfer through the killing of donorcells [40].

Conclusions

Thisshortreviewhighlightedsomeofthediversebiotic andabioticdriversofARGspreadbynaturaltransforma- tion(Figure1).ARGtransformationtakesplaceincom- plex microbial communities, including in host micro- biomes [44], and both donor and recipient cells are influenced bymyriad other strains, species andgenetic elements.For instance,transformationcanbeenhanced through the action of lytic phages [45] releasing donor DNA,and atthesametime canmediate thetransferof

geneticelementssuchaslysogens[46].Naturaltransfor- mationisanunderappreciatedmechanismofgenetrans- fer,which is demonstrated bythe fact it wasonly very recentlydiscoveredtooperateintheimportantopportu- nistic pathogen Pseudomonas aeruginosa [47]. Transfor- mationmight be especiallyimportant in thetransfer of plasmidsbetweenunrelatedspecies[10,19,22],playing animportant role in paving ‘highways of gene transfer’

[17].Itis also increasinglyclear that antimicrobialscan result in increased transformation rates, as has been shownbeforeforconjugation(e.g. Ref.[48]).

CaninterventionstopreventARGspreadbytransforma- tionbeimagined?Scopeforthisseemsverylimiteddue to the diverse and interconnected ways transformation governsgenetransfer.Somehaveevenarguedtheoppo- site: thedeletion of ARG-containing MGEs from chro- mosomescould in theorybe promoted byupregulating transformation [4]. Itis possible that disinfection mea- suresdesignedtoreducetheexposuretoAMRpathogens couldbecounterproductivebyselectingfor transforma- tion-mediated ARG transfer. The same goes for phage therapy, which could result in (plasmid-borne) ARG releasethroughcelllysis,enhancingtransformationeffi- ciency[45].Transformation was thefirst mechanism of horizontal gene transfer to beuncovered, but new dis- coveries continue to bemade at arapid pace. A better understandingofthecontextsin whichnaturaltransfor- mation takes places will provide valuable insights into bacterial evolutionin general and theconditions under whichAMRisdisseminatedspecifically.

Conflictofintereststatement Nothingdeclared.

Acknowledgements

KHisfundedbytheNorwegianResearchCouncil(grant275672).MV acknowledgessupportfromtheNationalEnvironmentResearchCouncil (NERC;grantNE/T008083/1).MWacknowledgessupportfromthe NationalEnvironmentResearchCouncil(NERC)andtheGW4+Doctoral TrainingPartnership(grantNE/S007504/1).Thisstudydidnotgenerate newdata.TheFigurewascreatedwithBioRender.com.Weapologiseto authorswhoserelevantworkcouldnotbefeaturedduetospaceconstraints.

Referencesand recommendedreading

Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:

ofspecialinterest ofoutstandinginterest

1. HuttnerA,HarbarthS,CarletJ,CosgroveS,GoossensH, HolmesA,JarlierV,VossA,PittetD:Antimicrobialresistance:a globalviewfromthe2013WorldHealthcare-associated InfectionsForum.AntimicrobResistInfectControl2013,2:1-13.

2. BlokeschM:Naturalcompetencefortransformation.CurrBiol 2016,26:R1126-R1130.

3. JohnstonC,MartinB,FichantG,PolardP,ClaverysJ-P:Bacterial transformation:distribution,sharedmechanismsand divergentcontrol.NatRevMicrobiol2014,12:181-196.

Figure1

Strain and Species Interactions

Interactions with MGEs

Upregulators of Transformation

Current Opinion in Microbiology

Asummaryofsometheprocessesinfluencingtransformation,with recipientcellsinblueanddonorcellsinyellow.Mechanismsinvolving conspecificcellsandotherspecies(toppanel)includeT6SS-mediated killing,lysogen-mediatedkillingand‘backtransfer’inconjunctionwith athirdpartnerandconjugation.MechanismsinvolvingMobileGenetic Elements(middlepanel)includetheuptakeofplasmidsand

transposons.Finally,anthropogenicpollutants(bottompanel)can upregulatetransformation.Mechanismsareexplainedinmoredetailin themaintext.

4. BlokeschM:Inandout—contributionofnaturaltransformation totheshufflingoflargegenomicregions.CurrOpinMicrobiol 2017,38:22-29.

5. VonWintersdorffCJ,PendersJ,VanNiekerkJM,MillsND, MajumderS,VanAlphenLB,SavelkoulPH,WolffsPF:

Disseminationofantimicrobialresistanceinmicrobial ecosystemsthroughhorizontalgenetransfer.FrontMicrobiol 2016,7:173.

6. PetersonE,KaurP:Antibioticresistancemechanismsin bacteria:relationshipsbetweenresistancedeterminantsof antibioticproducers,environmentalbacteria,andclinical pathogens.FrontMicrobiol2018,9:2928.

7. GodeuxA-S,LupoA,HaenniM,Guette-MarquetS,WilharmG, LaaberkiM-H,CharpentierX:Fluorescence-baseddetectionof naturaltransformationindrug-resistantAcinetobacter baumannii.JBacteriol2018,200.

8. VeeningJ-W,BlokeschM:Interbacterialpredationasastrategy forDNAacquisitioninnaturallycompetentbacteria.NatRev Microbiol2017,15:621.

9. BorgeaudS,MetzgerLC,ScrignariT,BlokeschM:ThetypeVI secretionsystemofVibriocholeraefostershorizontalgene transfer.Science2015,347:63-67.

10. CooperRM,TsimringL,HastyJ:Inter-speciespopulation dynamicsenhancemicrobialhorizontalgenetransferand spreadofantibioticresistance.eLife2017,6:e25950.

11. MajewskiJ,ZawadzkiP,PickerillP,CohanFM,DowsonCG:

Barrierstogeneticexchangebetweenbacterialspecies:

Streptococcuspneumoniaetransformation.JBacteriol2000, 182:1016-1023.

12. StefanicP,BelcijanK,KraigherB,NesmeJ,KovacJ,SorensenSJ, VosM,Mandic-MulecI:Kindiscriminationpromoteshorizontal genetransferbetweenunrelatedstrainsinBacillussubtili.Nat Commun2021,12:1

Demonstratesthattransformationefficiencyishigherbetweenlessclo- selyrelatedB.subtilisstrainsduetodivergenceintoxin-antitoxingenes facilitatinglysisofdonorstrains.

13. Molina-QuirozRC,DaliaTN,CamilliA,DaliaAB,Silva- ValenzuelaCA:Prophage-dependentneighborpredation fostershorizontalgenetransferbynaturaltransformation.

mSphere2020,5.

14.

VosM,BucklingA,KuijperB:Sexualselectioninbacteria?

TrendsMicrobiol2019,27:972-981

ExploreswhetherDNAreleaseandDNAuptakeviatransformationcould beanalysedintheframeworkofsexualselectiondevelopedforanimals.

15. PehrssonEC,TsukayamaP,PatelS,Mejı´a-BautistaM,Sosa- SotoG,NavarreteKM,CalderonM,CabreraL,Hoyos-ArangoW, BertoliMT:Interconnectedmicrobiomesandresistomesin low-incomehumanhabitats.Nature2016,533:212-216.

16. ForsbergKJ,PatelS,GibsonMK,LauberCL,KnightR,FiererN, DantasG:Bacterialphylogenystructuressoilresistomes acrosshabitats.Nature2014,509:612-616.

17. BeikoRG,HarlowTJ,RaganMA:Highwaysofgenesharingin prokaryotes.ProcNatlAcadSciUSA2005,102:14332-14337.

18. ForsbergKJ,ReyesA,WangB,SelleckEM,SommerMO, DantasG:Thesharedantibioticresistomeofsoilbacteriaand humanpathogens.Science2012,337:1107-1111.

19. JiangX,EllabaanMMH,CharusantiP,MunckC,BlinK,TongY, WeberT,SommerMO,LeeSY:Disseminationofantibiotic resistancegenesfromantibioticproducerstopathogens.Nat Commun2017,8:1-7

Provides genomic and experimentalevidence for DNA transfer from ProteobacteriatoActinomycetesandbackagaintoProteobacteriavia acombinationofconjugationandtransformation.

20. AmmannA,NeveH,GeisA,HellerKJ:Plasmidtransfervia transductionfromStreptococcusthermophilusto Lactococcuslactis.JBacteriol2008,190:3083-3087.

21. RumboC,Ferna´ndez-MoreiraE,MerinoM,PozaM,MendezJA, SoaresNC,MosqueraA,ChavesF,BouG:Horizontaltransferof theOXA-24carbapenemasegeneviaoutermembrane

vesicles:anewmechanismofdisseminationofcarbapenem resistancegenesinAcinetobacterbaumannii.Antimicrob AgentsChemother2011,55:3084-3090.

22. KothariA,SonejaD,TangA,CarlsonHK,DeutschbauerAM, MukhopadhyayA:Nativeplasmid-encodedmercuryresistance genesarefunctionalanddemonstratenaturaltransformation inenvironmentalbacterialisolates.mSystems2019,4.

23. DionisioF,MaticI,RadmanM,RodriguesOR,TaddeiF:Plasmids spreadveryfastinheterogeneousbacterialcommunities.

Genetics2002,162:1525-1532.

24. SikorskiJ,GraupnerS,LorenzMG,WackernagelW:Natural genetictransformationofPseudomonasstutzeriinanon- sterilesoil.Microbiology1998,144:569-576.

25. KnappS,BrodalC,PetersonJ,QiF,KrethJ,MerrittJ:Natural competenceiscommonamongclinicalisolatesofVeillonella parvulaandisusefulforgeneticmanipulationofthiskey memberoftheoralmicrobiome.FrontCellInfMicrobiol2017, 7:139.

26. DominguesS,HarmsK,FrickeWF,JohnsenPJ,DaSilvaGJ, NielsenKM:Naturaltransformationfacilitatestransferof transposons,integronsandgenecassettesbetweenbacterial species.PLoSPathog2012,8:e1002837.

27. Hu¨lterN,WackernagelW:Doubleillegitimaterecombination eventsintegrateDNAsegmentsthroughtwodifferent mechanismsduringnaturaltransformationofAcinetobacter baylyi.MolMicrobiol2008,67:984-995.

28. MajewskiJ,ZawadzkiP,PickerillP,CohanFM,DowsonCG:

Barrierstogeneticexchangebetweenbacterialspecies:

Streptococcuspneumoniaetransformation.JBacteriol2000, 182:1016-1023.

29. RayJL,HarmsK,WikmarkO-G,StarikovaI,JohnsenPJ, NielsenKM:SexualisolationinAcinetobacterbaylyiislocus- specificandvaries10,000-foldoverthegenome.Genetics 2009,182:1165-1181.

30. PrudhommeM,LibanteV,ClaverysJ-P:Homologous recombinationattheborder:insertion-deletionsandthe trappingofforeignDNAinStreptococcuspneumoniae.Proc NatlAcadSciUSA2002,99:2100-2105.

31. deVriesJ,WackernagelW:IntegrationofforeignDNAduring naturaltransformationofAcinetobactersp.byhomology- facilitatedillegitimaterecombination.ProcNatlAcadSciUSA 2002,99:2094-2099.

32. HarmsK,LunnanA,Hu¨lterN,MourierT,VinnerL,AndamCP, MarttinenP,FridholmH,HansenAJ,HanageWP:Substitutions ofshortheterologousDNAsegmentsofintragenomicor extragenomicoriginsproduceclusteredgenomic polymorphisms.ProcNatlAcadSciUSA2016,113:15066- 15071.

33.

KloosJ,JohnsenPJ,HarmsK:Tn1transpositioninthecourse ofnaturaltransformationenableshorizontalantibiotic resistancespreadinAcinetobacterbaylyi.Microbiology2021, 167:001003

Thispapershowsthatduringnaturaltransformation,transposonslocated inextracellularDNAcan insertthemselvesintotherecipient genome whenthetaken-upDNAinthecytoplasmistemporarilyprotectedfrom nucleotyticdegradationandexpressed.

34. CroucherNJ,MostowyR,WymantC,TurnerP,BentleySD, FraserC:HorizontalDNAtransfermechanismsofbacteriaas weaponsofintragenomicconflict.PLoSBiol2016,14:

e1002394.

35. DurieuxI,GinevraC,AttaiechL,PicqK,JuanP-A,JarraudS, CharpentierX:Diverseconjugativeelementssilencenatural transformationinLegionellaspecies.ProcNatlAcadSciUSA 2019,116:18613-18618.

36. DaliaAB,SeedKD,CalderwoodSB,CamilliA:Aglobally distributedmobilegeneticelementinhibitsnatural transformationofVibriocholerae.ProcNatlAcadSciUSA 2015,112:10485-10490.

37. VosM:Theevolutionofbacterialpathogensinthe Anthropocene.InfGenEvol2020,86:104611.

38. CharpentierX,PolardP,ClaverysJ-P:Inductionofcompetence forgenetictransformationbyantibiotics:convergent evolutionofstressresponsesindistantbacterialspecies lackingSOS? CurrOpinMicrobiol2012,15:570-576.

39. PrudhommeM,AttaiechL,SanchezG,MartinB,ClaverysJ-P:

Antibioticstressinducesgenetictransformabilityinthe humanpathogenStreptococcuspneumoniae.Science2006, 313:89-92.

40. JinM,LiuL,WangD-n,YangD,LiuW-L,YinJ,YangZ-W,WangH- R,QiuZ-G,ShenZ-Q:Chlorinedisinfectionpromotesthe exchangeofantibioticresistancegenesacrossbacterial generabynaturaltransformation.ISMEJ2020,14:1847-1856.

41. LuJ,WangY,ZhangS,BondP,YuanZ,GuoJ:Triclosanat environmentalconcentrationscanenhancethespreadof extracellularantibioticresistancegenesthrough transformation.SciTotalEnviron2020,713:136621.

42. Mantilla-CalderonD,PlewaMJ,MichoudGG,FodelianakisS, DaffonchioD,HongP-Y:Waterdisinfectionbyproducts increasenaturaltransformationratesofenvironmentalDNAin AcinetobacterbaylyiADP1.EnvironSciTechnol2019,53:6520- 6528.

43. WangY,LuJ,Engelsta¨dterJ,ZhangS,DingP,MaoL,YuanZ, BondPL,GuoJ:Non-antibioticpharmaceuticalsenhancethe transmissionofexogenousantibioticresistancegenes throughbacterialtransformation.ISMEJ2020,14:2179-2196 Demonstratesthatfrequentlyprescribednon-antibioticpharmaceuticals cansignificantlyfacilitatetransformationofARGsatclinicallyandenvir- onmentallyrelevantconcentrations.

44.

DingC,YangD,MaJ,JinM,ShenZ,ShiD,TianZ,KangM,LiJ, QiuZ:Effectsoffreeantibioticresistancegenesinthe environmentonintestinalmicroecologyofmice.Ecotoxicol EnvironSaf2020,204:111119

Providesthefirstexperimentalevidenceoftransformationinthegastro- intestinaltract.

45. KeenEC,BliskovskyVV,MalagonF,BakerJD,PrinceJS, KlausJS,AdhyaSL:Novel“superspreader”bacteriophages promotehorizontalgenetransferbytransformation.mBio 2017,8.

46. TragliaGM,PlaceK,DottoC,FernandezJS,Montan˜aS,dos SantosBahienseC,Soler-BistueA,IriarteA,PerezF, TolmaskyME:InterspeciesDNAacquisitionbyanaturally competentAcinetobacterbaumanniistrain.IntJAntimicrob Agents2019,53:483-490.

47.

NolanLM,TurnbullL,KatribM,OsvathSR,LosaD,LazenbyJJ, WhitchurchCB:Pseudomonasaeruginosaiscapableofnatural transformationinbiofilms.Microbiology2020,166:995 DemonstratesthattheimportanthumanpathogenPseudomonasaeru- ginosaiscapableofnaturaltransformation.

48. WangY,LuJ,MaoL,LiJ,YuanZ,BondPL,GuoJ:Antiepileptic drugcarbamazepinepromoteshorizontaltransferofplasmid- bornemulti-antibioticresistancegeneswithinandacross bacterialgenera.ISMEJ2019,13:509-522.

49. LeidyG,HahnE,AlexanderHE:Onthespecificityofthe desoxyribonucleicacidwhichinducesstreptomycin resistanceinHemophilus.JExpMed1956,104:305-320.

50. JanoirC,PodglajenI,KitzisM-D,PoyartC,GutmannL:Invitro exchangeoffluoroquinoloneresistancedeterminants betweenStreptococcuspneumoniaeandviridans streptococciandgenomicorganizationoftheparE-parC regioninS.mitis.JInfectDis1999,180:555-558.

51. BowlerLD,ZhangQ-Y,RiouJ-Y,SprattBG:Interspecies recombinationbetweenthepenAgenesofNeisseria meningitidisandcommensalNeisseriaspeciesduringthe emergenceofpenicillinresistanceinN.meningitidis:natural eventsandlaboratorysimulation.JBacteriol1994,176:333- 337.