Characterisation of OXA-244, a chromosomally-encoded OXA-48-like β-lactamase from Escherichia coli

CharacterisationofOXA-244,a chromosomally-encoded OXA-48-like ␤-lactamase from Escherichiacoli

Sir,

During the last decade, the carbapenem-hydrolysing ␤-lactamaseOXA-48hasrapidlyandwidelydisseminated,nowbeing themostcommonlyidentifiedcarbapenemaseinmostEuropean andMediterraneancountries[1].Sinceitsdiscovery,tenvariants of OXA-48 have beenreported [1]. In mostcases, bla_OXA-48-like genes are plasmid-borne and have been identified associated with insertion sequences involved in their acquisition and expression[1,2].

Escherichia coli strain VAL was recovered from a urine sample of an 85-year-old patient with no history of travel abroad.Theisolatewasresistanttopenicillinsandpenicillin/ ␤-lactamase inhibitor combinations but remained susceptible to broad-spectrumcephalosporins,imipenemandmeropenem,being ofintermediatesusceptibilitytoertapenem.Multilocussequence typing(MLST)showedthatE.coliVALbelongedtosequencetype ST38[1],knowntobeasuccessfulinternationalclone[3].UsingPCR experimentsfollowedbysequencing[2],E.coliVALwasfoundto harbourablaOXA-48-likegene,termedblaOXA-244(http://www.lahey.

org/studies/).ComparedwithOXA-48,OXA-244exhibitsasingle Arg214Glnsubstitution.Tocomparethehydrolyticprofileof OXA-244withthatof OXA-48,thecorrespondinggeneswerecloned intothevectorpCR-BluntII-TOPOasdescribedpreviously[2]and wereexpressedinE.coliHB4,whichlacksporinsOmpFandOmpC. Expression of both carbapenemase genes conferred high-level resistancetoimipenem,meropenemand ertapenem [minimum inhibitoryconcentrations(MICs)≥32mg/L].Noteworthy,theMICs of imipenem and temocillin were lower for OXA-244 (32mg/L and96mg/L,respectively)thanthoseforOXA-48(>32mg/Land >1024mg/L,respectively), suggestinga weakeractivitytowards thesesubstratesforOXA-244.Similarresultshavebeenobserved withOXA-232,differingfromOXA-181byasingleaminoacid sub-stitutionatposition214,whichislocatedneartheactivesiteof theenzyme[2].Thesetwoexampleshighlighttheimportanceof theintegrityofthisresidueatposition214inthehydrolytic capac-itiesofOXA-48-like␤-lactamases.SpecificactivitiesofOXA-244 for ertapenem and meropenem(2.2 and 4.3mU/mg of protein, respectively)wereclosetothoseofOXA-48(3.5and5.4mU/mg ofprotein,respectively)[1].However,thespecificactivityof OXA-244forimipenem(4.1mU/mgofprotein)wasmuchlowerthan thatdeterminedforOXA-48(111mU/mgofprotein),showinga weakhydrolysis ofimipenembyOXA-244.Plasmid DNA analy-sisshowedthatE.coliVALharbouredthreeplasmidsof120,80 and10kb(datanotshown).Despiteseveralattempts,no electro-transformantortransconjugant couldbeobtained,suggesting a chromosomallocationoftheblaOXA-244gene.Thegenetic

environ-mentoftheblaOXA-244 genewasdeterminedbyshotguncloning

performedasdescribedpreviously[1].Sequenceanalysisofthe DNAfragmentsurroundingtheblaOXA-244generevealedthatitwas

partofa truncatedTn1999.2transposon,madeoftwo copiesof insertionsequencesIS1999andasingleIS1Relementinsertedinto oneoftheIS1999copies[4].Comparedwiththestructure identi-fiedinpOXA-48a,aninvertedorientationofthetruncatedTn1999.2 transposonwasfoundinE.coliVAL(Fig.1).Furtheranalysisshowed thattheblaOXA-244genewasbracketedbytwoIS1Rcopiesforming

anIS1R-madecompositetransposon.This21852-bptransposon wasinsertedintoageneencodinganintrinsicendonucleasefrom E.coli,furthersupportingachromosomalintegrationofthis IS1R-madetransposon.Identificationofa9-bptargetsiteduplication (TGAATTGCT) at both extremities of the IS1R-madetransposon wasthesignatureofatranspositionevent.IS1R-madecomposite transposonsharbouringtheblaOXA-48geneandintegratedintothe

chromosomeofE.coliisolatesfromLebanonhavebeenrecently described[5].

ThisstudycharacterisedOXA-244possessingaweakerabilityto hydrolyseimipenemandtemocillincomparedwithOXA-48. Iden-tificationofsuchavariantraisesagaintheissueofthethreshold tobechosen for classifying a ␤-lactamaseas a carbapenemase ornot. Along withOXA-232, OXA-244is another OXA-48 vari-ant possessing a weaker ability to hydrolyse temocillin. Since thismoleculehasbeenintegratedinscreeningculturemediafor detecting carbapenemase-producers, it might be interesting to evaluatethe performances for detection of allthose producers of OXA-48-likevariants. ST38-type E.coli isolates harbouring a chromosomalblaOXA-48genehavebeenrecoveredfromLebanon,

Egypt,Turkey,SwitzerlandandFrance[3].Disseminationof OXA-48-like-producingE.coliisolatesmightthereforebelinkedtothe disseminationoftheST38cloneatleastinseveralcountries[3,5]. Nevertheless,thisdiffusionhasalsobeenshowntoberelatedtothe mobilityofblaOXA-48-carryingIS1R-madecompositetransposons,

insertedintodifferentlociamongvariousE.colibackgrounds[5]. Thenucleotidesequencedatareportedinthisworkhasbeen depositedintheGenBanknucleotidedatabaseunderaccessionno. KR364794.

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which should be cited to refer to this work.

Fig.1.Schematicmapof(A)thetransposonstructureandsurroundingsequencesinEscherichiacoliVALand(B)thetransposonTn1999inreferenceplasmidpOXA-48a.Open readingframes(ORFs)areshownasarrowsorashorizontalboxeswithanarrowindicatingtheorientationofthecodingsequence.Targetsiteduplications(TGAATTGCT)are representedbyblackbars.orf1,orf2andorf3weresimilartoORFsidentifiedonE.colichromosomes.

Funding

ThisworkwasfinancedbyINSERMU914(Paris,France)andthe UniversityofFribourg(Fribourg,Switzerland).

Competinginterests Nonedeclared. Ethicalapproval

Notrequired. References

[1]DortetL,OueslatiS,JeannotK,TandéD,NaasT,NordmannP.Geneticand biochemicalcharacterizationofOXA-405,anOXA-48-typeextended-spectrum ␤-lactamasewithoutsignificantcarbapenemaseactivity.AntimicrobAgents Chemother2015;59:3823–8.

[2]PotronA,RondinaudE,PoirelL,BelmonteO,BoyerS,CamiadeS,etal.Genetic andbiochemicalcharacterizationofOXA-232,acarbapenem-hydrolysingclass D␤-lactamase fromEnterobacteriaceae. IntJ AntimicrobAgents 2013;41: 325–9.

[3]PotronA,PoirelL,RondinaudE,NordmannP.IntercontinentalspreadofOXA-48 ␤-lactamase-producingEnterobacteriaceaeovera11-yearperiod,2001to2011. EuroSurveill2013;18(pii:20549).

[4]PoirelL,BonninRA,NordmannP.Geneticfeaturesofthewidespread plas-midcodingforthecarbapenemase OXA-48.AntimicrobAgentsChemother 2011;56:559–62.

[5]BeyrouthyR, RobinF,DelmasJ, GiboldL,Dalmasso G,DabboussiF, etal. IS1R-mediatedplasticityofIncL/MplasmidsleadstotheinsertionofblaOXA-48

intotheEscherichiacolichromosome.AntimicrobAgentsChemother2014;58: 3785–90.

AnaïsPotrona,b

LaurentPoirelc,∗

LaurentDorteta

PatriceNordmanna,c,d a_{Centrenationalderéférencedelarésistanceauxantibiotiques,}

K.-Bicêtre,France

b_{CentrehospitalierrégionalUniversitairedeBesanc¸on,Universitéde}

Franche-Comté,Besanc¸on,France

c_{EmergingResistancetoAntibioticsUnit,MedicalandMolecular}

Microbiology,DepartmentofMedicine,FacultyofScience, UniversityofFribourg,Fribourg,Switzerland

d_{HFR,HôpitalCantonal,Fribourg,Switzerland}

∗_{Correspondingauthor.Tel.:+41263009582;}

fax:+41263009585. E-mailaddress:laurent.poirel@unifr.ch(L.Poirel)

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