The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

The

role

of

gyrA

and

parC

mutations

in

fluoroquinolones-resistant

Pseudomonas

aeruginosa

isolates

from

Iran

Roghayeh

Nouri

_,

_Mohammad

_Ahangarzadeh

_Rezaee

_,

_Alka

_Hasani

_,

Mohammad

Aghazadeh

_,

_Mohammad

_Asgharzadeh

a_{TabrizUniversityofMedicalSciences,InfectiousandTropicalDiseasesResearchCenter,Tabriz,Iran} b_{TabrizUniversityofMedicalSciences,FacultyofMedicine,DepartmentofMicrobiology,Tabriz,Iran} c_{TabrizUniversityofMedicalSciences,StudentResearchCommittee,Tabriz,Iran}

d_{TabrizUniversityofMedicalSciences,BiotechnologyResearchCenter,Tabriz,Iran}

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Articlehistory:

Received15July2015 Accepted25March2016 Availableonline26July2016 AssociateEditor:AfonsoLuísBarth

Keywords: Pseudomonasaeruginosa Fluoroquinoloneresistance gyrA parC

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Theaimofthisstudywastoexaminemutationsinthequinolone-resistance-determining region(QRDR)ofgyrAandparCgenesinPseudomonasaeruginosaisolates.Atotal of100 clinicalP.aeruginosaisolateswerecollectedfromdifferentuniversity-affiliatedhospitals inTabriz,Iran.Minimuminhibitoryconcentrations(MICs)ofciprofloxacinandlevofloxacin wereevaluatedbyagardilutionassay.DNAsequencesoftheQRDRofgyrAandparCwere determinedbythedideoxychainterminationmethod.Ofthetotal100isolates,64were resistanttociprofloxacin.NoaminoacidalterationsweredetectedingyrAorparCgenesof theciprofloxacinsusceptibleorciprofloxacinintermediateisolates.Thr-83→Ile substitu-tioningyrAwasfoundinall64ciprofloxacinresistantisolates.Forty-four(68.75%)ofthem hadadditionalsubstitutioninparC.Acorrelationwasfoundbetweenthenumberofthe aminoacidalterationsintheQRDRofgyrAandparCandthelevelofciprofloxacinand levo-floxacinresistanceoftheP.aeruginosaisolates.Ala-88→ProalterationinparCwasgenerally foundinhighlevelciprofloxacinresistantisolates,whichweresuggestedtoberesponsible forfluoroquinoloneresistance.ThesefindingsshowedthatinP.aeruginosa,gyrAwasthe primarytargetforfluoroquinoloneandadditionalmutationinparCledtohighlyresistant isolates.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthorat:InfectiousandTropicalDiseasesResearchCenter,TabrizUniversityofMedicalSciences,Tabriz,Iran.} E-mail:rezaee@tbzmed.ac.ir(M.AhangarzadehRezaee).

http://dx.doi.org/10.1016/j.bjm.2016.07.016

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Pseudomonas aeruginosa is an important opportunistic pathogen1–3_{causinglife-threateninginfections,especiallyin}

immunocompromisedpatients.4–6_{Oftentheseinfectionsare}

difficulttotreatduetotheintrinsicresistanceofthespecies7

aswell as its remarkable abilitytoacquire resistanceto a wide range of antimicrobial agents.8 _{Fluoroquinolones are}

the only accessible antibiotics for effective oral treatment of infections caused by this organism.9 _Among

fluoro-quinolones,ciprofloxacinandlevofloxacinarewidelyusedin thetreatmentofP.aeruginosainfections.10

Fluoroquinolones act by inhibiting the action of tar-getenzymes,DNA gyraseand topoisomerase IV,with both enzymesplayingaprincipal roleinDNAreplication.11_DNA

gyraseandtopoisomeraseIVare heterotetramericenzymes thatarecomposedoftwosubunitsencodedbythegyrA,gyrB

andparC, parE,respectively.12 _{ThegyrAand gyrBgenesare}

homologstoparCandparE,respectively.13_{Themechanismsof}

fluoroquinoloneresistanceinP.aeruginosaincludemutations inthe DNA gyrase and topoisomerase IV,14,15

overexpress-ion ofefflux pump system and the innate impermeability ofthemembrane.16 _{Alterations intheso-called}

quinolone-resistance-determining region (QRDR) within DNA gyrase andtopoisomeraseIVarethemajormechanismsfor fluoro-quinoloneresistanceinP.aeruginosa.17–20

Moreover,isolateswith mutationsin QRDRofgyrA and

parCshowthehighestlevelsoffluoroquinoloneresistance.21

AlthoughaminoacidalterationsinthegyrBandparEgenes havebeendescribed,but thefrequencyofthesemutations islow, withonly acomplementary role influoroquinolone resistance.18,21

SeveralstudiesfromIranhavereportedthehighprevalence ofMDRstrainsamongIranianhospitalsandstrainsisolated from hospitalized patients; especially, burn patients have showhighlevelresistancetomostavailableantibiotics.22,23

Theprevalence of mutationsin DNA gyrase and topoi-somerase IV has not been well studied in Iran. This is

the largest analysis of the QRDR of gyrA and parC in the clinical isolates ofP. aeruginosafrom Iran.The aimof this studywastoexaminethemutationsingyrAandparCgenes and characterize their correlation with ciprofloxacin and levofloxacin resistance, as well as the evaluation of their effectonciprofloxacinandlevofloxacinMinimuminhibitory concentrations(MICs)amongtheclinicalisolatesofP. aerugin-osa.

Materials

and

methods

Bacterialisolates

Inaprospectivestudy,atotalof100non-repetitiveclinical isolates of P. aeruginosa were collected, between December 2013,andJuly2014,fromfourEducational-HealthCare Cen-tersofTabrizUniversityofMedicalSciencesinNorthwestIran. Thebacterialisolateswererecoveredfromdifferentclinical specimenssuchasurine(29%),wounddischarge(25%), tra-chealaspirates(21%),blood(8%)andotherclinicalspecimens

(Table1).Allisolateswereidentifiedbystandardconventional

biochemicaltests.24

Antimicrobialsusceptibilitytesting

Antimicrobialsusceptibilitytoticarcillin(75␮g), piperacillin-tazobactam(100/10␮g),ceftazidime(30␮g),cefepime(30␮g), aztreonam (30␮g), imipenem (10␮g), meropenem (10␮g), gentamicin (10␮g), amikacin (30␮g), ciprofloxacin (5␮g), levofloxacin (5␮g) and ofloxacin(5␮g) (Mast,UK) was per-formed by employing the standard disk diffusion method. MICsofciprofloxacinandlevofloxacin(Sigma-Aldrich)were determined by the standard agar dilution assay. The results of disk diffusion assay as well as MIC were inter-preted according to the clinical and laboratory standard institute (CLSI) guidelines.25 _{Pseudomonas aeruginosa ATCC}

27853 was used as the control strain for susceptibility testing.

Table1–DistributionofPseudomonasaeruginosaisolatesbythesiteofisolationandhospitalorigin.

Sourceofisolates Hospitalorigin Totalno.ofisolates

ImamReza Sina Koodakan ShahidMadani

Trachealaspirate 7 4 6 4 21 Wounddischarge 7 8 4 6 25 Urine 7 6 9 7 29 Blood 1 4 1 2 8 Throatculture 1 0 3 0 4 Catheter 0 0 2 2 4 Sputum 0 1 1 0 2 Bronchialwashing 0 0 0 2 2

Pleuralfluidculture 1 0 0 0 1

Urethraldischarge 1 0 0 0 1

Peritonealfluid 1 0 0 0 1

Eardischarge 0 1 0 0 1

Stool 1 0 0 0 1

Table2–AminoacidalterationsingyrAandparCinciprofloxacinresistantisolatesofPseudomonasaeruginosa.

Groups No.ofisolates ReplacementinQRDR

GyrAatposition ParCatposition

83 87 87 88

PAO1 Thr(ACC) Asp(GAC) Ser(TCG) Ala(GCC)

I 20 Ile(ATC) – – –

II 8 Ile(ATC) – – Pro(CCC)

III 31 Ile(ATC) – Leu(TTG) –

IV 5 Ile(ATC) Asn(AAC) Leu(TTG) –

PCRamplificationandDNAsequencing

Chromosomal DNA from the isolates of P. aeruginosa was extracted by DNA extraction kit (Yekta Tajhiz Azma, Iran) accordingtothemanufacturer’sinstructions.ThePCR reac-tion was performed in a 50␮Lmixture containing 1.5mM MgCl2,0.5pmolofeachprimer,0.2mMdNTPs(YektaTajhiz Azma,Iran),1UofPfuDNApolymerase(YektaTajhizAzma, Iran),1XPfuDNApolymerasebufferand10–100ngofthe tem-plateDNA.

The amplification of gyrA and parC genes was carried outusingthepolymerasechainreactionandspecificprimer sets as described previously.26 _{Purified PCR products were}

sequenced using the Applied Biosystems 3730/3730xl DNA analyzerssequencing(ABI)system,(BioneerCo.,Korea). Statisticalanalysis

Categoricalvariableswere comparedbytheChi-squaretest orFisher’sexacttestusingSPSS16.0statisticalsoftware(SPSS Inc.,Chicago,IL).Astatisticallysignificantdifferencewas con-sideredasaP-value<0.05.

Results

Antimicrobialsusceptibilitytesting

Ofthe100P.aeruginosaclinicalisolatestestedfor antimicro-bialsusceptibility,71ofthemwerefoundtoshowmultidrug resistance(MDR).MDRwasdefinedasresistancetothreeor moreunrelatedantibiotics.27 _{Thehighestrateofresistance}

was observed against ticarcillin (84%) and ofloxacin (82%). Moreover,thehighestsusceptibilityratewasobtainedagainst ceftazidime(45%),followedbygentamicin(44%).Ofthetotal isolates, 64 (64%) were resistant, 2 (2%) were intermediate and34(34%)wereobservedtobesusceptibletociprofloxacin. Moreover,63%ofisolateswereresistant,1%were intermedi-ate,and36%weresusceptibletolevofloxacin.

DNAsequencesanalysis

DNAsequencesofallP.aeruginosaisolateswerecomparedwith thecorrespondingsequencesofP.aeruginosaPAO1(Accession: NC002516.2GI:110645304).Asidefromciprofloxacin suscep-tible,levofloxacinsusceptibleandciprofloxacinintermediate isolateswhichhadnoaminoacidalterationsintheirgyrAor

parCgenes,theaminoacidalterationswererecognizedingyrA

andparCQRDRof64ciprofloxacinresistant,63levofloxacin

resistantand1levofloxacinintermediateisolates,asdescribed

inTable2.Thetotalmutationsfoundintheseisolateswere

classified into4distinct groupsaccordingtothe patternof aminoacidalteration.GroupI:isolatescontainedsingle muta-tion Thr-83→ Ile ingyrA.Group II: isolatescontainedone mutationThr-83→IleingyrAandonemutationAla-88→Pro inparC.GroupIII:IsolatescontainedonemutationThr-83→ IleingyrAandonemutationSer-87→LeuinparC.GroupIV: isolatescontainedtwomutationsThr-83→IleandAsp-87→ AsningyrAandonemutationSer-87→LeuinparC.

DNAsequencesofQRDRgyrAshowedThr-83→Ile substi-tutionforall64ciprofloxacinresistantisolates.Of64isolates, 20(31.25%)hadamutation(Thr-83→Ile)ingyrAalone(group I). Adoublemutationin gyrA (Thr-83→ Ile and Asp-87→ Asn)wasdetectedin5of64isolates.Aminoacidalteration intheQRDRparCwasobservedin44(68.75%)of64isolates. AlloftheseisolatespossessedadditionalmutationsingyrA.

NodoublemutationsinparCwerefound.TheSer-87→Leu substitutionwasfoundin31(48.4%)of64isolates.Moreover, thesubstitutionofProforAla-88wasobservedin8(12.5%)of 64isolates.

CorrelationbetweenfluoroquinolonesMICandQRDRs mutations

TheMICvaluesofciprofloxacinandlevofloxacinfor64 resis-tant isolates and their correlation with different types of mutations in gyrA and parC genes are shown in Table 3. Asshown, ciprofloxacinMICforisolateswithasinglegyrA

substitution(Thr-83 →Ile)rangedfrom4–64␮g/mLandfor levofloxacin, the 4–32␮g/mL range was observed. Isolates with a single gyrA (Thr-83 → Ile) substitution and a sin-gle parC substitution (Ala-88 → Pro or Ser-87 → Leu) had ciprofloxacinMICsrangingfrom8to128or16to256␮g/mL andlevofloxacinMICsvariedfrom8to64or8to256␮g/mL. Moreover,the isolateswithdouble gyrAsubstitutions (Thr-83 →IleandAsp-87→Asn)and asingleparCsubstitution (Ser-87→Leu)hadciprofloxacinandlevofloxacinMICs ran-gingfrom32to256␮g/mL.Ourresultsshowedthatthetwo concurrentmutationsin gyrAand parC geneswere associ-atedwithahigherlevelofciprofloxacinandlevofloxacinMICs, ascomparedtoasinglemutationingyrA.(Geometricmean MICs ofciprofloxacin,29.34(group2) and 32(group3) ver-sus16.56(group1)␮g/mL,p<0.05;geometricmeanMICsof levofloxacin,24.67(group2)and28.6 (group3) versus14.42 (group 1)␮g/mLp<0.05). Moreover,three concurrent muta-tionsingyrAandparCgeneswere associatedwithahigher levelofciprofloxacinandlevofloxacinMICs,ascomparedto

Table3–CorrelationofmutationsingyrAandparCgenesandMICsdistributionofciprofloxacinandlevofloxacin.

Group No.ofisolates Antimicrobial agents

No.ofisolateswithMICs(␮g/mL)

0.5 1 2 4 8 16 32 64 128 256 I 20 Ciprofloxacin 2 2 10 5 1 GyrA(83) Levofloxacin 1 4 12 3 II 8 Ciprofloxacin 1 1 5 1

GyrA(83),ParC(88) Levofloxacin 1 3 2 2

III

31 Ciprofloxacin 12 11 5 2 1

GyrA(83),ParC(87) Levofloxacin 4 8 12 4 2 1

IV

5 Ciprofloxacin 2 1 1 1

GyrA(83and87),ParC(87) Levofloxacin 2 1 2

twoconcurrentmutationsingyrAandparCgenes(Geometric meanMICsofciprofloxacin,73.5(group4)versus29.3(group 2)␮g/mL, and 32 (group 3)␮g/mL p<0.05; geometric mean MICsoflevofloxacin,64(group4)versus24.6 (group2)and 28.61(group3)␮g/mLp<0.05)orsinglemutationingyrA. (Geo-metricmeanMICsofciprofloxacin,73.51(group4)versus16.56 (group1)␮g/mL,p<0.05;geometricmeanMICsoflevofloxacin, 64(group4)versus14.42(group1)␮g/mLp<0.05).

Discussion

Fluoroquinolonessuchasciprofloxacinandlevofloxacinare animportantclassofantibioticsforthetreatmentofP. aeru-ginosa infections.26 _{However, P. aeruginosa rapidly becomes}

resistanttothesedrugsduringantibiotictherapy.15_The

princi-plemechanismoffluoroquinolonesresistanceinP.aeruginosa

involvesmutationsinthegenesofDNAgyraseand topoiso-meraseIV.18

In the present study, the alteration of Thr-83 to Ile in

gyrA was found inall ciprofloxacin and levofloxacin resis-tant P. aeruginosa isolates.Moreover, a doubleconcomitant mutation in gyrA (Thr-83 → Ile and Asp-87 → Asn) was observed in five ciprofloxacin and levofloxacin resistant isolates.More noteworthy,theMIC valuesoftested fluoro-quinolonesamong theseisolates were significantly higher. However,noaminoacidchangewasdetectedinciprofloxacin susceptible,levofloxacinsusceptibleandciprofloxacin inter-mediate isolates. So Thr-83 → Ile was shown to be the chief mechanism of fluoroquinolones resistance. This was consistentwiththe resultsofother studies.26,28–30

Further-more, the amino acid sequences analysis in the QRDR of

parCshowedthatmorethanhalfofciprofloxacin and levo-floxacinresistantisolateshadanalterationinparCandthe Ser-87→Leusubstitutionwasthepredominantaminoacid change (48.4%). Lee et al.18 _{and Mouneimne et al.}16 _have

previously reported this amino acid change in 35.9% and 25%oftheciprofloxacinresistantisolates,respectively.Also, another alteration in parC, Ala-88→ Pro substitution, was highlyfrequentinourisolates,incomparisontotheresults ofAkasakaet al.,17 _{and Sekiguchiet al.}31 _{They foundthis}

aminoacidchangeonlyinoneofallstudiedisolates.More importantly,thissubstitutionwasobservedgenerallyamong high level ciprofloxacin resistant isolates suggested to be responsibleforfluoroquinoloneresistance.However,wedid

notdetectaminoacidalterationatpositionsPro-83,Gly-85,31

Glu-91andLeu-9517_{inparC,asreportedintheprevious}

stud-ies.

Based on the analysis of sequencing results, all of the isolates with parC mutationhad one or two mutationsin

gyrA.Thisobservation confirmedthat theDNA gyrasewas the primary target for fluoroquinolone resistance in the clinical isolatesof P. aeruginosa.However, isolatesthat had doublemutationingyrAand parChad higherciprofloxacin and levofloxacin MICs than those with a single mutation ingyrA,therebysuggestingthatalterationinparCoccurred after gyrA, leading to higher level fluoroquinolone resis-tance in P. aeruginosa. Moreover, the addition of a second

gyrA alteration to gyrA and parC mutations had a signif-icant effect on ciprofloxacin and levofloxacin MICs. Our resultssuggestedthattherecouldbeacorrelationbetween the number of gyrA and parC alterations and the level of fluoroquinolone resistance. This has been reported by Lee et al.18 _{for the clinical isolates of P. aeruginosa.}

Dif-ferences in the MICs of ciprofloxacin and levofloxacin for isolates had a single alteration in QRDR of gyrA with or without an alteration in QRDR of parC, revealing that other resistance factors were involved in fluoroquinolone resistance. Mechanisms such as overexpression of efflux pumps(MexAB-OprM32,33 _MexCD-OprJ,28 _MexEF-OprN21 _and

MexXY-OprM34_{) and mutation in gyrB and parE have been}

describedforfluoroquinoloneresistanceinP.aeruginosa.17,18

However, the impact of these mechanisms on MICs of ciprofloxacinandlevofloxacincanbeclarifiedbyfurther stud-ies.

Toconclude, mechanisms other thanmutationsin gyrA

and parC (such as activeeffluxpumps, alterations ingyrB, parE orinnate impermeabilityofthe membrane)may con-tributetotheleveloffluoroquinoloneresistanceintheclinical isolatesofP. aeruginosa, but asingleaminoacid alteration, Thr-83→Ile,ingyrA,issufficienttocauseclinicallyimportant levels of resistance to fluoroquinolones, and the simul-taneous presence of mutation in parC (Ser-87 → Leu or Ala-88 → Pro) mediates significantly higher level fluoro-quinoloneresistance.

Finally,ourresultsrevealedthatthemutationsingyrAand

parCwerethemainmechanismoffluoroquinoloneresistance amongtheclinicalisolatesofP.aeruginosainTabriz,Iran.To thebestofourknowledge,thisstudyisthelargestanalysisof

theQRDRofgyrAandparCintheclinicalisolatesofP. aerugin-osafromIran.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisworkwasfullysupportedbyInfectiousandTropical Dis-easesResearchCenter(grantNo.93-02),TabrizUniversityof MedicalSciences.Itisalsoareportorginiatingfromadatabase developedforthe thesisoffirst author registered in Infec-tiousandTropicalDiseasesResearchCenter,TabrizUniversity ofMedicalSciences,Tabriz,Iran.TheauthorsalsothankDr. HosseinSamadiKafilforhiskindhelpinanalyzing sequenc-ingresults,andMrs.MaryamRasoliandMr.JaberKamranfor collectingtheclinicalisolates.

r

e

f

e

r

e

n

c

e

s

1.AhangarzadehRezaeeM,BehzadiannezhadQ,

Najjar-PirayehS,OuliaP.Invitroactivityofimipenemand

ceftazidimeagainstmucoidandnon-mucoidstrainsof

PseudomonasaeruginosaisolatedfrompatientsinIran.Arch

IranMed.2002;5:251–254.

2.LihuaL,JianhuitW,JialiniY,YayinL,GuanxinL.Effectsof

allicinontheformationofPseudomonasaeruginosabiofinm

andtheproductionofquorum-sensingcontrolledvirulence

factors.PolJMicrobiol.2013;62:243–251.

3.WolskaK,SzwedaP.GeneticfeaturesofclinicalPseudomonas

aeruginosastrains.PolJMicrobiol.2009;58:255–260.

4.AhangarzadehRezaeeM,BehzadiannezhadQ,NajjarPS,

OuliaP.Higheraminoglycosideresistanceinmucoid

Pseudomonasaeruginosathaninnon-mucoidstrains.Arch IranianMed.2002;5:108–110.

5.OliveiraACd,MalutaRP,StellaAE,RigobeloEC,MarinJM,

ÁvilaFAd.IsolationofPseudomonasaeruginosastrainsfrom

dentalofficeenvironmentsandunitsinBarretos,stateof

SãoPaulo,Brazil,andanalysisoftheirsusceptibilityto

antimicrobialdrugs.BrazJMicrobiol.2008;39:579–584.

6.WolskaK,KotB,JakubczakA.Phenotypicandgenotypic

diversityofPseudomonasaeruginosastrainsisolatedfrom

hospitalsinSiedlce(Poland).BrazJMicrobiol.2012;43:274–282.

7.PerezLRR,LimbergerMF,CostiR,DiasCAG,BarthAL.

Evaluationofteststopredictmetallo-␤-lactamaseincystic

fibrosis(CF)andnon-(CF)Pseudomonas.BrazJMicrobiol.

2014;45:835–839.

8.StratevaT,YordanovD.Pseudomonasaeruginosa–a

phenomenonofbacterialresistance.JMedMicrobiol.

2009;58:1133–1148.

9.KugelbergE,LöfmarkS,WretlindB,AnderssonDI.Reduction

ofthefitnessburdenofquinoloneresistanceinPseudomonas

aeruginosa.JAntimicrobChemother.2005;55:22–30.

10.LlanesC,KöhlerT,PatryI,DehecqB,VanDeldenC,PlésiatP.

RoleoftheeffluxsystemMexEF-OprNinlowlevelresistance

ofPseudomonasaeruginosatociprofloxacin.AntimicrobAgents Chemother.2011;55:5676–5684.

11.DalhoffA.Globalfluoroquinoloneresistanceepidemiology

andimplicationsforclinicaluse.InterdiscPerspectInfectDis.

2012:1–37.

12.WydmuchZ,Skowronek-CiolekO,CholewaK,MazurekU,

PachaJ,KepaM.gyrAmutationsinciprofloxacin-resistant

clinicalisolatesofPseudomonasaeruginosainaSilesian

HospitalinPoland.PolJMicrobiol.2005;54:201–206.

13.AkasakaT,OnoderaY,TanakaM,SatoK.Cloning,

expression,andenzymaticcharacterizationofPseudomonas

AeruginosatopoisomeraseIV.AntimicrobAgentsChemother.

1999;43:530–536.

14.AgnelloM,Wong-BeringerA.Differentiationinquinolone

resistancebyvirulencegenotypeinPseudomonasaeruginosa.

PLoSONE.2012;7:e42973.

15.JalalS,CiofuO,HøibyN,GotohN,WretlindB.Molecular

mechanismsoffluoroquinoloneresistanceinPseudomonas

aeruginosaisolatesfromcysticfibrosispatients.Antimicrob AgentsChemother.2000;44:710–712.

16.MouneimnéH,RobertJ,JarlierV,CambauE.TypeII

topoisomerasemutationsinciprofloxacin-resistantstrains

ofPseudomonasaeruginosa.AntimicrobAgentsChemother.

1999;43:62–66.

17.AkasakaT,TanakaM,YamaguchiA,SatoK.TypeII

topoisomerasemutationsinfluoroquinolone-resistant

clinicalstrainsofPseudomonasaeruginosaisolatedin1998and

1999:roleoftargetenzymeinmechanismoffluoroquinolone

resistance.AntimicrobAgentsChemother.2001;45:2263–2268.

18.LeeJK,LeeYS,ParkYK,KimBS.AlterationsintheGyrAand

GyrBsubunitsoftopoisomeraseIIandtheParCandParE

subunitsoftopoisomeraseIVinciprofloxacin-resistant

clinicalisolatesofPseudomonasaeruginosa.IntJAntimicrob

Agents.2005;25:290–295.

19.NakanoM,DeguchiT,KawamuraT,YasudaM,KimuraM,

OkanoY.MutationsinthegyrAandparCgenesin

fluoroquinolone-resistantclinicalisolatesofPseudomonas

aeruginosa.AntimicrobAgentsChemother.1997;41:2289–2291.

20.SalmaR,DabboussiF,KassaaI,KhudaryR,HamzeM.gyrA

andparCmutationsinquinolone-resistantclinicalisolates

ofPseudomonasaeruginosafromNiniHospitalinnorth

Lebanon.JInfectChemother.2013;19:77–81.

21.ListerPD,WolterDJ,HansonND.Antibacterial-resistant

Pseudomonasaeruginosa:clinicalimpactandcomplex

regulationofchromosomallyencodedresistance

mechanisms.ClinMicrobiolRev.2009;22:582–610.

22.JaponiA,FarshadS,AlborziA.Pseudomonasaeruginosa:burn

infection,treatmentandantibacterialresistance.IranRed

CrescentMedJ.2009;2009:244–253.

23.RanjbarR,OwliaP,SaderiH,MansouriS,Jonaidi-JafariN,

IzadiM.CharacterizationofPseudomonasaeruginosastrains

isolatedfromburnedpatientshospitalizedinamajorburn

centerinTehran,Iran.ActaMedIran.2011;49:675–679.

24.HallGS.Nonfermentingandmiscellaneousgram-negative

bacilli.In:MahonCR,LemanDC,ManyselisG,eds.Textbook

ofDiagnosticMicrobiology.3thed.Ohio:Saunders-Elsevier; 2007:564–585.

25.ClinicalandLaboratoryStandardsInstitute(CLSI).

PerformanceStandardsforAntimicrobialSusceptibilityTesting DocumentApprovedStandardM100-S20.PA,USA:Wayne;2010.

26.GorganiN,AhlbrandS,PattersonA,PourmandN.Detection

ofpointmutationsassociatedwithantibioticresistancein

Pseudomonasaeruginosa.IntJAntimicrobAgents.

2009;34:414–418.

27.AhangarzadehRezaeeM,SheikhalizadehV,HasaniA.

Detectionofintegronsamongmulti-drugresistant(MDR)

Escherichiacolistrainsisolatedfromclinicalspecimensin

northernwestofIran.BrazJMicrobiol.2011;42:1308–1313.

28.HigginsP,FluitA,MilatovicD,VerhoefJ,SchmitzF-J.

MutationsinGyrA,ParC,MexRandNfxBinclinicalisolates

ofPseudomonasaeruginosa.IntJAntimicrobAgents.

2003;21:409–413.

29.KureishiA,DiverJM,BecktholdB,SchollaardtT,BryanLE.

CloningandnucleotidesequenceofPseudomonasaeruginosa

quinolone-resistantclinicalisolates.AntimicrobAgents Chemother.1994;38:1944–1952.

30.TakenouchiT,SakagawaE,SugawaraM.DetectionofgyrA

mutationsamong335Pseudomonasaeruginosastrains

isolatedinJapanandtheirsusceptibilitiesto

fluoroquinolones.AntimicrobAgentsChemother.

1999;43:406–409.

31.SekiguchiJ-I,AsagiT,Miyoshi-AkiyamaT,etal.Outbreaksof

multidrug-resistantPseudomonasaeruginosaincommunity

hospitalsinJapan.JClinMicrobiol.2007;45:979–989.

32.LambertP.Mechanismsofantibioticresistancein

Pseudomonasaeruginosa.JRSocMed.2002;95:22–26.

33.PooleK.Efflux-mediatedresistancetofluoroquinolonesin

gram-negativebacteria.AntimicrobAgentsChemother.

2000;44:2233–2241.

34.VanBambekeF,GlupczynskiY,PlesiatP,PechereJ,Tulkens

PM.Antibioticeffluxpumpsinprokaryoticcells:occurrence,

impactonresistanceandstrategiesforthefutureof

antimicrobialtherapy.JAntimicrobChemother.