Bacteremia and meningitis caused by OXA-23-producing Acinetobacter baumannii – molecular characterization and susceptibility testing for alternative antibiotics

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

Clinical

Microbiology

Bacteremia

and

meningitis

caused

by

OXA-23-producing

Acinetobacter

baumannii

–

molecular

characterization

and

susceptibility

testing

for

alternative

antibiotics

Kamile

Francine

Schuertz

_,

_Felipe

_Francisco

_Tuon

_,

_Jussara

_Kasuko

_Palmeiro

_,

Danieli

Conte

_,

_João

_Paulo

_Marochi

_Telles

_,

_Lucas

_Eduardo

_Trevisoli

_,

Libera

Maria

Dalla-Costa

a_{InstitutodePesquisaPelePequenoPrincipe,Curitiba,PR,Brazil}

b_{PontifíciaUniversidadeCatólicadoParaná,EscoladeMedicina,Curitiba,PR,Brazil} c_{UniversidadeFederaldoParaná,HospitaldeClinicas,Curitiba,PR,Brazil}

d_{HospitaldeInfectologiaEmilioRibas,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

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o

Received1August2017 Accepted3April2018 Availableonline24April2018 AssociateEditor:AfonsoBarth

Keywords: Acinetobacterbaumannii Oxacillinases Carbapenem Multidrugresistance Doxycycline

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Background:Carbapenem-resistantAcinetobacterbaumanniiinfectionisaconcernin devel-opingcountriesduetohighincidence,fewtherapeuticoptions,andincreasingcosts.

Objective:Characterizeandanalyzetheantibioticsusceptibilitypatternsof carbapenem-resistant A. baumanniiisolatesandevaluate clinicaldata ofmeningitisandbacteremia causedbythismicroorganism.

Methods:Twenty-sixA.baumanniiisolatesfrom23patientswereidentifiedbyMALDI-TOF andautomatedmethodsandgenotypedusingpulsedfieldgenotypingelectrophoresis. Clin-icaldata andoutcomeswereevaluated.Susceptibilityofisolatestocolistin,tigecycline, meropenem,imipenem,anddoxycyclinewasdetermined.

Results:MortalityduetoA.baumanniiinfectionswas73.91%;allpatientswithmeningitisand 7/8patientswithventilator-associatedpneumoniadied.Allisolatesweresusceptibilityto polymyxin(100%;MIC50,MIC90:1␮g/mL,1␮g/mL)andcolistin(100%;MIC50,MIC90:2␮g/mL, 2␮g/mL),and92%weresusceptibletotigecycline(MIC50,MIC90:1␮g/mL,1␮g/mL)and doxy-cycline(MIC50,MIC90:2␮g/mL,2␮g/mL).blaOXA-23wasidentifiedin24isolates.Molecular typingshowed8differentpatterns:13isolatesbelongedtopatternA(50%).

Conclusion: Carbapenem-resistant A. baumannii infections mortality is high. Alternative antimicrobialtherapy(doxycycline)forselectedpatientswithcarbapenem-resistantA. bau-manniiinfectionshouldbeconsidered.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:[email protected](F.F.Tuon).

1 _{AllauthorsmeettheICMJEauthorshipcriteria.}

https://doi.org/10.1016/j.bjm.2018.04.002

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Acinetobacter baumannii healthcare associated infections (HAIs)areconsideredaseriouspublichealthproblemowing totheirprevalenceandmortalityindevelopingcountries.1,2

ThesusceptibilityofA.baumanniitothemostcommonlyused antibioticshasdecreased.3

Thetreatmentofcarbapenem-resistantA.baumannii (CR-AB) has been a challenge because carbapenem is the first option in most hospitals for empirical treatment of HAI. Consequently, older drugs with increased toxicity, includ-ingpolymyxinsandaminoglycosides,havebeenusedbased solelyonempiricalevidence.4 _{Doxycycline isanoral}

tetra-cyclinewithactivityagainstA.baumanniiandthisdrugcan achievetherapeuticlevels incerebrospinalfluidwhen ade-quatedosageisused.5_{Tigecyclineisanotheroptionapproved}

forintra-abdominalandsoft tissueinfections thathasalso beenusedextensivelyforthetreatmentofmultidrugresistant infectionsatotheranatomic locations.Unfortunately, clini-calandantimicrobialsusceptibilitydataarerarelyevaluated together,sothetrueefficacyofnon-carbapenemantibiotics against CR-AB is unknown. The aims of this study were tocharacterizethemoleculartypesofA.baumanniiisolates from cerebrospinal fluidand blood cultureand toevaluate clinicaldataand susceptibilitypatternsoftheseisolatesto treatmentwithpolymyxin,colistin,tigecycline,doxycycline, meropenem,andimipenem.

Material

and

methods

Studysetting

AtransversalstudywascarriedoutatHospitalUniversitario EvangelicodeCuritiba(HUEC),atertiary-care,trauma refer-encehospital,inSouthernBrazil,withatotalnumberof660 beds.ThestudyincludedpatientsadmittedfromApril2010 throughApril 2013.Thisstudy wasapprovedbythe Ethics CommitteeofFEPARandFPP(311.769).

Clinicaldata

Datawerecollectedfrommedicalchartsand/orhospital com-putersystemdatabases.Theclinicaldataevaluatedincluded age, gender, site of infection, comorbidities, admission at intensive care unit, antimicrobial therapy, and outcome. Antimicrobial therapy was included in the analysis when it wasstarted within72hofinfection diagnosis.Infections whereclassifiedaccordingtoCDCcriteria.6_Thirty-day

mor-talitywasalsoanalyzed.

Microbiologicalstudies

Twenty-sixisolatesofA.baumanniifrom23patientsadmitted atHUECfromApril2010throughApril2013wereevaluated. Nineteen isolateswere obtained from blood cultureand 7 fromcerebrospinalfluidculture.All isolateswereidentified using the Vitek2® Compact System (bioMérieux, Durham, USA)and matrix-assistedlaser desorption ionization

time-of-flightmassspectrometry(MALDI-TOF)usingBiotyper2.0 software(Bruker151DaltonikInc,Bremen,Germany).

Antibioticsusceptibilitytests

Minimal inhibitory concentration (MIC)of colistin, doxycy-cline, imipenem, meropenem,polymyxinBand tigecycline wasestimatedbytheagardilutionmethodaccordingtothe CLSI, M7-A9 approved standard.7 _{Polymyxin B and colistin}

werealsotestedbyabrothmicrodilutionmethodaccording toCLSIM7-A9.8_{InterpretationofMICwasperformed}

accord-ingtoCLSI,M100-S24,9_{exceptfortigecycline.}10_TheMIC50and

MIC90werecalculatedforboththeagarandbrothmethods. Wealso comparedbrothand agar dilution forcolistin and polymyxinusingaconcordancelevelof±≤1log2.11

Moleculardetectionofoxacillinasegenes

To determine oxacillinase-encoding genes, polymerase chain reaction (PCR) was performed for all isolates by using the following primers: blaOXA-23 (F-5 GATCG-GATTGGAGAACCAGA 3; R-5 ATTTCTGACCGCATTTCCAT 3), blaOXA-51 (F-5 TAATGCTTTGATCGGCCTTG 3; R-5 TGGATTGCACTTCATCTTGG 3), blaOXA-58 (F-5 AAG-TATTGGGGCTTGTGCTG 3; R-5 CCCCTCTGCGCTCTACATAC 3), blaOXA-143 (F-5 TGGCACTTTCAGCAGTTCCT 3; R-5 TAATCTTGAGGGGGCCAACC3).PCRwasperformedinafinal volumeof25␮LwithafinalDNAconcentrationof100ng/mL. Theamplificationconditionswere94◦Cfor25s,followedby 52◦Cfor40sand72◦Cfor50s.12

Pulsedfieldgelelectrophoresis(PFGE)

All26isolatesweregenotypedbyPFGEandDNAwasprepared as described previously.13 _{Theentirechromosomal DNAof}

thestrainswasdigestedwith30UofApaI(Fermentas–Life Science,Glen Burnie,MD). PFGEwasperformed byusinga clamped homogeneouselectric field electrophoresis(CHEF) DRIII apparatus (Bio-Rad Laboratories, Hercules, USA). The conditionsemployedwere asfollows:temperatureof12◦C, voltageof6V/cm,runtimeof30h,andswitchtimeof5–35s. Thepatternofbandswasinterpretedaccordingtopreviously describedcriteria.14_{PFGEclusteringwasdeterminedbyusing}

theunweighted-pairgroupmethodwitharithmeticaverages (UPGMA)andbyusingDice’scoefficient.Thetolerancewasset at1%.Identicalisolateswereassignedthesamecapitalletter. Thosewith80%similaritywereassignedasasubtypeofthe majortype,followedbyanArabicnumber(e.g.A1,A2,A3,A4).

Results

Patientdata

Twenty-six isolates from 23 patients were evaluated. The medianageofpatientswas42years(10–60years).Allpatients werehospitalizedintheintensivecareunitwhenclinical sam-pleswerecollected.Isolateswereobtainedfrombloodculture orcerebrospinalfluid.Samplesfromotherbodysites(sputum

Table1–Clinicalandlaboratorialdatafrom23patientswithAcinetobacterbaumanniicausinginfection. Ne _{Sex Age Siteof}

infection

Disease HAIc _{Outcome Treatment}b,d _{Brothdilutionmethod Agardilution}

IPM MEM DC TGC COL POL COL POL PFGE C3 F 54 Meningitis Neoplasm Yes Death MEM,VAN,SXT,AK >32 >32 1 1 0.5 ≤0.25 0.5 0.125D1 C6 M 23 Meningitis Trauma Yes Death AK,TGC >32 >32 1 2 0.5 0.5 2 0.25 A11 C8 F 41 Meningitis Neoplasm Yes Death MEM,VAN,POL >32 >32 ≤0.5 2 0.5 ≤0.25 1 0.25 A5 C7 M 22 Meningitis Trauma Yes Death AK,TGC,IPM >32 >32 ≤0.5 2 1 0.5 1 0.5 C3

C9 >32 >32 1 2 1 0.5 1 1 A6

H14 >32 >32 ≤0.5 2 0.5 ≤0.25 1 0.5 G1

C10 M 41 Meningitis Hydrocephalus Yes Death RIF,COL 0.5 0.5≤0.5 0.5 1 0.5 2 1 E2 C11 F 20 Meningitis Headtrauma Yes Death MEM,VAN,POL >32 >32 1 2 1 0.5 1 0.5 B1 H2 M 42 VAPa _Acute

Lymphocytic Leukemia

Yes Survival IPM,VAN 32 >32 1 2 1 0.5 1 0.5 F3

H7 M 49 VAP Acute Lymphocytic Leukemia

Yes Death IPM,VAN >32 >32 1 2 1 2 1 0.5 A9

H8 M 49 SSTI Hemorragic stroke

Yes Death RIF,TZP,VAN,POL >32 >32 ≤0.5 2 1 0.5 2 1 H1 H9 F 10 Bacteremia Burn Yes Survival AK 0.1250.25≤0.5 0.5 1 ≤0.25 1 0.5 C1 H10 F 46 VAP Ureteral

calculus

Yes Survival LZD,MEM >32 >32 ≤0.5 2 1 1 1 0.5 C2

H18 >32 >32 ≤0.5 2 0.5 ≤0.25 1 0.25 A10

H11 M 41 Bacteremia Myelodysplasic syndrome

Yes Death LZD,MEM >32 >32 1 2 0.5 0.5 1 0.5 A3 H13 M 42 Bacteremia Renal

transplant

No Survival CIP >32 >32 1 2 1 0.5 1 1 A3 H15 M 75 VAP Trauma Yes Death ETP >32 >32 1 2 1 0.5 2 0.25 A7 H16 M 88 Bacteremia Trauma Yes Death TZP >32 >32 1 2 0.5 ≤0.25 1 0.5 A7 H17 F 51 VAP Trauma Yes Death IPM,LZD,SXT,POL >32 >32 ≤0.5 2 0.5 ≤0.25 1 0.5 A8 H19 M 52 VAP Acuterenal

failure

Yes Death IPM,VAN >32 >32 1 1 1 0.5 1 0.5 E1 H20 M 31 SSTI Trauma Yes Death MEM,VAN,POL >32 >32 1 2 1 ≤0.25 1 0.25 A1 H21 M 68 VAP Sarcoidosis Yes Death IPM,LZD >32 >32 1 2 1 ≤0.25 1 0.25 A4 H22 M 32 SSTI Trauma Yes Survival RIF,LZD,MEM,POL >32 >32 ≤0.5 2 0.5 0.5 1 0.25 A2 H24 M 78 UTI Acuteinfarct Yes Survival TZP,MEM,POL >32 >32 1 2 1 1 1 1 F1 H25 M 68 VAP Chronicrenal

failure

Yes Death MEM,LZD >32 >32 8 4 0.5 ≤0.25 1 0.5 F1 H26 M 38 VAP HIVinfection Yes Death POL,MEM >32 >32 8 4 1 0.5 1 0.5 F2

a _{VAP,ventilator-associatedpneumonia;UTI,urinarytractinfection;SSTI,skinandsofttissueinfection.} b _{Therapywithin72hofdiagnosisofHAI.}

c _{HAI,hospital-acquiredinfection.}

d _{IPM,imipenem;MEM,meropenem;COL,colistin;POL,polymyxin;DC,doxycycline;AK,amikacin;LZD,linezolid;ETP,ertapenem;VAN,}

vancomcycin;TZP,piperacillin-tazobactam;SXT,sulfamethoxazole/trimetoprim;CIP,ciprofloxacin;RIF,rifampin.

e _{H,bloodsample;C,cerebrospinalfluid.}

orbronchoalveolar lavage,urine ortissuebiopsy) were not evaluated.ClinicalcharacteristicsarepresentedinTable1.

Meningitis

Six patients presented with nosocomial meningitis. The medianagewas33years(15–56years)with3femalesand3 males.All patientspresentedwithmeningitissecondary to externalventricularshuntduetoheadtraumaorneoplasm withamortalityrate of100%.All strains inthesepatients weresusceptibletopolymyxins(colistinandpolymyxin)and doxycycline.Whenbacterialsusceptibilitytopolymyxinswas takenintoaccount,3patientsreceivedthecorrecttreatment. The3remainingpatientsweretreatedwithcarbapenems,and theMICofthisantibioticwasdeterminedtobe>32mg/Lin

all strainsisolated fromthesepatients.Therewasno clon-alitysharedamongthemeningitiscases.Inonepatientwith

A.baumanniimeningitis,thecloneisolatedfromcerebrospinal fluidwasidenticaltoonefoundinbloodcultureandanother cerebrospinal fluid sample that was obtained 5 days later (CloneA).

Bacteremia

SeventeenpatientspresentedwithA.baumanniibacteremia, 4ofthemprimaryand13ofthemsecondarytoinfectionat anothersite.Ventilator-associatedpneumonia(VAP)with sec-ondarybacteremiawasthemostcommoninfection(9cases), followedbysecondarybacteremiacausedbyskinandsoft tis-sueinfection(3cases)andurinarytractinfection(1case).The

medianageofpatientswas50years(10–60year),with14males and3females.Therewasnoclonalassociationwithaspecific siteofinfection.Consideringthetherapeuticand microbiolog-icalaspectofbacteremicpatients,7patientsreceivedcorrect therapywithin72h(41.17%).ThemortalityofpatientswithA. baumanniibacteremiawas64.70%;specifically,7of9patients withVAP,and1of3withskin-softtissueinfectionsdied.There wasnocorrelationofsurvivalwithcorrectantibiotictherapy withinfirsthoursofculturesample.Therewasnoclonality amongcasesofA.baumanniibacteremia.

Antimicrobialsusceptibility

Morethan90%ofisolateswereresistanttocarbapenem(MIC50 andMIC90>32␮g/mL).

Isolates were susceptibility to polymyxin and colistin (MIC50andMIC90=1␮g/mLforpolymyxin;MIC50=0.5␮g/mL andMIC90=1␮g/mLforcolistin).Brothandagardilutionsfor

colistinandpolymyxinsusceptibilityshowedahigh concor-dancelevel(100%).

Bacterialisolatesalsohadsusceptibilitytotigecyclineand doxycycline(MIC50andMIC90=1␮g/mLfortigecycline;MIC50 andMIC90=2␮g/mLfordoxycycline).Onlytwosampleswere resistanttothesedrugs.

PCRamplifiedblaOXA-51 inallisolates.TheblaOXA-23 gene was amplified in all 24 isolates resistant to carbapenem, but was not detected in the two isolates susceptible to carbapenem.

PFGE

MoleculartypingwithPFGErevealed8distinctpatternswith 80% similarity (see dendrogram, Fig. 1). Thirteen isolates belongtopatternA(50%),including11subtypes.Other pat-terns (B, C, D,E, F, Gand H) were found in1, 3,1, 2, 4,1 and 1 samples, respectively. Both isolates of

carbapenem-PFGE PFGE 70.3 H G F E D A B C 66.9 73.0 83.3 87.2 96.6 92.6 88.9 57.7 96.6 93.8 87.3 96.8 91.9 90.4 84.5 75.4 72.7 92.9 C3 C2 C1 B1 A11 A10 A9 A8 A7 A7 A6 A5 A4 A3 A3 A2 A1 D1 E2 E1 F3 F2 F1 F1 G1 H1 85.2 97.9 65.6 60 65 70 75 80 85 90 95 100

Fig.1–Dendrogramof26isolatesfrom23patientswithAcinetobacterbaumanniicausinginfection.SeeTable1tocorrelation ofisolatesandsiteofinfection.

susceptibleA.baumanniishowed70.3%similarity(patternsG andH)

Discussion

Carbapenem-resistantA.baumanniiinfectionsaresevereand resultinhighmortalityincriticallyillpatients.Allpatients with meningitis ultimately died despite adequate therapy, a mortality rate previously described by our group.15 _The

outcome is not different for bacteremia, although previ-ousstudieshaveshownthatdelayintheadequatetherapy canreducethemortality.16_{Carbapenem-resistantA.}

bauman-nii is becoming increasingly prevalent in Brazil, and more than 60% of strains in some areas are resistant. OXA-23 carbapenemase hasbeen described asendemic since2003 inSouthern Brazil17 _{and wasidentified inall}

carbapenem-resistant A. baumannii in other publications.18 _Thecurrent

studyconfirmsthatthisoxacillinaseisprevalentand proba-blythemainmechanismofcarbapenemresistance.However, isolates from some hospitals have a different carbapene-mase profile, including a low prevalence of OXA-23 in A. baumannii.19

Outof26isolates,24weresusceptibletodoxycyclineand two isolatesshowed intermediate resistance.Timurkaynak et al. found asimilar doxycyclinesusceptibility pattern in

A.baumannii,20_{thisdrughasabactericidaleffectagainstA.}

baumanniiinsomeisolates,includingcarbapenem-resistant strains.21_{Althoughminocyclinehasbeenmorepotentagainst}

Acinetobacter,thistetracyclineisnotavailableinBrazil. Inourstudy,therewasahighconcordancebetween sus-ceptibility to colistin and polymyxin, but we did not test resistantisolates.SimilarresultsweredescribedbyGalesetal., who reported 94.3% concordance in susceptibility tothese drugs.11 _{Furthermore,amongthe 26 isolatesofA.}

bauman-nii,5showedskippedwellphenomenaforcolistinand8for polymyxin(datanotshown).Thisphenomenonoccurswhen thesedrugsaretestedinbrothmicrodilutionandisassociated withhetero-resistance.22

PFGE demonstrated a predominant clone (A) with the remainingisolatescharacterizedasrelatedclones.The mech-anism of carbapenem resistance was associated with the presenceofthegeneblaOXA-23,althoughwecannotexclude combinedmechanismssuchaspumpeffluxandporinloss.23

Thequestionofwhethertousecombinedtherapytotreat

Acinetobacterinfectionsremainsunanswered.Invitrostudies arecontroversialandclinicalstudiesarecomprisedof retro-spectivecohortswithsmallsamplenumbers.However,inthe contextofmultidrug-resistantAcinetobacter,fewoptionsare available,mainlyforhard-to-treatinfections,likemeningitis. Tigecyclinehasalowpenetrationintocerebrospinalfluid.24

Tigecyclineisalsocontra-indicatedforthetreatmentof pri-marybacteremiaaspeakconcentrationoflessthan1mg/Lcan beachieved,aninsufficientlevelfortreatmentofbloodstream infection. Furthermore, sub-therapeutic concentrations are associatedwithincreasingresistance.25_{Polymyxinisthedrug}

primarilyusedfortreatmentofCR-AB.Theuseofpolymyxin Bislessreportedthanthatofcolistin,althoughthisdrughas shown successfulresultswith less acuterenal injury than colistin.26 _{However,todate, thereare no reports regarding}

thepenetrationofpolymyxinB,themostcommonpolymyxin used inBrazil, inthe central nervous system. Colistin has been usedforthe treatmentofmeningitiswithreasonable outcomes.26_{Despitethepresenceofasusceptibilitypattern}

to polymyxinsin mostof ourisolates, these drugs should becautiouslyusedduetoincreasingresistance, nephrotox-icity,andneurotoxicity.Whenthesedrawbacksaretakeninto consideration,doxycyclineisanattractivealternativeforor potentialadditiontootheractivedrugstotreatnosocomial meningitis.

Insummary,A.baumanniiinfectionsinourpatientcohort resultedinhighmortality,andcarbapenemresistancewas fre-quentinthesesevereinfections.OXA-23continuestobethe mainmechanismofresistanceofA.baumanniiinthisregion oftheBrazil,withapredominantclonalpattern.Fortunately, alternative drugsare availableforuseunderspecific condi-tionsthatmustbedecidedbyexperts.

Ethical

approval

Informed

consent

Funding

Conflict

of

interest

FelipeTuonisaCNPQresearcher. Thereisnotconflictofinterest.

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1.ClimacoEC,OliveiraML,Pitondo-SilvaA,etal.Clonal

complexes104,109and113playingamajorroleinthe

disseminationofOXA-carbapenemase-producing

AcinetobacterbaumanniiinSoutheastBrazil.InfectGenetEvol.

2013;19:127–133.

2.FournierPE,RichetH.Theepidemiologyandcontrolof

Acinetobacterbaumanniiinhealthcarefacilities.ClinInfectDis.

2006;42(5):692–699.

3.MaragakisLL,PerlTM.Acinetobacterbaumannii:epidemiology,

antimicrobialresistance,andtreatmentoptions.ClinInfect

Dis.2008;46(8):1254–1263.

4.Fernandez-CuencaF,Tomas-CarmonaM,Caballero-Moyano

F,etal.[Invitroactivityof18antimicrobialagentsagainst

clinicalisolatesofAcinetobacterspp.:multicenternational

studyGEIH-REIPI-Ab2010].EnfermInfecMicrobiolClin.

2013;31(1):4–9.

5.KarlssonM,HammersS,Nilsson-EhleI,MalmborgAS,

WretlindB.ConcentrationsofdoxycyclineandpenicillinG

inseraandcerebrospinalfluidofpatientstreatedfor

neuroborreliosis.AntimicrobAgentsChemother.

6. EmoriTG,CulverDH,HoranTC,etal.Nationalnosocomial

infectionssurveillancesystem(NNIS):descriptionof

surveillancemethods.AmJInfectControl.1991;19(1):19–35.

7. CLSI(ClinicalandLaboratoryStandardsInstitute).Methods

forDilutionAntimicrobialSusceptibilityTestsforBacteriathat GrowAerobically;ApprovedStandard—TenthEdition(M07-A9).

ClinicalandLaboratoryStandardInstitute;2012.

8. CLSI.ClinicalandLaboratoryStandardsInstitute.Methodsfor

DilutionAntimicrobialSusceptibilityTestsforBacteriathatGrow Aerobically;ApprovedStandard—TenthEdition(M07-A10).

ClinicalandLaboratoryStandardInstitute;2015.

9. CLSI(ClinicalandLaboratoryStandardsInstitute).

PerformanceStandardsforAntimicrobialSusceptibilityTesting. ApprovedStandardM100-S24.ClinicalandLaboratory

StandardInstitute;2014.

10.MarchaimD,PogueJM,TzumanO,etal.Majorvariationin

MICsoftigecyclineinGram-negativebacilliasafunctionof

testingmethod.JClinMicrobiol.2014;52(5):1617–1621.

11.GalesAC,ReisAO,JonesRN.Contemporaryassessmentof

antimicrobialsusceptibilitytestingmethodsforpolymyxinB

andcolistin:reviewofavailableinterpretativecriteriaand

qualitycontrolguidelines.JClinMicrobiol.2001;39(1):183–190.

12.WoodfordN,EllingtonMJ,CoelhoJM,etal.MultiplexPCRfor

genesencodingprevalentOXAcarbapenemasesin

Acinetobacterspp.IntJAntimicrobAgents.2006;27(4):351–353.

13.KaufmannME.Pulsed-fieldgelelectrophoresis.In:Woodford

N,JohnsonAP,eds.MolecularBacteriology:ProtocolsandClinical

Applications.Vol15.HumanaPress;1998:33–51.

14.TenoverFC,ArbeitRD,GoeringRV,etal.Interpreting

chromosomalDNArestrictionpatternsproducedby

pulsed-fieldgelelectrophoresis:criteriaforbacterialstrain

typing.JClinMicrobiol.1995;33(9):2233–2239.

15.TuonFF,Penteado-FilhoSR,AmaranteD,AndradeMA,Borba

LA.MortalityrateinpatientswithnosocomialAcinetobacter

meningitisfromaBrazilianhospital.BrazJInfectDis.

2010;14(5):437–440.

16.TuonFF,RymszaAM,Penteado-FilhoSR,PilonettoM,Arend

LN,LevinAS.Shouldpolymyxinbeusedempiricallytotreat

infectionsinpatientsunderhighriskfor

carbapenem-resistantAcinetobacter?JInfect.

2011;62(3):246–249.

17.SchimithBierKE,LuizSO,SchefferMC,etal.Temporal

evolutionofcarbapenem-resistantAcinetobacterbaumanniiin

Curitiba,southernBrazil.AmJInfectControl.

2010;38(4):308–314.

18.CieslinskiJM,ArendL,TuonFF,etal.Molecularepidemiology

characterizationofOXA-23carbapenemase-producing

Acinetobacterbaumanniiisolatedfrom8Brazilianhospitals

usingrepetitivesequence-basedPCR.DiagnMicrobiolInfect

Dis.2013;77(4):337–340.

19.WerneckJS,PicaoRC,GirardelloR,etal.Lowprevalenceof

blaOXA-143inprivatehospitalsinBrazil.AntimicrobAgents

Chemother.2011;55(9):4494–4495,authorreply5.

20.TimurkaynakF,CanF,AzapOK,DemirbilekM,ArslanH,

KaramanSO.Invitroactivitiesofnon-traditional

antimicrobialsaloneorincombinationagainst

multidrug-resistantstrainsofPseudomonasaeruginosaand

Acinetobacterbaumanniiisolatedfromintensivecareunits.Int JAntimicrobAgents.2006;27(3):224–228.

21.BantarC,SchellC,PosseG,LimanskyA,BalleriniV,Mobilia

L.Comparativetime-killstudyofdoxycycline,tigecycline,

sulbactam,andimipenemagainstseveralclonesof

Acinetobacterbaumannii.DiagnMicrobiolInfectDis.

2008;61(3):309–314.

22.HawleyJS,MurrayCK,GriffithME,etal.Susceptibilityof

acinetobacterstrainsisolatedfromdeployedU.S.military

personnel.AntimicrobAgentsChemother.2007;51(1):

376–378.

23.ZarrilliR,CrispinoM,BagattiniM,etal.Molecular

epidemiologyofsequentialoutbreaksofAcinetobacter

baumanniiinanintensivecareunitshowstheemergenceof

carbapenemresistance.JClinMicrobiol.2004;42(3):

946–953.

24.PallottoC,FiorioM,D’AvolioA,etal.Cerebrospinalfluid

penetrationoftigecycline.ScandJInfectDis.2014;46(1):

69–72.

25.Navon-VeneziaS,LeavittA,CarmeliY.Hightigecycline

resistanceinmultidrug-resistantAcinetobacterbaumannii.J

AntimicrobChemother.2007;59(4):772–774.

26.FalagasME,KasiakouSK.Colistin:therevivalofpolymyxins

forthemanagementofmultidrug-resistantgram-negative