Identification of a Stable Chromosomal Tandem Multicopy of bla(VIM-63), a New bla(VIM-2) Carbapenemase
Abstract
This study characterizes a new genetic structure containing a multicopy of a bla(VIM-2) variant with an A676C substitution, bla(VIM-63). This gene was detected on the chromosome of two carbapenem-resistant clinical strains of Citrobacter freundii ST22 recovered from two patients, separated by a 6-month period, and previously in Pseudomonas aeruginosa ST2242 from the same hospital unit. Short-read sequencing was used to characterize the new variant in both species, and long-read sequencing was used to characterize the genome of C. freundii. On the P. aeruginosa chromosome, the bla(VIM-63) gene was inserted between ISPsy 42-type sequences, flanked by an intl1 sequence, nearby aph(3′)-VI, and sul1. On the C. freundii chromosome, the bla(VIM-63) gene was inserted into a Tn6230-like transposon as a stable five-tandem-repeat multimer, flanked by the same intl1 as in P. aeruginosa. This structure was stable across subcultures and did not change in the presence of carbapenems. The bla(VIM-63) gene was cloned into the pCR-Blunt plasmid to study antimicrobial susceptibility patterns and into pET29a for kinetic activity analysis. VIM-63 showed higher K(m) values than VIM-2 for ceftazidime and cefepime and higher k(cat) values for cefotaxime, ceftazidime, imipenem, and ertapenem, without differences in MIC values. This is the first study to describe this new variant, VIM-63, in two different species with a chromosomal location integrated into different mobile elements and the first to describe a stable multimer of a metallo-β-lactamase. Despite the amino acid substitution, the susceptibility pattern of the new variant was similar to that of VIM-2. IMPORTANCE: VIM group metallo-β-lactamases are usually captured by IntI1 integrases. This work describes the detection for the first time of a novel, previously unknown variant of VIM-2, VIM-63. This carbapenemase has been found on the chromosome of two different species, Citrobacter freundii and Pseudomonas aeruginosa, from the same hospital. The adjacent genetic environment of the bla(VIM-63) gene would indicate that the capture of this gene by IntI1 has occurred in two different genetic events in each of the species, and in one there has been a stable integration of tandem copies of this gene.
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1 Identification of a stable chromosomal tandem multicopy of blaVIM‐63, a new blaVIM‐2 1 carbapenemase.2 Authors: MarinaR.Pulidoa,b, #,AndreaGarcía‐Montanerb #, LorenaLópez‐Cereroa,b,c,d*,Felipe3 Fernández‐Cuencab,c,d,JoséGutiérrez‐Fernándeze,f,ÁlvaroPascuala,b,c,d4 aDepartamentodeMicrobiología,UniversidaddeSevilla,Sevilla,Spain.5 bInstitutodeBiomedicinadeSevillaIBIS,HospitalUniversitario Virgen6 Macarena/CSIC/UniversidaddeSevilla,Sevilla,Spain.7 cUnidad Clínica de Enfermedades Infecciosas y Microbiología, Hospital Universitario Virgen8 Macarena,Sevilla,Spain.9 dCentro de Investigación Biomédica en Red en Enfermedades Infecciosas (CIBERINFEC),10 InstitutodeSaludCarlosIII,Madrid,Spain.11 eDepartamentodeMicrobiología,UniversidaddeGranada,Spain.12 fHospitalUniversitarioVirgendelasNieves,Granada,Spain.13 14 #Bothauthorscontributedequallytothiswork.15 16 *Correspondingauthor:LorenaLópez‐Cerero:,ll[email protected],Phonenumber:+34954558263,17 18 Runningtitle:CharacterizationoftandemmulticopyofblaVIM‐63.19 20 Keywords21 VIM‐63,VIM‐2variant,carbapenemase,multimer,WGS22 23 24
2 Abstract25 This study characterizes a newgenetic structure containing a multicopy of a blaVIM‐2variant26 with an A676C substitution,blaVIM‐63. This genewas detected on the chromosome of two27 carbapenem‐resistantclinicalstrainsofCitrobacterfreundiiST22recoveredfromtwopatients,28 separatedbya6‐monthperiod,andpreviouslyinPseudomonasaeruginosaST2242fromthe29 samehospitalunit.Short‐read‐sequencingwasusedtocharacterizethenewvariantinboth30 species, and long‐ read‐sequencing to characterize the genome ofC. freundii. On theP.31 aeruginosachromosome, theblaVIM‐63genewasinsertedbetweenISPsy42‐type sequences,32 flanked by anintl1sequence, nearbyaph(3´)‐VIandsul1. On theC. freundiichromosome,33 theblaVIM‐63genewasinsertedintoaTn6230‐liketransposonasastablefive‐tandemrepeat34 multimer, flanked by the sameintl1as inP. aeruginosa. This structure was stable across35 subculturesanddidnotchangeinthepresenceofcarbapenems.TheblaVIM‐63genewascloned36 intothepCR‐Bluntplasmidtostudyantimicrobialsusceptibilitypatterns,andintopET29afor37 kinetic activity analysis. VIM‐63 showed higherKmvalues than VIM‐2 for ceftazidime and38 cefepimeandhigherkcatvalues for cefotaxime, ceftazidime, imipenem and ertapenem,39 withoutdifferencesinMICvalues.Thisisthefirststudytodescribethisnewvariant,VIM‐63,in40 twodifferentspecieswithachromosomallocationintegratedintodifferentmobileelements,41 andthefirsttimetodescribeastablemultimerofametallo‐betalactamase.Despitetheamino42 acidsubstitution,thesusceptibilitypatternofthenewvariantwassimilartoVIM‐2.43 IMPORTANCEVIMgroupmetallo‐betalactamasesareusuallycapturedbyIntI1integrases.This44 workdescribesthedetectionforthefirsttimeofanovel,previouslyunknownvariantofVIM‐45 2,VIM‐63.Thiscarbapenemasehasbeenfoundonthechromosomeoftwodifferentspecies,46 Citrobacter freundii and Pseudomonas aeruginosa, from the same hospital. The adjacent47 geneticenvironmentoftheblaVIM‐63genewouldindicatethatthecaptureofthisgenebyIntI148
3 hasoccurredintwodifferentgeneticeventsineachofthespeciesandinonetherehasbeena49 stableintegrationoftandemcopiesofthisgene.50 51 52 53 54
4 Introduction.55 Inrecentyears,theemergenceandprevalenceofGram‐negativebacteriaexpressingmetallo‐56 β‐lactamases(MBLs)hasbecomeamajorhealthproblemworldwide,sincetheyconferbroad‐57 spectrumβ‐lactamresistance,includingresistancetocarbapenemsandnewantibioticsactive58 against other types of carbapenemases (1). In Andalusia, a region in the south of Spain, a59 significanttemporalchangeintheepidemiologyofMBLwasrecentlyobserved,withaclear60 upwardtrendassociatedwithseveraloutbreaks(2).OneofthemaingroupsofMBLsisthe61 Verona‐integron‐encodedmetallo‐β–lactamase(VIM)(3).Morethan70variantsofVIMhave62 been described over a 20‐year period63 (https://www.ncbi.nlm.nih.gov/pathogens/isolates#/refgene/gene_family:(blaVIM)). 64 VIM‐type enzymes were widespread in Pseudomonas aeruginosa isolates in Mediterranean65 countries in the late twentieth century (4).blaVIMgenesaretypicallyinsertedintoclassI66 integrons(5)andcanbemobilizedbydifferentplasmidlineages (IncN, IncI1, IncX) (6).67 Enterobacterales such as Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii and68 otherscanacquireVIM‐containingplasmidsbyhorizontaltransferofhighlyconjugative,broad‐69 host‐rangeplasmids(7).70 One VIM‐producing P. aeruginosaandtwoC. freundii isolates were recently detected in 371 patientsinthesameunitinahospitalinthesouthofSpain(Andalusia).Thisstudydescribesa72 new variant of blaVIM‐2, named blaVIM‐63, detected for the first time on the chromosome of73 thoseisolates,aswellasitsgeneticenvironment.74 Methods75 Bacterialisolates.76 A VIM-producing P. aeruginosa isolate (named 20190031), was recovered in 2019 from a rectal 77 swab of a hematological patient admitted to University Hospital Virgen de las Nieves (Granada, 78 Spain). The blaVIM was identified as a new variant of VIM-2.Thisnewvariantwasidentifiedon79
5 two subsequent occasions in two Citrobacter freundii isolates detected in the same ward:80 isolate20200334,recovered18monthslaterfromaurinesampleinadifferentpatient,and81 isolate20200723,recovered6monthsafterthatfromthebloodsampleofanotherpatient.82 For comparative analysis of sensitivity and enzyme activity, a clinical isolate ofVIM‐2‐83 producingP.aeruginosa(20181090)wasalsoselected.84 Sequencingandgenomeassembly85 To characterize the genome of the isolates, genomic DNA from P. aeruginosa(isolates86 20190031 and 20181090) and C. freundii (isolates 20200334 and 20200723) was analyzed87 using the Nextera XT DNA sample preparation kit (Illumina San Diego, CA, USA), then88 sequenced using Illumina MiSeq 300‐bp paired‐end sequencing technology and assembled89 usingtheCLCWorkbench9.01.1(Qiagen).GenomicDNAfromisolate20200334(C.freundii)90 wasalsoobtainedwiththeDNeasyBlood&TissueKit(Qiagen)andsequencedwiththePacBio91 Sequel system. A genome assembly was obtained with Canu v2.1.1 (option ‐‐92 genomeSize=4.5MB)(8)and furtherpolishedwithPilonv.1.24(defaultparameters)(9).The93 representative species genome of the FDAARGOS_549 strain (accession number94 GCA_003812345.1)wasusedasareferencetomanuallysortthechromosome contigs95 according to origin of replication (defined by dnaAgeneposition),usingtheArtemis96 Comparison Tool (ACT) v18.1.0 (10). The sorted PacBio genome assembly was used as a97 referencetore‐assembletheIlluminareadsfromC.freundiiisolates20200334and20200723,98 using the SPAdes genome assembler v3.13.0 (option ‐‐trusted‐contigs) (11, 12) and Pilon.99 Finally,QUASTv5.0.2wasusedforqualitycontrolofallgenome assemblies (13). Assembly100 statisticsareaddedasasupplementaryfile(TableS1).101 Assembled genomes were annotated using the Rapid Annotations Subsystems Technology102 (RAST)server(http://rast.nmpdr.org/),togetherwithISfinder(14),Resfinder (15),103 ComprehensiveAntibioticResistanceDatabase(CARD)(16)andPlasmidFinderdatabases(17)104
6 for resistance and plasmid replicon genes, and the MLSTfinder (18) for ST assignment. P.105 aeruginosasequencesinvolvedintheregulationofAmpCandeffluxpumpswerecompared106 withthewild‐typeP.aeruginosareferencestrainPAO1(www.pseudomonas.com).107 GenomicislandswereidentifiedinP.aeruginosa20190031andC.freundii20200334isolates108 withtheIslandViewer4webserver(19).Inthecaseofisolate20190031,thereferencegenome109 (P.aeruginosastrainPAO1)wasrequired,duetothehighfragmentationofourdraftgenome.110 Sequencehomologysearches(percentageBLASTidentity>95%)wereperformedagainstthe111 positive reference dataset (i.e. confirmed genomic islands) in the integrated IslandPick112 database(20)toidentifytheoriginofthepredictedislandsandtheirencodedgenes.113 Burrows‐WheelerAligner(BWA)softwarev0.7.17‐r1188(21,22)andSamtoolsv1.11(23)were114 usedtomapsequencingreadstotheassembledgenomesandtoevaluatecoverageacrossthe115 assemblies.116 Allnucleotide‐basedcomparisonsbetweenassemblieswereperformedwithBLAST+v2.10.1117 andvisualizedwithArtemisv18.1.0(10)andEasyFigv2.2.5(24).118 Finally,3DproteinstructureswereobtainedusingtheSwiss‐Modelplatform(25).119 SNP‐basedphylogenetictrees120 GeneticdistancebetweenthetwoC.freundiiisolateswasassessedbyidentifyingthenumber121 ofsinglenucleotidepolymorphisms(SNPs)betweenthem.Snippysoftwarev4.6.0(26)was122 usedtomapthereadsofeachisolatetotheassembledgenomeoftheother.Anassemblyof123 strainCF8_ST22belongingtocloneST22(accessionnumberGCA_001880825.1)wasusedas124 reference(withthesnippy‐multi‐inputoption‐‐ref)toproducebothacoreSNPalignmentand125 awholegenomeSNPalignment.EachalignmentwasusedtobuildaphylogenywithIQ‐Tree126 v2.0.3(withdefaultoptions)(27)showingtherelationshipandgeneticdistancesbetweenthe127 isolatesandthereferencestrainCF8_ST22,whichwerecalculated with snp‐dists software128 v0.8.2(https://github.com/tseemann/snp‐dists). 129 StabilityofblaVIM‐63platforminC.freundii130
7 Toanalyzethestabilityofthe5tandemrepeatsinthe geneticorganizationofblaVIM‐63, we131 usedtwoapproaches.First,C.freundiiisolateswereseriallysubculturedonMHBagarplates132 withoutantimicrobial pressure(15passages).Minimuminhibitoryconcentrations(MICs)for133 meropenem,cefepimeandpiperacillin/tazobactamweremonitoredevery3passages,andthe134 strains obtained after all 15 passages were also sequenced using Illumina sequencing135 technology.Second,toanalyzewhetherexposuretocarbapenemsaffectsthecopynumberof136 blaVIM‐63,thetwoC. freundiiisolateswereexposedbysubculturingfor24hoursonMueller137 Hintonagarsupplementedwithvariousconcentrationsofmeropenem(0.25,0.5,1,2and4138 µg/ml)andsequencingtheresultingcolonies.139 Inbothapproaches,blaVIM‐63genecoveragewasanalyzedasdescribedabove.140 Cloningexperiments141 InordertoexpressblaVIMgenesinanidenticalbackground,bacterialDNAwasextractedusing142 theDNeasyBloodandTissuekit(Qiagen),followingthemanufacturer´sinstructions.TheblaVIM143 geneswereclonedintoE.coliTOP10cellsusingpCR‐Blunt.Cloneswereselectedon0.06,0.12,144 0.25and0.5µg/mlmeropenemagarplates.Thecorrespondingrecombinantstrainswereused145 forMICdetermination.146 Susceptibilitytesting147 TheMICsofampicillin,cefotaxime,ceftazidime,cefepime,aztreonam,piperacillin/tazobactam,148 ertapenem, imipenem and meropenem were determined by gradient strip method149 (Liofilchem,Spain)anddiskdiffusionassaysaccordingtoEuropeanCommitteeon150 Antimicrobial Susceptibility Testing (EUCAST) guidelines.151 (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_10.0_ 152 Breakpoint_Tables.pdf). 153 Proteinpurification154
8 TheblaVIM genes werecloned into thepET29avector. Therecombinants,pET29a‐blaVIM‐2or155 pET29a‐ blaVIM‐63, were used to transform electrocompetent E. coliBL21(DES)bacteria.156 TransformedcellsweregrownovernightinLBagarsupplementedwith50µg/mlkanamycinat157 37ºC. Colonies were used to sequence plasmidsand confirm transformation. Bacteria were158 grownin60mlLBcontaining50µg/mlkanamycintoOD600nm=0.6.VIMexpressionwasthen159 inducedwith1mMIPTGfor3hours.ThebacterialcellswerepelletedandVIMproteinswere160 purifiedusingtheNi‐NTASpinKit(Qiagen),followingthemanufacturer´sinstructions.Purified161 proteinsweredialyzedagainst100mMphosphatebuffer(pH7.2).TheBCAassaywasusedto162 determineproteinconcentration.ThepurityofVIMproteinextractswasdeterminedbySDS‐163 polyacrylamidegelelectrophoresis.164 Carbapenemaseactivitywasevaluatedin100mMphosphatebuffer(pH7.2)supplemented165 with50µMZnCl2 anddifferentantimicrobialsassubstrates.Kineticmeasurements(kcatand166 Km) of purified carbapenemases were performed spectrophotometrically, as described167 previously(28,29).168 Forinhibitionassays,thehalf‐maximalinhibitoryconcentration(IC50)wasdeterminedusing169 EDTA.Briefly,enzymeswerepre‐incubatedwithdifferentconcentrationsofEDTAfor10minat170 25ºC and hydrolysis was quantified spectrophotometrically with 100 mM imipenem as the171 reportersubstrate(30).172 173 Resultsanddiscussion.174 CharacterizationofthegeneticenvironmentofblaVIM‐63175 WholegenomesequencingoftheP.aeruginosa20190031isolaterevealedthatthisstrainhad176 a novel blaVIM variant, designated blaVIM‐63, which differed from blaVIM‐2 by an Ile226Leu177 substitutionduetoanA676Cmutation.ThepredictedaminoacidsequenceofblaVIM‐63showed178 asingleaminoacidsubstitution(Iso226Leu)ascomparedtoblaVIM‐2.Theisolatewasassigned179 toST2242,asinglelocusvariantofST762,bothlowfrequencySTs. Analysis of the genetic180
9 environmentofblaVIM‐63inP.aeruginosashowedthatitwaslocatedonthechromosomeclose181 toISPsy42andaISPsy30,bothbelongingtotheTn3family.NexttotheblaVIM‐63gene,anintl1182 sequencewasfound, followed byresistance genes suchas aph(3´)‐VIandsul1,andaVapC183 toxin‐antitoxinsystem(Figure1,A).Somestudieshavereportedthatothermembersofthe184 blaVIMfamilyarelocatedinintegronswithasimilarstructuretothesequencedescribedabove,185 whileotherstudieshavefoundthattheseintegronsareflankedbytwoinsertionsequences186 (ISs)(31–34),although,inthiscase.onlyonecompleteISwasfound.187 AnidenticalblaVIM‐63genewasfoundinthetwoC.freundiiisolates.Tocomparethegenetic188 environmentsofthetwospecies,weperformedpairwisenucleotideBLASTcomparisonsofthe189 P.aeruginosa20190031andC.freundii20200334(PacBioassembly)genomesandvisualized190 thegeneticregioncontainingtheblaVIM‐63gene(Figure1,B).Theclosegeneticenvironmentsof191 theblaVIM‐63geneinbothspeciesincludedthesameintl1sequencefoundinP.aeruginosa,as192 well as the sul1 andaph(3’)‐VIgenes, although C. freundiialsoharboredant(2’’)‐Ia in this193 location.AccordingtoGuerinetal.(35), the P2 promoter of the class 1 integron wasat137bp194 andcontainedsequencesforbindingoftranscriptionfactorsrpoD17/cpxR.195 ThemaindifferencebetweentheP.aeruginosaandC.freundiiisolateswasthatP.aeruginosa196 20190031encodedonecopyoftheblaVIM‐63 gene,whereastheC.freundiiisolatesharbored197 fivetandemcopies.ThisscenariowasobservedbothinthePacBiogenomeassemblyandin198 the hybrid assemblies. To rule out potential assembly artifacts,wealignedthesequencing199 readswiththerespectiveassembliesproduced(SupplementaryfigureS2).Inthecaseofthe200 PacBiogenomeassembly(isolate20200334),weidentifiedseveralreadscoveringtheentire201 chromosomal region encoding the five gene copies (supplementary figure S2, A). Next, we202 assessed the read coverage of the Illumina assemblies of isolates 20200334 and 20200723203 (supplementaryfigureS2,BandC).Amarkedincreaseofcoverageintheregionencodingonly204 theblaVIM‐63genewasobserved,whichwasconsistentwiththenumberofcopiesobservedin205 thePacBioassembly(anincreaseof4.91and5.22timescomparedtomeancoverageofthe206
16 References.345 1. BoydSE,LivermoreDM,HooperDC,HopeWW.2020.Metallo‐β‐lactamases:Structure,346 function,epidemiology,treatmentoptions,andthedevelopmentpipeline.Antimicrob347 AgentsChemother64.348 2. López‐HernándezI,Delgado‐ValverdeM,Fernández‐CuencaF,López‐CereroL,349 MachucaJs.,PascualÁ.2020.Carbapenemase‐ProducingGram‐negativebacteriain350 andalusia,Spain,2014‐2018.EmergInfectDis26.351 3. BushK,BradfordPA.2020.Epidemiologyofβ‐lactamase‐producingpathogens.Clin352 MicrobiolRev.353 4. NordmannP,NaasT,PoirelL.2011.Globalspreadofcarbapenemaseproducing354 Enterobacteriaceae.EmergInfectDis17:1791–1798.355 5. PartridgeSR,KwongSM,FirthN,JensenSO.2018.Mobilegeneticelementsassociated356 withantimicrobialresistance.ClinMicrobiolRev.357 6. TaggarG,RhemanMA,BoerlinP,DiarraMS.2020.Molecularepidemiologyof358 carbapenemasesinenterobacterialesfromhumans,animals,foodandthe359 environment.Antibiotics.360 7. ArcariG,DiLellaFM,BibbolinoG,MengoniF,BeccaccioliM,AntonelliG,FainoL,361 CarattoliA.2020.AmultispeciesclusterofVIM‐1carbapenemase‐producing362 enterobacteraleslinkedbyanovel,highlyconjugative,andbroad‐host‐rangeIncA363 plasmidforebodesthereemergenceofVIM‐1.AntimicrobAgentsChemother64.364 8. KorenS,WalenzB,BerlinK,MillerJ,BergmanN,PhillippyA.2016.Canu:scalableand365 accuratelong‐readassemblyviaadaptivek‐merweightingandrepeatseparation.366 bioRxivhttps://doi.org/10.1101/071282.367
17 9. WalkerBJ,AbeelT,SheaT,PriestM,AbouellielA,SakthikumarS,CuomoCA,ZengQ,368 WortmanJ,YoungSK,EarlAM.2014.Pilon:Anintegratedtoolforcomprehensive369 microbialvariantdetectionandgenomeassemblyimprovement.PLoSOne9.370 10. CarverT,BerrimanM,TiveyA,PatelC,BöhmeU,BarrellBG,ParkhillJ,RajandreamMA.371 2008.ArtemisandACT:Viewing,annotatingandcomparingsequencesstoredina372 relationaldatabase.Bioinformatics24.373 11. PrjibelskiA,AntipovD,MeleshkoD,LapidusA,KorobeynikovA.2020.UsingSPAdesDe374 NovoAssembler.CurrProtocBioinforma70.375 12. AntipovD,KorobeynikovA,McLeanJS,PevznerPA.2016.HybridSPAdes:Analgorithm376 forhybridassemblyofshortandlongreads.Bioinformatics32.377 13. GurevichA,SavelievV,VyahhiN,TeslerG.2013.QUAST:Qualityassessmenttoolfor378 genomeassemblies.Bioinformatics29.379 14. SiguierP,PerochonJ,LestradeL,MahillonJ,ChandlerM.2006.ISfinder:thereference380 centreforbacterialinsertionsequences.NucleicAcidsRes381 https://doi.org/10.1093/nar/gkj014.382 15. ZankariE,HasmanH,CosentinoS,VestergaardM,RasmussenS,LundO,AarestrupFM,383 LarsenMV.2012.Identificationofacquiredantimicrobialresistancegenes.JAntimicrob384 Chemotherhttps://doi.org/10.1093/jac/dks261.385 16. AlcockBP,RaphenyaAR,LauTTY,TsangKK,BouchardM,EdalatmandA,HuynhW,386 NguyenALV.,ChengAA,LiuS,MinSY,MiroshnichenkoA,TranHK,WerfalliRE,Nasir387 JA,OloniM,SpeicherDJ,FlorescuA,SinghB,FaltynM,Hernandez‐KoutouchevaA,388 SharmaAN,BordeleauE,PawlowskiAC,ZubykHL,DooleyD,GriffithsE,MaguireF,389 WinsorGL,BeikoRG,BrinkmanFSL,HsiaoWWL,DomselaarGV.,McArthurAG.2020.390 CARD2020:Antibioticresistomesurveillancewiththecomprehensiveantibiotic391
18 resistancedatabase.NucleicAcidsReshttps://doi.org/10.1093/nar/gkz935.392 17. CarattoliA,HasmanH.2020.PlasmidFinderandInSilicopMLST:Identificationand393 TypingofPlasmidRepliconsinWhole‐GenomeSequencing(WGS)MethodsinMolecular394 Biology.395 18. LarsenMV.,CosentinoS,RasmussenS,FriisC,HasmanH,MarvigRL,JelsbakL,396 Sicheritz‐PonténT,UsseryDW,AarestrupFM,LundO.2012.Multilocussequence397 typingoftotal‐genome‐sequencedbacteria.JClinMicrobiol398 https://doi.org/10.1128/JCM.06094‐11.399 19. BertelliC,LairdMR,WilliamsKP,LauBY,HoadG,WinsorGL,BrinkmanFSL.2017.400 IslandViewer4:Expandedpredictionofgenomicislandsforlarger‐scaledatasets.401 NucleicAcidsRes45.402 20. LangilleMGI,HsiaoWWL,BrinkmanFSL.2008.Evaluationofgenomicislandpredictors403 usingacomparativegenomicsapproach.BMCBioinformatics9.404 21. LiH,DurbinR.2009.FastandaccurateshortreadalignmentwithBurrows‐Wheeler405 transform.Bioinformatics25.406 22. LiH,DurbinR.2010.Fastandaccuratelong‐readalignmentwithBurrows‐Wheeler407 transform.Bioinformatics26.408 23. LiH,HandsakerB,WysokerA,FennellT,RuanJ,HomerN,MarthG,AbecasisG,Durbin409 R.2009.TheSequenceAlignment/MapformatandSAMtools.Bioinformatics25.410 24. SullivanMJ,PettyNK,BeatsonSA.2011.Easyfig:Agenomecomparisonvisualizer.411 Bioinformatics27.412 25. WaterhouseA,BertoniM,BienertS,StuderG,TaurielloG,GumiennyR,HeerFT,De413 BeerTAP,RempferC,BordoliL,LeporeR,SchwedeT.2018.SWISS‐MODEL:Homology414
19 modellingofproteinstructuresandcomplexes.NucleicAcidsRes415 https://doi.org/10.1093/nar/gky427.416 26. TorstenSeemann.2015.Snippy,Rapidhaploidvariantcallingandcoregenome417 alignment.GitHubRepos.418 27. MinhBQ,SchmidtHA,ChernomorO,SchrempfD,WoodhamsMD,VonHaeselerA,419 LanfearR,TeelingE.2020.IQ‐TREE2:NewModelsandEfficientMethodsfor420 PhylogeneticInferenceintheGenomicEra.MolBiolEvol37.421 28. Rodriguez‐MartinezJM,NordmannP,FortineauN,PoirelL.2010.VIM‐19,ametallo‐β‐422 lactamasewithincreasedcarbapenemaseactivityfromEscherichiacoliandKlebsiella423 pneumoniae.AntimicrobAgentsChemotherhttps://doi.org/10.1128/AAC.00458‐09.424 29. BorgianniL,VandenameeleJ,MatagneA,BiniL,BonomoRA,FrèreJM,RossoliniGM,425 DocquierJD.2010.MutationalanalysisofVIM‐2revealsanessentialdeterminantfor426 metallo‐β‐lactamasestabilityandfolding.AntimicrobAgentsChemother427 https://doi.org/10.1128/AAC.01336‐09.428 30. MakenaA,DüzgünA,BremJ,McDonoughMA,RydzikAM,AbboudMI,SaralA,ÇiçekA,429 SandalliC,SchofieldCJ.2016.Comparisonofveronaintegron‐bornemetallo‐β‐430 lactamase(VIM)variantsrevealsdifferencesinstabilityandinhibitionprofiles.431 AntimicrobAgentsChemotherhttps://doi.org/10.1128/AAC.01768‐15.432 31. BotelhoJ,GrossoF,QuinteiraS,BrilhanteM,RamosH,PeixeL.2018.Twodecadesof433 blaVIM‐2‐producingPseudomonasaeruginosadissemination:Aninterplaybetween434 mobilegeneticelementsandsuccessfulclones.JAntimicrobChemother435 https://doi.org/10.1093/jac/dkx517.436 32. PilatoVDi,AntonelliA,GianiT,DeAngelisLH,RossoliniGM,PolliniS.2019.437 Identificationofanovelplasmidlineageassociatedwiththedisseminationofmetallo‐438
20 β‐lactamasegenesamongpseudomonads.FrontMicrobiol439 https://doi.org/10.3389/fmicb.2019.01504.440 33. JahanMI,RahamanMM,HossainMA,SultanaM.2020.OccurrenceofintI1‐associated441 VIM‐5carbapenemaseandco‐existenceofallfourclassesofβ‐lactamasein442 carbapenem‐resistantclinicalPseudomonasaeruginosaDMC‐27b.JAntimicrob443 Chemotherhttps://doi.org/10.1093/jac/dkz426.444 34. JuanC,BeceiroA,GutiérrezO,AlbertíS,GarauM,PérezJL,BouG,OliverA.2008.445 Characterizationofthenewmetallo‐β‐lactamaseVIM‐13anditsintegron‐bornegene446 fromaPseudomonasaeruginosaclinicalisolateinSpain.AntimicrobAgentsChemother447 https://doi.org/10.1128/AAC.00465‐08.448 35. GuérinE,JovéT,TabesseA,MazelD,PloyMC.2011.High‐levelgenecassette449 transcriptionpreventsintegraseexpressioninclass1integrons.JBacteriol193.450 36. AbeR,AkedaY,SugawaraY,MatsumotoY,MotookaD,KawaharaR,YamamotoN,451 TomonoK,IidaT,HamadaS.2021.EnhancedCarbapenemResistancethrough452 MultimerizationofPlasmidsCarryingCarbapenemaseGenes.MBio12.453 37. HuangTW,ChenTL,ChenYT,LauderdaleTL,LiaoTL,LeeYT,ChenCP,LiuYM,LinAC,454 ChangYH,WuKM,KirbyR,LaiJF,TanMC,SiuLK,ChangCM,FungCP,TsaiSF.2013.455 CopyNumberChangeoftheNDM‐1SequenceinaMultidrug‐ResistantKlebsiella456 pneumoniaeClinicalIsolate.PLoSOne8.457 38. PetersJE,FrickerAD,KapiliBJ,PetassiMT.2014.Heteromerictransposaseelements:458 GeneratorsofgenomicIslandsacrossdiversebacteria.MolMicrobiol.459 39. CabotG,Ocampo‐SosaAA,DomínguezMA,GagoJF,JuanC,TubauF,RodríguezC,460 MoyàB,PeñaC,Martínez‐MartínezL,OliverA.2012.Geneticmarkersofwidespread461 extensivelydrug‐resistantPseudomonasaeruginosahigh‐riskclones.AntimicrobAgents462
21 Chemother56.463 40. LeirosHKS,EdvardsenKSW,BjergaGEK,SamuelsenØ.2015.Structuralandbiochemical464 characterizationofVIM‐26showsthatLeu224hasimplicationsforthesubstrate465 specificityofVIMmetallo‐β‐lactamases.FEBSJhttps://doi.org/10.1111/febs.13200.466