Spread of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clones in patients with ventilator-associated pneumonia in an adult intensive care unit at a university hospital

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The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Spread

of

multidrug-resistant

Acinetobacter

baumannii

and

Pseudomonas

aeruginosa

clones

in

patients

with

ventilator-associated

pneumonia

in

an

adult

intensive

care

unit

at

a

university

hospital

Sabrina

Royer

_,

_Ana

_Luiza

_Souza

_Faria

_,

_Liliane

_Miyuki

_Seki

_,

Thiago

Pavoni

Gomes

Chagas

_,

_Paola

_Amaral

_de

_Campos

_,

Deivid

William

da

Fonseca

Batistão

_,

_Marise

_Dutra

_Asensi

_,

Paulo

P.

Gontijo

Filho

_,

_Rosineide

_Marques

_Ribas

a_{LaboratóriodeMicrobiologiaMolecular,InstitutodeCiênciasBiomédicas,UniversidadeFederaldeUberlândia,Uberlândia,MG,Brazil} b_{LaboratóriodePesquisaemInfecc¸ãoHospitalar,InstitutoOswaldoCruz-Fiocruz,RiodeJaneiro,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16October2014 Accepted29March2015 Availableonline19May2015

Keywords:

Acinetobacterbaumannii

Intensivecareunit Molecularepidemiology Multidrug-resistant

a

b

s

t

r

a

c

t

Background:InBrazil,ventilator-associatedpneumonia(VAP)causedbycarbapenem resis-tant Acinetobacter baumannii and Pseudomonas aeruginosa isolates are associated with significantmortality,morbidityandcosts.Studiesontheclonalrelatednessoftheseisolates couldlaythefoundationforeffectiveinfectionpreventionandcontrolprograms.

Objectives:WesoughttostudytheepidemiologicalandmolecularcharacteristicsofA. bau-manniivs.P.aeruginosaVAPinanadultintensivecareunit(ICU).

Methods:ItwasconductedacohortstudyofpatientswithVAPcausedbycarbapenem resis-tantA.baumanniiandP.aeruginosaduring14monthsinanadultICU.Genomicstudieswere usedtoinvestigatetheclonalrelatednessofcarbapenemresistantOXA-23-producingA. baumanniiandP.aeruginosaclinicalisolates.TheriskfactorsforacquisitionofVAPwerealso evaluated.Clinicalisolateswerecollectedforanalysisasweresamplesfromthe environ-mentandweretypedusingpulsedfieldgelelectrophoresis.

Results:Multivariatelogisticregressionanalysisidentifiedtraumadiagnosedatadmission andinappropriateantimicrobialtherapyasindependentvariablesassociatedwiththe devel-opmentofA.baumanniiVAPandhemodialysisasindependentvariableassociatedwithP. aeruginosaVAP.AllcarbapenemresistantclinicalandenvironmentalisolatesofA.baumannii

wereOXA-23 producers. NoMBL-producerP.aeruginosawasdetected. Moleculartyping

∗ _{Correspondingauthorat:UniversidadeFederaldeUberlândia,InstitutodeCiênciasBiomédicas,LaboratóriodeMicrobiologiaMolecular,}

Av.Amazonas,s/n,Bloco4C,Umuarama,Uberlândia,MG38405-302,Brazil. E-mailaddress:sabriroyer@yahoo.com.br(S.Royer).

http://dx.doi.org/10.1016/j.bjid.2015.03.009

revealedapolyclonalpattern;however,cloneA(clinical)andH(surface)werethemost frequentamongisolatesofA.baumanniitested,withagreaterpatternofresistancethan otherisolates.InP.aeruginosathemostfrequentcloneIwasmulti-sensitive.

Conclusion:Thesefindingssuggesttherequirementofconstantmonitoringofthese microor-ganismsinordertocontrolthespreadoftheseclonesinthehospitalenvironment.

©2015ElsevierEditoraLtda.Allrightsreserved.

Introduction

Ventilator-associatedpneumonia(VAP)isoneofthemost fre-quentintensive-care-unit (ICU)-acquired infection, with an incidencerangingfrom6to52%,1_{andcontinuestobeamajor}

cause of morbidity, mortality and increased financial bur-deninICUs.2_{TheoverallrateofVAPishigherindeveloping}

countriesICUsthanthatreportedfromtheUSICUs,withrate of13.6vs.3.3per1000ventilator-days,respectively.3

Non-fermentativeGram-negativebacilli(NF-GNB)mainly

A.baumanniiandP.aeruginosa,haveemergedasmajoragents ofVAP4andtheresistanceoftheseorganismstoantibiotics, particularlytocarbapenems,hasposedimportant therapeu-tic challenges. Currently, carbapenems are considered the antimicrobialsofchoicefortreatment ofserious infections caused by A. baumannii and P. aeruginosa5 _{but their}

effi-cacyisincreasingly compromisedbyresistanceasreported worldwide.6 _{Thisresistancehasbeenattributedtothe}

pro-ductionofcarbapenem-hydrolysing-␤-lactamaseenzymesof Ambler molecularclass D (oxacillinases) and B(metallo- ␤-lactamases).7

Acinetobacterspecies, considered organisms oflow viru-lence, have become one of the most difficult nosocomial pathogenstocontrolandtreatwithanassociatedmortalityof approximately30%.6_{P.aeruginosaisahighlyvirulent}

microor-ganismandmortalityratesrangefrom40%tomorethan60%.8

Ithasbeenreportedthatmultidrug-resistant(MDR)strainsof thisorganismareassociatedwithahigherrateofmortality andthatmortalityattributedtoinfectionbyP.aeruginosais considerable.9

Theobjectivesofthepresentstudyweretoinvestigatethe riskfactorsofA.baumanniivs.P.aeruginosaVAPinanadult ICU.Furthermore,dataonspreadofMDRA.baumanniiandP. aeruginosaisolatesandtheirphenotypicandgenotypic char-acteristicsarepresentedinthisstudy.

Material

and

methods

TheUberlandiaFederalUniversity-HospitalClinic(UFU-HC)is a530-bedteachinghospitalanda30-bedclinical-surgicalICU foradults.

Studydesign

ThisprospectivecohortstudywasconductedfromApril2011 toJune2012,ina30-bedclinical-surgicalICUforadults.All

themedicalrecordsofthepatientswithVAPbyA. bauman-niiandP.aeruginosaadmittedtoICUatthetimeofthestudy were analyzed. Adultpatients (≥18 years old) with a first episodeofVAPbyA.baumanniiorP.aeruginosawereincluded inthestudy.ClinicalisolatesforA.baumanniiandP. aerugin-osawereobtainedfromculturesofendotrachealaspirate(EA) ofpatientsadmittedtotheICU.Patientsidentifiedashaving VAPbyA.baumanniiwereselectedforenvironmental samp-ling, and sampleswere collectedon surfacesnearpatients withVAP,beforeandaftercleaning.

Definitions

All patients included inthis study were monitoredfor the developmentofVAPusingclinicalandmicrobiologicalcriteria, until discharge or death. VAP was defined as a pneumo-nia occurring 48h or more after endotracheal intubation, with new and/or progressive radiological infiltrate, and at least twoofthe followingfeatures: purulentsputum, tem-perature higherthan 38.5◦Cor lower than 35◦C,leukocyte counthigherthan10000/␮Lorlowerthan3000/␮L;and pos-itive quantitativeculture ofthe EA (count ≥106_CFU/mL).10

TheClinicalPulmonaryInfectionScore(CPIS),11_basedonsix

clinical assessments, was calculated for patients clinically suspectedofVAPonthesamedayofendotrachealsecretions collection,andallthosewithCPIS≥6wereincluded.Patients developingVAPwithinthefirstfourdaysofmechanical venti-lation(MV)wereclassifiedashavingearly-onsetVAP,while those developingVAP fiveormoredaysafterthe initiation of MVwere classified as having late-onset VAP.12 _Previous

antimicrobial therapy was defined as receiving a systemic antimicrobialagentforatleast48hwithintwoweeks preced-ingcollectionsputumforculture.13_{Theantimicrobialtherapy}

wasconsideredappropriateifallpathogensisolatedfrom cul-tureweresensitivetoitandinappropriateifanypathogenwas resistant to it.13 _{Multidrug-resistant (MDR)pathogens were}

definedasresistanttothreeormoreclassesofantibiotics.14

Datacollection,clinicalandenvironmentalsamples

Thedemographiccharacteristicsincludingage,gender, under-lying disease or condition, admission diagnosis, surgery, Average SeverityofIllnessScore(ASIS),15 _{lengthofhospital}

stay, previoususe ofantibiotics,and dischargestatuswere obtainedfrommedicalcharts.Thecollectionofclinical speci-mensobtainedfromEAwasperformedbythenursingstaff and physiotherapists,early inthemorning,byprobe num-ber12,andtransportedinasteriletubetotheMicrobiology LaboratoryofUFUforprocessing.EnvironmentalisolatesofA.

baumanniiwereobtainedwhilethepatientoccupiedtheroom. Threesurfacesineachroomweresampled,beforeandafter cleaning:bedsidetable,bedrail,anddoorknob.Ateachsite, anareawassampledusingapre-moistenedsterilewipecloth (7×7cm).

Speciesidentificationandantimicrobialsusceptibilities

Isolates were identified by conventional techniques (envi-ronment/surfaces) and automated systems (clinical spec-imen), including the Vitek2® _{system (bioMérieux Vitek}

SystemsInc.,Hazelwood,MO).Susceptibilitytestingfor clin-ical specimens was performed using the Vitek2® _system,

and the disk diffusion method was employed to evalu-ate susceptibility of isolates from surfaces. The following antimicrobial agents were used to evaluate susceptibility of A. baumannii isolates: amikacin, ampicillin/sulbactam, cefepime, ciprofloxacin, polymyxin B or colistin, genta-micin, imipenem, and tigecycline. Imipenem, meropenem, ceftazidime,ciprofloxacin, gentamicin,cefepime, amikacin, polymixinB,andpiperacillin/tazobactamwereusedto eval-uatesusceptibility ofP. aeruginosa. All the testswere done in accordance with the Clinical and Laboratory Standards Institute16_{recommendedpractices.Sincethereareno}

break-pointsavailablefortigecyclineforAcinetobacterspp.,USFood andDrugAdministration(FDA)tigecyclinebreakpointslisted forEnterobacteriaceae(≤2,4and ≥8␮g/mLforsusceptible, intermediateandresistantstrains,respectively)wereapplied toAcinetobacterspp.inthisstudy.PseudomonasaeruginosaATCC 27853 and Escherichia coliATCC 25922were used asquality controlstrains.AllisolatesofA.baumanniiwerescreenedfor carbapenemaseproducingphenotypebytheModifiedHodge Test(MHT)16 and allisolatesofP.aeruginosawere screened formetallo-␤-lactamasesproducingphenotypebythe double-disksynergytest(DDST).17

PCRamplification

ToassessthepresenceofblaOXAgenesinA.baumanniistrains,

themultiplexPCRwasperformedaspreviouslydescribedby Higginsetal.18 _{and Woodfordet al.}19_{and foramplification}

ofthe genesencodingMetallo-Beta-lactamases(MBLs)inP. aeruginosastrains,themultiplexPCRwasperformedaccording toWoodford.20_{Amplificationofthegeneswascarriedoutwith}

PCRusingprimersdescribedinTable1.

Moleculartyping

Isolates were typed by pulsed-field gel electrophoresis21

(PFGE),followingdigestionofintactgenomicDNAwithApaI

(Invitrogen)forA.baumanniiandSpeI(Fermentas)forP. aeru-ginosa.DNA fragmentswereseparated on1%(w/v)agarose gels in0.5%TBE [Tris–borate–ethylene diamine tetra-acetic acid (EDTA)] buffer using aCHEF DRIIIapparatus (Bio-Rad, Hercules, CA)with6V/cm,pulsed from 5s to15s, for18h at14◦Cfor A. baumanniiand with 6V/cm,pulsed from 5s to35s,for17hat14◦C,forP.aeruginosa. Gelswerestained withethidiumbromideandphotographedunderultraviolet light.Computer-assistedanalysiswasperformedusing BioN-umericsv.4.0(AppliedMaths,Sint-Martens-Latem,Belgium).

Table1–Specificprimersusedinthisstudy.

Gene Sequence Reference

blaOXA-51 F5-TAATGCTTTGATCGGCCTTG-3

R5-TGGATTGCACTTCATCTTGG-3

19

blaOXA-23 F5-GATCGGATTGGAGAACCAGA-3

R5-ATTTCTGACCGCATTTCCAT-3

19

blaOXA-24 F5-GGTTAGTTGGCCCCCTTAAA-3

R5_{-AGTTGAGCGAAAAGGGGATT-3}

19

blaOXA-58 F5-AAGTATTGGGGCTTGTGCTG-3

R5-CCCCTCTGCGCTCTACATAC-3

19

blaOXA-143 F5-TGGCACTTTCAGCAGTTCCT-3

R5-TAATCTTGAGGGGGCCAACC-3

18

blaIMP F5-GAATAGRRTGGCTTAAYTCTC-3

R5-CCAAACYACTASGTTATC-3

20

blaVIM F5-GTTTGGTCGCATATCGCAAC-3

R5-AATGCGCAGCACCAGGATAG-3

20

blaSPM F5-CTAAATCGAGAGCCCTGCTTG-3

R5-CCTTTTCCGCGACCTTGATC-3

20

blaGIM F5-TCAATTAGCTCTTGGGCTGAC-3

R5-CGGAACGACCATTTGAATGG-3

20

blaSIM F5-GTACAAGGGATTCGGCATCG-3

R5-TGGCCTGTTCCCATGTGAG-3

20

Comparison ofthe banding patterns was accomplished by theunweightedpair-groupmethodwitharithmeticaverages (UPGMA)usingtheDicesimilaritycoefficient.

Statisticalanalysis

TheChi-squaretestsorFisher’sexacttestwereusedto com-parediscretevariables.Fisher’sexacttestwasusedinstead oftheChi-squaretestwhenoneormoreexpectedvaluesin the 2×2 contingencytablewere equalor less than 5.The comparisonoftwoquantitativevariableswasmadeusingthe Mann–Whitneytestfornonparametricvariablesandthe Stu-dentttestforparametricvariables.Two-sidedtestswereused forall analyses.Multivariate analysiswas performedusing multiple logistic regression and the values were included whensignificancewas<0.05inunivariateanalysis.Allp-value <0.05wasconsideredstatisticallysignificant.The epidemio-logicaldatawere analyzedthroughtheprogramsGraphPad Prism®_{5.0(LaJolla,CA,USA)andBioEstat5.0(Tefé,AM,Brazil).}

Ethicalconsiderations

TheEthicsCommitteeforHumanResearchofUFUapproved theprojectundertheprotocolnumber228/11.

Results

During the study period,weenrolled 30 subjects withVAP causedbyA.baumanniiandasimilarnumberofsubjectswith VAP caused by P. aeruginosa. Three patients were excluded from the epidemiological study because the VAP were due A.baumanniiassociatedtoothermicroorganisms.The demo-graphicandclinicalcharacteristics,includingriskfactorsand outcomeofpatientsinfectedbythesebacteria,areshownin

Table2.

TheriskfactorsforacquiringA.baumanniiVAPby univari-ate analysis(p≤0.05)were:traumadiagnosis atadmission,

Table2–Baselinepatientcharacteristics,riskfactorsandoutcomeofVAPcausedbyA.baumanniivs.P.aeruginosainthe adultICUoftheUFU-HC.

Characteristics Microorganisms p-Value RR(95%CI)

A.baumannii P.aeruginosa N=27(%) N=30(%) Age[mean];±SDa _{49.89;±16.84 58.97;±19.46 0.064 –} Gender,n(%) Male 21(77.78) 24(80.00) 0.837 0.9333(0.4890–1.781) Female 6(22.22) 6(20.00) Admissiondiagnosis Clinical 7(25.92) 21(70.00) 0.001 0.3625(0.1824–0.7203) Surgical 2(7.41) 1(3.33) 0.599 1.440(0.6153–3.370) Trauma 18(66.67) 8(26.67) 0.003 2.385(1.299–4.377) Cormorbidities Smoking 7(25.92) 11(36.67) 0.384 0.7583(0.3939–1.460) Alcoholism 7(25.92) 6(20.00) 0.594 1.185(0.6511–2.155) Diabetesmellitus 2(7.41) 6(20.00) 0.258 0.4900(0.1430–1.679) Heartfailure 6(22.22) 15(50.00) 0.030 0.4898(0.2359–1.017) Lungdisease 5(18.52) 4(13.33) 0.722 1.212(0.6262–2.346)

Chronicrenalfailure 5(18.52) 4(13.33) 0.722 1.212(0.6262–2.346)

Oldstroke 0(0.0) 2(6.66) 0.493 0.0000(–)

Malignancy 0(0.0) 1(3.33) 1.000 0.0000(–)

Traumaticbraininjury 8(29.63) 4(13.33) 0.195 1.579(0.9328–2.673)

ASISb_{(>4) 13(48.15) 6(20.00) 0.024 3.714(1.152–11.98)}

CPISc_≥6d _{8(6–10) 7(6–10) 0.342 –}

Tracheostomy 21(77.78) 17(56.67) 0.091 1.750(0.8508–3.600)

Hemodialysis 3(11.11) 15(50.00) 0.002 0.2708(0.09358–0.7838)

Anysurgicalprocedure 20(74.07) 19(63.33) 0.384 1.319(0.6849–2.539)

MDRe_{pathogen 14(51.85) 7(23.33) 0.026 1.846(1.087–3.135)}

Antimicrobialtherapy

Inappropriate 21(77.78) 8(26.67) 0.000 3.379(1.606–7.112)

Appropriate 6(22.22) 22(73.33)

Empiricantimicrobialtherapy

≥2 25(92.59) 25(83.33) 0.427 1.750(0.5250–5.833)

Carbapenems 10(37.04) 13(43.33) 0.629 0.8696(0.4895–1.545)

Cephalosporins(3rdand4thgeneration) 25(92.59) 26(86.67) 0.673 1.471(0.4583–4.719)

Aminoglycoside 0(0.0) 1(3.33) 1.0000 0.0(–) Fluoroquinolones 8(29.63) 7(23.33) 0.590 1.179(0.6609–2.103) Glycopeptides 19(70.37) 19(63.33) 0.779 1.188(0.6415–2.198) Colistin 0(0.0) 4(13.33) 0.114 0.0(–) VAP Late 20(74.07) 24(80.00) 0.594 0.8442(0.4640–1.536) Early 7(25.93) 6(20.00)

LengthinICUcarebeforeVAP(>7days) 11(40.75) 20(66.67) 0.050 0.5766(0.3281–1.013)

LengthinICUcareafterVAP(>20days) 11(40.75) 11(36.67) 0.752 1.094(0.6296–1.900)

ICUlengthofstayindays(>30) 9(33.33) 13(43.33) 0.439 0.7955(0.4380–1.445)

HClengthofstayindays(>60) 8(29.63) 11(36.67) 0.574 0.8421(0.4549–1.559)

Mortality 12(44.44) 19(63.33) 0.153 0.6710(0.3863–1.165)

a_{SD,standarddeviation;}b_{AverageSeverityofIllnessScore;}c_{ClinicalPulmonaryInfectionScore;}d_{Variablesexpressedasmedian(range);}

e_{Multidrug-resistant;VAP,ventilatorassociatedpneumonia;ICU,intensivecareunit;UFU-HC,UberlandiaFederalUniversity-HospitalClinic;}

RR(CI),relativerisk(confidenceinterval).

score ASIS >4, MDR, and inappropriate antimicrobial ther-apy.However,aftermultiplelogisticregressionanalysisonly traumadiagnosisatadmission(OR7.21,95%CI:1.62–32.10, p=0.001)andinappropriateantimicrobialtherapy(OR17.29, 95% CI:2.61–114.50, p=0.003)remained as an independent variablesassociatedwiththedevelopmentofA.baumanniiVAP

(Table3).ForacquiringP.aeruginosaVAP,onlyhemodialysis(OR

0.14,95%CI:0.03–0.70,p=0.017)remainedasanindependent variableassociatedwiththedevelopmentofVAP(Table4).

Overall,outof29clinicalisolatesofVAPduetoA. bauman-nii,17(58.62%)wereimipenem-resistantandmost(12,70.59%) were positiveforclassDcarbapenemaseproduction(MHT). Screeningforresistancegenes wasperformedbymultiplex PCRandallthe12isolateswereOXA-23producers.No MBL-producerP.aeruginosawasdetected(Table5).

Thirty one(29.5%)ofthe 126 samplesobtainedfrom 21 rooms occupiedbyan A.baumanniiVAP patientwere posi-tiveforA.baumanniiandtheisolateswererecoveredmainly

Table3–MultivariableregressionanalysisofriskfactorsforVAPcausedbyA.baumanniiinpatientsinternedintheadult ICUofUFU-HC.

Variables OR 95%CI p-Value

Traumadiagnosisadmission 7.2122 1.62–32.10 0.001

ASISa_{(>4) 3.7154 0.85–16.20 0.080}

MDRb_{pathogen 0.6736 0.11–4.02 0.664}

Inappropriateantimicrobialtherapy 17.2911 2.61–114.50 0.003

a_{AverageSeverityofIllnessScore;}b_{Multidrug-resistant;VAP,Ventilatorassociatedpneumonia;ICU,intensivecareunit;UFU-HC,Uberlandia}

FederalUniversity-HospitalClinic;OR,oddsratio;CI,confidenceinterval.

from the bed rail (29.0%)after cleaning.The prevalenceof MDRorganismswas74.2%(23/31)witheight(25.8%)imipenem resistantandsix(75.0%)positivebytheMHT.ByPCR,allsix isolatesrecoveredfromsurfacewerepositiveforthepresence ofblaOXA-51andblaOXA-23.

Foroperationalreasons,only23A.baumanniiand19P. aeru-ginosastrainsweretyped.Thisisthefirststudythatevaluated theclonalrelationshipamongsamplesofA.baumanniifrom patientsandhospitalenvironmentattheICUstudied.

Twenty-three isolates ofA. baumannii were analyzed by PFGE.Mostofthem wereimipenem-resistant(20/23,86.9%) andrecoveredfromVAPepisodes(17/23,73.9%).Eight geno-typeswith80%similaritywereobservedandgenotypeA(n=9, 52.9%ofclinicalisolates)andH(n=4,66.6%ofsurfaceisolates) werethemostfrequentintheunit.Ingeneral,weobserved different genotypes among environmental (H) and clinical (A) imipenem-resistantA.baumannii isolatesand the three imipenem-susceptibleisolatesbelongedtotwootherdistinct genotypes(1,Fand2,G)(Table5).

Basedontheantibioticsusceptibilitytesting,eight antibio-types (R1–R8) were identified among A. baumannii isolates thatweregenotypicallypositiveforoxacillinasesgenes.Five isolates were assigned as antibiotype R1 and were resis-tanttoallantibioticstested, withtheexceptionofcolistin. Theotherantibiotypes(R2–R5)fitintheMDRpattern.These antibiotypes presented major pulsotypes characterized as typeAinEAisolatesand typeHinenvironmentalisolates

(Table5).

Atotalof10PFGEpatternsofP.aeruginosawereobserved amongthe19isolatesanalyzed,alsowithapolyclonalpattern, butwiththreemajorclones:I(n=4,21.1%),A(n=3,15.8%), H(n=3,15.8%).Basedontheantibioticsusceptibilitytesting, nineantibiotypes(R1–R9)wereidentifiedamongP.aeruginosa

isolatesthatweregenotypicallynegativeforMBLsgenesand themainpulsotypeIpresenteditselfasmulti-sensitiveoronly resistanttocarbapenems(Table5).

Discussion

Ventilator-associatedpneumoniacausedbyA.baumanniiand

P. aeruginosa, particularly those resistant to the carbapen-ems,areassociatedwithsignificantmortalityandmorbidity, addingconsiderablecoststohospitalcare.2,4

RiskfactorsforVAPduetoA.baumanniiaremainlyhead trauma,prolongedhospitalstayand priorantibioticuse,1,10

whereas VAP duetoP. aeruginosaisdiagnosed in immuno-compromisedpatients,especiallythosewithneutropenia.22

In our study, the univariate analysis identified some vari-ablesassignificantriskfactorsforacquiringA.baumanniiVAP, but onlytrauma diagnosis atadmission and inappropriate antimicrobialtherapywereindependentrisksfactorsforthe developmentofVAPbyA.baumannii.Likewise,foracquiringP. aeruginosaVAP,onlyhemodialysiswasanindependentfactor. Therapeutic options are limited forA. baumannii and P. aeruginosa VAP and the antimicrobial resistance in these organisms are associated with several mechanisms, but the most importantisthe productionof␤-lactamases and has been attributed to the production of carbapenem-hydrolysing-␤-lactamaseenzymesofAmblermolecularclass D(oxacillinases)forA.baumannii,andclassB(MBLs) associ-atedwithlossofporinsandeffluxsystemoverexpressionfor

P.aeruginosa.7

Currently,carbapenemsaretheantimicrobialsofchoicefor treatingseriousinfectionscausedbyA.baumannii.6_InBrazil

and elsewhere7,23 _{carbapenemresistantisolatesare mostly}

relatedto␤-lactamaseOXA-23,asdetectedinallA.baumannii

imipenemresistantisolatesrecoveredinourstudy.

AlthoughthemostimportantsourceofA.baumanniiisthe infected or colonized patient, a widespreadenvironmental contaminationisoftendemonstrated24_{asseeninourstudy,}

withamoreexpressivecontaminationofthebed raileven aftercleaning(29.0%),questioningtheroutineofthisservice

Table4–MultivariableregressionanalysisofriskfactorsforVAPcausedbyP.aeruginosainpatientsinternedintheadult ICUofUFU-HC.

Variables OR 95%CI p-Value

Clinicaldiagnosisadmission 0.2671 0.06–1.11 0.069

Heartfailure 0.6279 0.14–2.88 0.549

Hemodialysis 0.1424 0.03–0.70 0.017

LengthinICUcarebeforeVAP(>7) 0.2913 0.08–1.11 0.071

VAP,ventilatorassociatedpneumonia;ICU,intensivecareunit;UFU-HC,UberlandiaFederalUniversity-HospitalClinic;OR,oddsratio;CI,

b r a z j i n f e c t d i s . 2 0 1 5; 1 9(4) :350–357

355

N(%) bp/gene(isolates) (isolates) profile(isolates) pulsotypes/N(%) pulsotype/N(%)

Endotrachealaspirate 17/29(58.6)/ 12/29(41.4) 17(100.0)/12 (70.6) a_{12/17(70.6) 353pb/bla}

OXA-51(12) R1(5) AN;SAM;GEN;IPM;TGC A/4(80.0) G/1(20.0)

501pb/blaOXA-23(12) R2(3) SAM;GEN;IPM;TGC A/2(66.7) D/1(33.3)

b_{02/17(11.8) R3(1) AN;SAM;GEN;IPM A/1(100.0) –}

353pb/blaOXA-51(02) R4(1) AN;SAM;IPM – C/1(100) c_{03/17(17.6) 501pb/bla}

OXA-23(02) R5(2) SAM;GEN;IPM A/2(100.0) –

R6(2) SAM;IPM B/1(50.0);C/1(50.0)

353pb/blaOXA-51(03) R7(1) SAM G/1(100.0)

R8(2) Multi-sensitive F/1(50.0);G/1(50.0)

Environment

8/31(25.8)/

23/31(74.2) 8(100.0)/6(75.0)

6(75.0) 353pb/blaOXA-51(6) R2(1) SAM;GEN;IPM;TGC H/1(100.0) –

501pb/blaOXA-23(6) R5(4) SAM;GEN;IPM H/3(75.0) G/1(25.0)

R6(1) SAM;IPM – E/1(100.0)

Pa4 carbapenem-resistant/susceptible N(%) DDST5_isolates ana-lyzed/positive N(%) PCR3_analyzed N(%) PCRproductsize bp/gene(isolates) Antibiotype (isolates) Resistance profile(isolates) MajorPa4 pulsotypes/N(%) Non-majorPa4 pulsotype/N(%) Endotrachealaspirate 15/30(50.0)/ 15/30(50.0) 14(93.3)/11(78.6) 14(100.0) Negative

R1(1) AN;FEP;CIP;GEN;IPM;MEM;TZP – L/1(100.0) R2(1) FEP;CIP;GEN;IPM;MEM;TZP – C/1(100.0) R3(1) FEP;CIP;IPM;MEM;TZP – E/1(100.0) R4(2) FEP;CIP;GEN;IPM;MEM A/2(100.0) – R5(1) FEP;GEN;IPM;MEM A/1(100.0) –

R6(6) IPM;MEM H/2(33.3),I/1(16.7) C/1(16.7),E/1(16.7), F/1(16.7)

R7(2) IPM I/1(50.0) G/1(50.0)

R8(1) MEM – B/1(100.0)

R9(4) Multi-sensitive H/1(25.0),I/2(50.0) J/1(25.0)

1_{Acinetobacterbaumannii;}2_{ModifiedHodgeTest;}3_{Polymerasechainreaction;}4_{Pseudomonasaeruginosa;}5_{DoubleDiskSynergyTest;}a_{12isolatespositiveforcarbapenemaseproduction(MHT);}b_2isolates

negativeforcarbapenemaseproduction(MHT);c_{3isolatescarbapenem-susceptible;AN,amikacin;SAM,ampicillin/sulbactam;CIP,ciprofloxacin;FEP,cefepime;GEN,gentamicin;IPM,imipenem;}

intheICU.Asthemicroorganismcansurviveindryconditions andpersistforprolongedperiodsoftime,surfacesmaybea reservoirofepidemicstrainsinoutbreaksinICUsthatceased onlyafterclosingforterminalcleaninganddisinfection.25

ThediversityofMDRA. baumanniiandP. aeruginosa iso-latesinourICUcouldprovideusefulinformationforhospital infectioncontrol.OneormoreA.baumanniiclonesoften coex-istinICUs,asseeninourinvestigation,but differentfrom otherauthors26,27 _{wehavedemonstratedthecoexistenceof}

two major PFGE-typed clones (A – clinical and H – envi-ronmental)ofA. baumanniiand three ofP. aeruginosa (I,A, H–clinical),reinforcing the clonal diversity ofA. baumannii

andP.aeruginosa.Furthermore,carbapenem-resistancecould betransmittedhorizontallyhighlightingtheinfectioncontrol measures,suchashandhygiene,thatshouldbestrengthened toreducethefurtherspreadofbothmicroorganisms.9,27_There

isnotasinglemechanismofpolyclonaldisseminationand althoughthisworkhasnotanalyzedcolonizedpatients,this isapuzzlethatcouldindicateendogenousacquisition.28

Person-to-persontransmissionofcarbapenemresistantA. baumanniicarryingblaOXA-23wasindeedidentifiedinisolates

belongingtothesamepulsotype(genotypeA)recoveredfrom differentpatients.Thesedatasuggestthatrapididentification ofblaOXA-23bymolecularmethodsisnecessarytoimplement

rigorousinfectioncontrolprograms,justifyingmoreeffective contactprecautions forpatients withcarbapenem-resistant strains.6,27

In summary, this study demonstrates high VAP rates caused by A. baumannii and P. aeruginosa in an adult ICU ofour hospital mainly associated withthe severity ofthe underlyingdisease inpatients withP. aeruginosa and inap-propriatetherapy forpatients withA.baumannii. Moreover, PFGErevealedthepolyclonalspreadofOXA-23-producingA. baumannii,isolatedalsointhe environment,and P. aerugin-osaresistant tocarbapenems.Takentogether, theseresults suggesttherequirementofanepidemiologicalsurveillanceto controlthetransmissionoftheseresistancemechanismsin thehospitalenvironment.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TotheLAPIH(LaboratóriodePesquisaemInfecc¸ãoHospitalar –IOC/Fiocruz)bytrainingandsupplyofmaterialsneededfor themoleculartechniques. Thisworkwassupportedbythe BrazilianFundingAgencyCAPES.

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