Modulation of mgrB gene expression as a source of colistin resistance in Klebsiella oxytoca

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Modulation

of

mgrB

gene

expression

as

a

source

of

colistin

resistance

in

Klebsiella

oxytoca

Aurélie

Jayol

a

_,

_Laurent

_Poirel

a,∗

_,

_{Maria-Virginia}

_Villegas

b

_,

_Patrice

_Nordmann

a,c

a_Medical_and_Molecular_Microbiology_«Emerging_Antibiotic_Resistance»_Unit,_Department_of_Medicine,_Faculty_of_Science,_University_of_Fribourg,_Rue Albert-Gockel3,CH-1700Fribourg,Switzerland

b_{International}_Center_for_Medical_Research_and_Training,_CIDEIM,_Cali,_Colombia c_Hôpital_Fribourgeois_–_Hôpital_Cantonal_de_Fribourg,_Riaz,_Switzerland

GenemodiﬁcationsinthePmrABandPhoPQtwo-componentregulatorysystems,aswellasinactivation

ofthemgrBgene,areknowntobecausesofcolistinresistanceinKlebsiellapneumoniae.Theobjective

ofthisstudywastocharacterisethemechanisminvolvedincolistinresistanceinaKlebsiellaoxytoca

isolate.AK.oxytocaclinicalisolateshowingresistancetocolistinwasrecoveredinCali,Colombia.The

pmrA,pmrB,phoP,phoQandmgrBgeneswereampliﬁedandsequenced.Wild-typemgrBgenesfromK.

pneumoniaeandK.oxytocawerecloned,andcorrespondingrecombinantplasmidswereusedfor

com-plementationassays.ByanalysingthemgrBgeneoftheK.oxytocaisolateanditsﬂankingsequences,

aninsertionsequence(IS)of1196bpwasidentiﬁedinitspromoterregion.Theinsertionwaslocated

betweennucleotides−39and−38whenreferringtothestartcodonofthemgrBgene,thusnegatively

interferingwithexpressionofthemgrBgenebymodifyingitspromoterstructure.ThisISwasvery

simi-lartoISKpn26(99%nucleotideidentity)belongingtotheIS5family.ComplementationassayswithmgrB

genesfromwild-typeK.pneumoniaeorK.oxytocarestoredfullsusceptibilitytocolistin.Inconclusion,

hereweidentiﬁedthemechanisminvolvedincolistinresistanceinaK.oxytocaisolate.Modulationof

mgrBgeneexpressionwasthekeyfactorforthisacquiredresistancetocolistin.

1. Introduction

Klebsiella oxytoca and Klebsiella pneumoniae are frequent sourcesof nosocomialinfections andthesources ofnosocomial outbreaks[1,2].Theoccurrenceofmultidrugresistanceis increas-inglyobservedamongtheGram-negativepathogensAcinetobacter baumannii,PseudomonasaeruginosaandEnterobacteriaceae. Con-sequently, useof colistin, a polymyxin-typeantibiotic, is being reconsideredinparticularfortreatingcriticallyillpatientsinfected withthesepathogens[3–5].Unfortunately,theincreasingusageof colistinisbeingassociatedwiththeemergenceofcolistin resis-tance,inparticularinK.pneumoniae[6–11].

Alterationsinthetwo-componentregulatorysystemsPmrAB and PhoPQ [8,9,12] are known to be involved in polymyxin resistance in K. pneumoniae. Inactivation of the mgrB gene,

∗ Correspondingauthor.Tel.:+41263009582. E-mailaddress:[email protected](L.Poirel).

encoding a negative feedback regulator of the PhoPQ two-component system, is also a common mechanism involved in colistin resistancein K.pneumoniae[10,13].Thereported alter-ations in the mgrB gene include insertion of several insertions sequences(IS5-like,IS1F-like,ISKpn13,ISKpn14)atdifferent loca-tions in the coding sequence of mgrB and in its promoter region.Non-silentpointmutations,prematurestopcodons,small intragenic deletions and large deletion of the mgrB locus are also known. These alterations result in either reduced or even lack of expressionof themgrB gene leading toupregulationof the phoPQ and pmrHFIJKLM operons that confer resistance to colistin.

Lippa and Goulian have identiﬁed MgrB homologues in the genomesequencesofvariousenterobacterialspecies[14]. There-fore, alterations in mgrB gene expression might possibly be involvedinacquisitionofcolistinresistanceindifferentKlebsiella species.

Theaimofthisstudywastoidentifythemechanismresponsible forresistancetocolistininaK.oxytocaclinicalisolate.

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2. Materialsandmethods

2.1. Bacterialstrain

The colistin-resistant K. oxytoca clinical isolate was iden-tiﬁed using an API20E system and the matrix-assisted laser desorption/ionisation time-of-ﬂight (MALDI-TOF) technique (AB bioMérieux,LaBalme-les-Grottes,France). Acolistin-susceptible K.oxytocaclinicalisolatewasusedasawild-typecontrol. 2.2. Antimicrobialsusceptibility

AntimicrobialsusceptibilitytestingwasperformedusingEtest strips(ABbioMérieux) onMueller–Hintonagarplates (Bio-Rad, Marnes-la-Coquette, France) with a 0.5 McFarland inoculum. Minimuminhibitory concentrations (MICs) wereinterpreted as indicated by the European Committee on Antimicrobial Sus-ceptibility Testing (EUCAST) guidelines (Breakpoint tables for interpretation of MICs and zone diameters. Version 4.0, 2014; http://www.eucast.org).Isolates withcolistinMICs of≤2␮g/mL were categorised as susceptible whereas those with MICs of >2␮g/mLwereresistant.

2.3. PCRampliﬁcationandsequencing

Whole-cellDNAwasextractedusingaQIAquickKit(QIAGEN, Courtaboeuf,France)accordingtothemanufacturer’sinstructions. TheentirepmrA,pmrB,phoP,phoQandmgrBgeneswere ampli-fied with specific oligonucleotides primers (Table 1) designed withsequencesofK.oxytocaKCTC1686andE718genomes avail-ableontheNationalCenterforBiotechnologyInformation(NCBI) database(http://www.ncbi.nlm.nih.gov.gate2.inist.fr/). Amplified DNAfragmentswerepurifiedwithaQIAquickPCRPurificationKit (QIAGEN).Bothstrandsoftheamplificationproductsobtainedwere sequencedwithanABI3100Sequencer(AppliedBiosystems, Fos-terCity,CA).Thenucleotideanddeducedproteinsequenceswere analysedattheNCBIwebsite(http://www.ncbi.nlm.nih.gov)bythe BasicLocalAlignmentSearchTool(BLAST)programme.The inser-tionsequence(IS)wasanalysedusingtheISfinderwebsite(http:// www-is.biotoul.fr).

2.4. Complementationassays

Thewild-typemgrBgenefromacolistin-susceptibleK.oxytoca strainandaK.pneumoniaestrain,aswellasthenon-codingmdh sequence[15],wereampliﬁedbyPCRusing2XPhusion®HF Mas-terMix (Finnzymes;LifeTechnologies, Illkirch,France) and the primerslistedinTable 1.Theampliﬁedfragments werecloned intotheplasmidpCR®-BluntII-TOPO®(Invitrogen,Illkirch,France)

Table1

Primersusedinthisstudy.

Primer Sequence(5–3) Gene Ref. KoxpmrCextF GCCGATATCGCAGGGGTTAA pmrC Thisstudy KoxpmrCextR TAACAGGAGCGCATCGTCTT pmrC

KoxpmrAextF GCAAGCGTATTCGCAGGATA pmrA KoxpmrAextR CTGATGAGCTGACAAACGGC pmrA KoxpmrBextF CAACGACATTTACAGCTGGG pmrB KoxpmrBextR CTTTACTGAGGATAGCGCCA pmrB KoxphoPextF CCAAGAGACCGAGGTACAAA phoP KoxphoPextR GAGTGACAACACCAGCACTA phoP KoxphoQextF CCATACCATCGATGTGCTGA phoQ KoxphoQextR GCAGGTGTCTGTTAGGGATT phoQ KoxmgrBextF CGCGGTTTAAGAAGGTCATG mgrB KoxmgrBextR AGGCGTTTATTCTACCACCC mgrB mdhF CCCAACTCGCTTCAGGTTCAG mdh [15]

mdhR CCGTTTTTCCCCAGCAGCAG mdh

andtheresultingplasmidspTOPO-mgrBKox,pTOPO-mgrBKpand pTOPO-mdh(encodingresistancetoZeocinTM₎_were_respectively

transformed into an electrocompetent colistin-resistant K. oxy-tocaisolatebyelectroporation.Electrotransformantswereselected byovernightincubationat37◦ConMueller–Hintonagar supple-mentedwith100␮g/mLZeocinTM_._MICs_of_colistin_for_the_K._oxytoca

transformantsweredeterminedbyEtest(bioMérieux).

3. Resultsanddiscussion

3.1. Strainandpatientfeatures

The colistin-resistant K. oxytoca isolate C24 was recovered in February 2008from a soft-tissue secretion from a 66-year-old male patient hospitalised for an intra-abdominal infection in Cali, Colombia. The isolate was resistant to broad-spectrum cephalosporins(MICsofceftazidimeandcefotaxime=32␮g/mL), amikacin(MIC=16␮g/mL)andcolistin(MIC=24␮g/mL)andwas of intermediate susceptibility to ciproﬂoxacin (MIC=1␮g/mL). The isolate was susceptible to carbapenems (MIC<0.5␮g/mL), gentamicin(MIC=0.5␮g/mL)andtigecycline(MIC=0.5␮g/mL).It harbouredablaCTX-M-15genebutdidnotproduceany

carbapene-maseaccordingtotheCarbaNPtest.Toourknowledge,thepatient hadneverbeentreatedwithanypolymyxin.

3.2. ModiﬁcationofmgrBgeneexpressionthroughinsertionof aninsertionsequenceelement

Sequence analysis of thepmrA, pmrB, phoP and phoQ genes knowntobeinvolvedinlipopolysaccharide(LPS)synthesisshowed 100%identitywiththegenesidentiﬁedfromcolistin-susceptible wild-typeK.oxytocaisolates(datanotshown).Sequenceanalysisof themgrBgeneaswellasupstream-locatedsequencesrevealedthat anISof1196bphadtargetedtheupstreamvicinityofthemgrBgene betweennucleotides−38and−39whenreferringtothestartcodon ofmgrB.ThisISwassimilartoISKpn26(differingbyonly5bp)and belongedtotheIS5family.Itwasbracketedbya4-bpduplication (TTAT)beingthelikelysignatureofatranspositionprocess.The ori-ginofISKpn26isK.pneumoniae(https://www-is.biotoul.fr/index. html?isspecialname=ISKpn26)andithasalsobeenidentiﬁedin K.oxytocaKONIH1(GenBankaccessionno.CP008788.1).Insilico analysisofthesequencelocatedupstreamofawild-typemgrBgene revealeda putativepromoter sequence madeof −35[TTGAAA] and−10[TAAACT]boxesseparatedby15bp(Fig.1).Insertionof ISKpn26inisolateC24ledtodisplacementofthispromoterwith respecttothemgrBgeneposition,thecorresponding−35and−10 sequencesbeingconsequentlyverydistantfromit(Fig.1).This sug-gestsadecreaseorevenlackofexpressionofthemgrBgene,leading toloworabsenceofproductionofthecorrespondingproteinand consequentlytoacquiredresistancetocolistin.

3.3. Complementationexperiments

ToconﬁrmwhetherhigherexpressionofmgrBmightrestore susceptibility to colistin, complementation of isolate C24 with a plasmidoverexpressing mgrB wasperformed.Transformation assayswereeitherperformedwithplasmidsencodingtheMgrB proteinofK.oxytocaorK.pneumoniae,respectively.ThemgrBgene wasprovidedintranstogetherwithitsoriginalpromoterinorder toensureit wouldbeexpressedatawild-typelevel. Following complementation, isolate C24 recovered full susceptibility to colistinwiththewild-typeMgrBofK.oxytoca(MIC=0.047␮g/mL). Interestingly,thesameMICwasobtainedaftercomplementation withtheMgrBofK.pneumoniae(MIC=0.047␮g/mL).Asexpected,

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TGAACATCGTTTGAAACAAGTCTATGATTCCTAAACTTGCCTTTCGTATTACAGTTAGCCGCGGTT

–35 PROM –10

ISKpn26-like

TAAGAAGGTCATGTTATCCTGGCGACATCTGGTACTGATGCGGAGTGTGGAGTG

mgrB start codon

Fig.1.Sequenceslocatedatthe5_-extremity_of_the_mgrB_gene._The_putative_promoter_(PROM)_is_indicated,_with_its_respective₋₃₅_and₋₁₀_boxes_underlined._The_arrow indicatesthetargetsiteforinsertionoftheinsertionsequenceISKpn26-like.ThemgrBstartcodonisinbold.

transformation with plasmid pTOPO-mdh used as a negative controldidnotrestoresusceptibilitytocolistin(MIC=24␮g/mL). 3.4. ComparisonofmgrBgenesequencesofK.oxytocaandK. pneumonia

AlignmentofthemgrBsequencesfromwild-typeK. pneumo-niae MGH78578and K.oxytoca KCTC1686 strainsshowed that the sequenceswere almost identical[only a single amino acid substitution (CystoTyrat aminoacidposition28)].Thisresult explainswhycomplementationwiththemgrBgeneofK. pneumo-niaerestoredfullsusceptibilitytocolistinintheK.oxytocaisolate. Furthermore,itmaysuggestthatallpreviouslyreportedalterations ofthemgrBgene[10,13]thathavebeenshowntobethesources ofacquiredresistancetocolistininK.pneumoniae(suchas inser-tionsofISelementsatdifferentlocationsinthecodingsequence ofmgrB,non-silentpointmutations,prematurestopcodons, intra-genicdeletions,etc.)mightalsobeasourceofcolistinresistancein K.oxytoca.

4. Conclusion

Thisstudyreports onthemolecularidentificationof colistin resistanceinK.oxytoca.Thisresistancemechanismisassociated withmodificationofthebiosynthesispathwayofLPS.Itis simi-lartothatreportedinK.pneumoniae,underliningthatcommon mechanismsofcolistinresistancecouldbeidentifiedindifferent enterobacterialspecies.

Nucleotidesequenceaccessionnumber

ThenucleotidesequencesofthemutatedmgrBgenesidentiﬁed inthisstudyhavebeendepositedatDDBJ/EMBL/GenBankunder accessionno.KP748172.

Funding

This work was funded by the University of Fribourg (Fri-bourg,Switzerland)andbyagrantfromtheEuropeanCommunity [MAGIC-BULLET,FP7/HEALTH-F3-2001-278232]. Competinginterests Nonedeclared. Ethicalapproval Notrequired. References

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