Genotypic study documents divergence in the pathogenesis of bloodstream infection related central venous catheters in neonates

(1)

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

Genotypic

study

documents

divergence

in

the

pathogenesis

of

bloodstream

infection

central

venous

catheters

in

neonates

Cristiane

Silveira

Brito

a,∗

_,

_Rosineide

_Marques

_Ribas

a

_,

_Daiane

_Silva

_Resende

a

_,

Denise

Von

Dolinger

de

Brito

a

_,

_Vânia

_Olivetti

_Steffen

_Abdallah

b

_,

Kátia

Regina

Netto

dos

Santos

c

_,

_Fernanda

_Sampaio

_Cavalcante

c

_,

Pricilla

Dias

Moura

de

Matos

c

_,

_Paulo

_P.

_Gontijo

_Filho

a

a_Laboratório_de_{Microbiologia,}_Instituto_de_Ciências_Biomédicas,_Universidade_Federal_de_Uberlândia_(UFU),_Campus_Umuarama,

Uberlândia,MG,Brazil

b_Unidade_de_Terapia_Intensiva_Neonatal,_Hospital_de_Clínicas_da_Universidade_Federal_de_Uberlândia_(UFU),_Campus_Umuarama,

Uberlândia,MG,Brazil

c_Departamento_de_{Microbiologia}_Médica,_Instituto_de_{Microbiologia}_Prof._Paulo_de_Góes,_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de

Janeiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18September2013

Accepted15November2013

Availableonline29March2014

Keywords:

Neonates

Centralvenouscatheter

Pathogenesis

a

b

s

t

r

a

c

t

Objective:ToinvestigatethepathogenesisofbloodstreaminfectionbyStaphylococcus epider-midis,usingthemolecularepidemiology,inhigh-riskneonates.

Methods:Weconductedaprospectivestudyofacohortofneonateswithbloodstream

infec-tionusingcentralvenouscathetersformorethan24h.“NationalHealthcareSafetyNetwork”

surveillancewasconducted.GenotypingwasperformedbyDNAﬁngerprintingandmecA

genesandicaADweredetectedbymultiplex-PCR.

Results:FromApril2006to April2008,theincidence ofbloodstreaminfection and

cen-tral venous catheter-associated bloodstreaminfection was15.1 and 13.0/1000catheter

days, respectively,withS.epidermidisaccounting for42.9% ofepisodes.Molecular

anal-ysis wasused todocument the similarityamongsix isolatesofbloodstream infection

byS.epidermidisfromcaseswithpositivebloodandcentralvenouscathetertipcultures.

Fifty percentofneonateshadbloodstreaminfectionnotidentiﬁedasdeﬁniteor

proba-blecentralvenouscatheter-relatedbloodstreaminfection.Onlyonecasewasconsidered

asdeﬁnitecentralvenouscatheter-relatedbloodstreaminfectionandwasextraluminally

acquired;theremainingwereconsideredprobablecentralvenouscatheter-related

blood-streaminfections,withoneprobableextraluminallyandanotherprobableintraluminally

acquiredbloodstreaminfection.Additionally,amongmecA+andicaAD+samples,oneclone

(A)waspredominant(80%).Apolyclonalproﬁlewasfoundamongsensitivesamplesthat

werenotcarriersoftheicaADgene.

Conclusions: ThemajorityofinfectionscausedbyS.epidermidisinneonateshadanunknown

origin,although33.3%appearedtohavebeenacquiredintraluminallyandextraluminally.

∗ _{Corresponding}_author.

E-mailaddress:cristianebrito08@yahoo.com.br(C.S.Brito).

http://dx.doi.org/10.1016/j.bjid.2013.11.010

(2)

We observeda polyclonal proﬁlebetweensensitive samplesanda prevalentclone (A)

betweenresistantsamples.

Introduction

Centralvenouscatheters(CVCs)arewidelyusedinadultsand

newbornsandaretheleadingcauseofbloodstreaminfection

(BSI)inhospitals.1_Infections_associated_with_the_use_of_CVCs

represent10–20%ofhospitalinfections(IHs)inadultpatients2

andapproximatelyhalfofthoseobservedinneonates.3

Infec-tionsrelatedtotheuseofCVC(CR-BSI),i.e.thoseinwhichthe

microorganismdetectedinthebloodstreamisalsopresentin

thecathetertip,representup to29%oftheseinfections in

neonatalintensivecareunits(NICUs)withanincidence

vary-ingfrom2to49/1000daysCVC.4

ThepathogenesisofCR-BSIismultifactorialandcomplex.

Datafromstudiesinadultsshowedtwomainpotentialroutes

ofcontaminationofthecathetertipwithBSI:dissemination

ofintraluminalmicroorganismsthroughcontaminationofthe

hubinlong-termCVCs,andextraluminallyduetomigration

ofmicroorganismstotheskinatthesiteofinsertioninthose

shortdurationCVC.5,6

Althoughevaluationofsamplesbymoleculartechniques

isessential,duetotheirdiscriminatory power,theiruse in

investigatingthepathogenesisofBSIsassociated/relatedto

CVChasbeenscarce;moreover,therearefewstudiesonthe

pathogenesisofCVC-relatedBSIincriticalnewborns.7,8

Gram-negativerodsare themainpathogensofneonatal

sepsisindevelopingcountries.9_In_NICUs,_following_the

adop-tionofsophisticatedtertiaryneonatalcarewithahighrateof

invasivedeviceuse,coagulase-negativestaphylococci(CoNS)

standoutasthemainagentsofneonatalnosocomialsepsis,

withStaphylococcusepidermidisbeingthemostfrequently

iso-latedfromlateonsetsepsis,accountingfor70%ofcases.10,11

NeonatalinfectionbyCoNSislessseverebutcauses

signif-icant morbidity,especiallyamonginfantsofvery lowbirth

weight.12

ThemainfactorinthepathogenicityofS.epidermidisisits

abilitytoformbioﬁlmsmediatedbypolysaccharide

intercellu-laradhesin(PIA),encodedbytheicaoperon.13_This_bacterium

adherestothesurfaceofinvasivedevicessuchasCVCsand

formsbioﬁlms, which accountfor its greater resistance to

antibiotics,particularlytometicillin,increasingthelengthof

hospitalstayandcostingaround$2billionUSdollarsjustin

theUnitedStates.14_The_bacteria_that_form_bioﬁlms_are

dif-ﬁculttoeradicateanditisestimatedthatthismechanismis

associatedwith65.0%ofinfectionsinhospitals.15

Objective

ToinvestigatethepathogenesisofCR-BSIbyS.epidermidisin

high-riskneonatesthroughaprospectivenestedcohortstudy

inarandomizedtrialusingmolecularepidemiologyto

deter-minetheoriginofmicroorganismsresponsibleforBSI.

Materials

and

methods

Patients,studydesignandsetting

ThiswasaprospectivecohortstudyofpatientswithaCVC

withlaboratoryconﬁrmedprimaryBSIbyS.epidermidis

con-ducted inthe NICU with 10 level-3 beds withina 500-bed

teachinghospitalfromApril/2006toApril/2008.

The population analyzed was followed ﬁve times/week

from birth until discharge or death. Ethical approval was

obtained from the Ethics CommitteeofUberlândiaFederal

University accordingtothe requirementsofthe Ministryof

Health.

Thestudysampleconsistedofneonateswhohadatleast

oneCVCplacedforlongerthan24h,followed-upthrough

epi-demiologicvigilanceaspartofthe“NationalHealthcareSafety

Network”(NHSN).

Deﬁnitions

Laboratory-conﬁrmedprimaryBSI

Isolationofrecognizedpathogensfromtwobloodculturesthat

werenotrelatedtoinfectionatanothersite,withtemperature

>38◦Candwithclinicalsignsofsepsis,includingapnea,

tem-peratureinstability,lethargy,feedingintolerance,worsening

respiratorydistress,orhemodynamicinstability.16

Cathetertipcolonization

Absence ofsigns ofinfection atthe catheter insertionsite

andmicroorganismgrowth≥103_CFU/mL_at_the_catheter_tips

(by quantitativeculture) or≥15UFC/mL(bythe “roll-plate”

technique).17

CVC-associatedBSI

Bacteraemia(isolationofthesame organismwithidentical

antibiogram from blood drawn from peripheral veins and

CVC), clinical manifestationsof sepsis,defervescence after

removal ofthe implicated catheter, but without laboratory

conﬁrmationofCVCcolonization.18

Deﬁnitecatheter-relatedBSI

ForneonateswithBSIthatdidnothaveanidentiﬁedsource,

deﬁnite catheter-related BSI was deﬁned bya culture ofa

peripheral,percutaneously obtainedbloodsamplethatwas

positive forthe same organism foundtobe colonizingthe

catheter hub or tip (i.e., concordant colonization of the

catheterhubortip).19

Probablecatheter-relatedBSI

Probable catheter-related BSI was deﬁned as BSI without

an identiﬁedsourcethat met oneofthe followingcriteria:

(3)

centralcatheter(PICC)positiveoncultureforthesame

organ-ismrecoveredfromthecathetertiporfromthecatheterhub;

cultureofapercutaneouslyobtainedperipheralbloodsample

thateitheryieldednogrowthorwasnotperformed;and,if

CoNSwasisolated,clonalsimilarityconﬁrmedby

restriction-fragmentDNA subtyping;or (2) a peripheralblood sample

positive for CoNS on culture with similarity between the

organism recovered from peripheral blood and that

recov-ered from the catheter tip or from the catheter hub as

demonstrated by speciation and antibiogram results, but

restriction-fragmentDNAsubtypingwasnotdone.19

Determinationofpathogenesis

BSIwasclassiﬁedasextraluminallyacquirediftherewas

sim-ilaritybetweentheisolaterecoveredfromthecathetertipand

isolatesrecoveredfrombloodsamples,eithersimilarornotto

isolatesrecoveredfrominsertion-sitesamples.BSIwas

classi-ﬁedasintraluminallyacquiredifsimilaritywasdemonstrated

solelybetweenisolatesrecoveredfromhubsamplesand

iso-latesrecoveredfromtheblood.Ifresultssuggestedbothroutes

ofacquisition,thenthepathogenesisoftheBSIwasclassiﬁed

asindeterminate.19

Microbiologicaltechniques

Hemoculture

Blood specimens were obtained from peripheral puncture.

Hemocultureswereperformedbyinoculating1.0mLofblood

intoaﬂaskoftheautomaticcommercialsystemBactec/Alert

(VitekSystem).Positivecultureswerefurthersubculturedonto

plateswithBloodAgar.

CVCtip

Catheterswereremovedwhennolongerrequiredforpatient

care,whenthepatientexperiencedanadverseevent,orwhen

catheter exchange was deemed necessary. Catheters were

removedunderasepticconditions;theirtipswerecutoffwith

sterilescissorsandtransferredinto steriletubesand

trans-portedtothe Microbiology Laboratory. Quantitative culture

and“roll-plate”techniqueswerecarriedoutaccordingtothe

methodsofBrun-Buisson etal.20 _and _Maki_et_al.,21

respec-tively,andwereconsideredpositivewhen≥103_CFU/mL_and

≥15CFU/mLwerepresent,respectively.

SkinofCVCinsertionsite

Thecollectionofskinmaterialwasperformedsevenand14

daysaftercatheterinsertion,withasterilesalinepre-wetswab

overa20cm2_area._The_swab_was_put_into_a_tube_with₁_mL

ster-ilesalineandthenstirredinavortex;about0.1mLoftheliquid

wasinoculatedontobloodagarplates,whichwereincubated

at35◦Cfor24h.Cultureswereconsideredpositiveifgrowth

of≥200UFC/20cm2_was_seen.22

Nasalmucosaandcatheterhub

Qualitativeculturesofmaterialcollectedfromthenostrilsand

thecatheterhubwereperformed.Theswabwasputinto a

tubecontaining1mLsterilesalineandthenstirredina

vor-tex;about0.1mLoftheliquidwasinoculatedontobloodagar

platesfor24hat35◦C.

Microorganismidentiﬁcation

Theisolatesobtainedfromclinicalsampleswereinoculated

ontobloodagarandGram-stainedinordertodeterminetheir

purityandobservetheirmorphologyandspeciﬁccolor.After

determiningthesecharacteristics,thestrainsweresubmitted

tocatalaseandcoagulasetests.

To identify S. epidermidis, the tests described by

Bannerman23 _and _MAcFaddin24 _were _used: _production _of

bound coagulase factor (“clumping”) and coagulase-free

productionofhemolysis,pyrrolidonylarylamidaseenzyme,

urease, ornithine decarboxylase, novobiocin susceptibility

and fermentationofthe carbohydrates mannitol,mannose

andtrehalose.Molecularanalyseswereperformedon

coag-ulase negative Staphylococcus using the RapID STAPH PLUS

system(Oxoid)toconﬁrmtheiridentity.

Moleculartechniques

Bacterialstrains

Atotalof12S.epidermidisstrainsisolatedfromblood,catheter

tip,andnasalsecretionwereevaluated.Tworeferencestrains

wereused:(1)ATCC35984S.epidermidisasapositivecontrol

forthemecAandicaADgenes;(2)ATCC12228S.epidermidisas

anegativecontrolforicaADgene.Thestrainswerestoredat

−20◦_C_in_tryptic_soy_broth_(TSB)_(Difco_{Laboratories,}_MD,_USA)

with20%glycerol.

(a) DetectionofthemecAandicaADgenes:

To assess the presence of mecA and icaAD genes in S.

epidermidis a multiplex-PCR was performed that has

previously been described by Santos et al.,25 _Araújo

etal.26_and_McClure_et_al.27_The_primers_for_the_mecA_gene

detection were: mecA-MRS1 (5

TAGAAATGACTGAACGT-CCG3)andmecA-MRS2(5TTGCGATCAATGTTACCGTAG3),

and the primers for the icaAD gene were:

icaAD-F (5TAGTAATCACAGCCAACATCTT3) and icaAD-R

(5AAACAAACTCATCCATCCGAAT3). Cycling conditions

were asfollows: initialdenaturationat94◦C for3min,

followedby30cyclesofdenaturationat94◦Cfor1min,

annealingat55◦Cfor1min,and extensionat72◦Cfor

2min,followedbyaﬁnalextensionstepat72◦Cfor5min.

Theampliﬁedproductswereanalyzedbyelectrophoresis

on2.0%agarosegelsandstainedwithethidiumbromide

stain.

(b) Analysisofthemacrorestrictionproﬁlesofchromosomal

DNAaftercleavagewiththerestrictionenzymeSmaIand

pulsedﬁeldelectrophoresis.

AnalysisofmacrorestrictionproﬁlesofchromosomalDNA

aftercleavagewiththeSmaIrestrictionenzymeandpulsed

ﬁeldelectrophoresiswasperformedaccordingtothemethod

described by Nunes et al.28 _The _gel _was _processed _using

theContour-ClampedHomogeneousElectricField(CHEF)DR

IIISystem (Bio-RadLaboratoryPty-Ltd.), usingapulsetime

increasingfrom1to35sduring6V/cmelectrophoresis23h

and13◦Cwithanangleof120◦.Thegelwasstainedwith

ethid-ium bromide (0.5␮g/mL) for30min, destainedfor 1h with

distilledwaterandobservedunderultravioletlightandthen

(4)

Table1–Ratesofbloodstreaminfectionassociatedwith andrelatedtoCVC,cathetertipcolonizationand mortalityin318neonatesbetweenApril2006andApril 2008.

Rates n(%)

Laboratory-conﬁrmedprimaryBSIper1000 patient-days

12.9 Laboratory-conﬁrmedprimaryBSIper1000

catheter-days

15.1 Cathetertipcolonizationper1000

catheter-days

17.3 CVC-associatedBSIper1000catheter-days 13.0 CVC-relatedBSIper1000catheter-days 2.1 Overallmortality 43(13.5) MortalityofCVC-associatedBSI 30(69.8) MortalityofCVC-relatedBSI 1(2.3) BSI,bloodstreaminfection;CVC,centralvenouscatheter.

Differencesbetween isolateswere determined byvisual inspectionof the bands, as recommended byVan Belkum

etal.29_and_by_the_Molecular_Analyst_{Fingerprinting}_Plus

soft-ware package(version 1.12) of the Image Analysis System

(Bio-Rad, Hercules,CA, USA),using theDiceindexand the

unweightedpairgroupmethodwitharithmeticaveragesfor

estimationsofsimilarityandclustering.

Results

Atotalof63of318neonatesfromtherandomizedtrialwere

includedinthiscohortstudy ofthepathogenesisofCR-BSI

inhigh-risk neonateswithCVCwho had atleastoneCVC

placedformorethan24h,totalling5674patient-daysand4845

catheter-days.Therewasanaverageof1.5catheters/patient,

with461episodesofCVCcatheterization.

The incidence of laboratory-conﬁrmed primary BSI per

1000catheterdayswas15.1,and12.6forneonatesweighing

≤1500g.TheincidenceofCVC-associatedtoBSIwas13.0per

1000CVCdays,respectively. Theoverallmortalityratewas

13.5%,andinneonateswithlowweight(≤1500g)itwas16.1%

(Table1).

Theoverallcathetertipcolonizationratewas18.2%.S.

epi-dermidiswasthemicroorganismisolatedinhalf(48.7%)ofthe

positive culturesfrom the CVC tip (Table 2)followed by S.

aureus(33.3%).On average,colonized CVCswere inuse for

21.9days.Themainagentsofmicrobiologicallyconﬁrmed

pri-maryBSI were S. epidermidis(39.7%), S. aureus(24.6%), and

Gram-negative bacilli (12.2%). S. epidermidis was associated

with43.0%ofCVC-associatedBSI.

Only one case was considered to be deﬁnite CR-BSI,

and it was extraluminally acquired. The others were

considered probable CR-BSIs, with one probable

extralu-minally and another probable intraluminally acquired BSI

(Table3).

ResultsfromDNAtypingofS.epidermidisisolatesbyPFGE

are summarized in Fig. 1. No temporal relationship was

observed.MacrorestrictionanalysesofDNAsingled outthe

presenceofoneprevalentcloneintheunit(cloneA).Among12

isolatesanalyzed,8PFGEpatternswereobserved.PFGEtypeA

waspositiveforthemecAandicaADgenes.Inonlyonepatient

the same clone(A) was detectedinblood and onthe CVC

tip.Differentcloneproﬁlesofisolateswereobservedin

col-onized andinfectedpatients,andinCVCofsomepatients.

AmongsensitivesamplesthatdidnotcarrytheicaADgene,

weobservedapolyclonalproﬁlewithoutpredominanttypes.

CloneAwasrelatedtothesamesamplesrecoveredfromblood

andCVCtipofthepatient(Table4).

MoststrainsofS.epidermidis(66.7%)isolatedfrom blood

and cathetertipswere resistanttooxacillin,withthemecA

genedetectedin50.0%ofthesamples.Approximately67.0%

ofthesamplesobtainedfrombloodand50.0%ofthosefrom

CVC tips had the ability to form bioﬁlms, and the icaAD

gene was detected in two and three of six samples

cul-tured from blood and catheter tip, respectively. Multiplex

PCRforthemecAand icaADgeneswasperformedfor14S. epidermidis resistant to oxacilin,and 50% and 42.9% of the

isolates presented an amplicon consistent with the mecA

andicaADgenes,respectively.Allisolatesshowedresistance

to a 1mg/mL oxacilin disk diffusion test and were

con-sidered resistant by agar screeningwith oxacilin (datanot

shown).

Discussion

OrganismsresponsibleforCR-BSIcanaccessacathetereither

extraluminallyorintraluminally,fromtheskinattheinsertion

siteofthecatheterorbyhematogenousspreadoforganisms

from adistant focusofinfection andcontaminationofthe

catheterhubor,inthecaseofanoutbreak,viacontaminated

intravenoussolution,respectively.Accordingtotheliterature,

in adults,extraluminal acquisition appearstobethe most

frequentlyfoundinnon-cuffedcathetersandnon-tunneled

shorttermCVC.5,6

Unlike thestudyconductedbyGarlandet al.19 _in_which

the intraluminal pathway was the main source of

con-tamination of CVCs (67%), in our study with neonates,

considering the genotypic analysis, we demonstrated that

most BSI was indetermined and only one was from a

conﬁrmed extraluminal source.This failure toconﬁrm the

contamination routes of CVC strongly suggests signiﬁcant

differences in the pathogenesisofCR-BSI inneonates and

adults.

Inthepresentstudy,high-riskneonateswithBSIcaused

byS. epidermidishad aCVCinserted foranaverage of12.3

days and wedemonstrated thatmost CVCinsertions were

via long-term phlebotomy (39.5%). The high percentageof

phlebotomyuseinthisseriesandthehighpercentageof

con-taminationofthemucosa(40%)intheneonatescouldexplain

the results.Basedonthisstudy,ourneonatalunit reduced

theuseofphlebotomyconsiderablyandincreasedtheuseof

PICC.

Cathetercolonizationratewaslowerinourstudythanthe

ratereportedbyGarlandetal.,19_in_which_22%_of₁₀₆_cultures

obtainedgrewanorganism.Inourstudythecatheterhubwas

foundtobealsocolonizedinonly4(10.5%)of38hubcultures

whenaCVCtipwas colonized.Differencesinstudydesign

andoursterilesalinemoistenedswabofthehubmayexplain

someofthediscrepanciesincolonizationratesbetweenthese

(5)

Table2–PathogenesisofbloodstreaminfectionbyS.epidermidisinhigh-riskneonateswithcentralvenouscatheter.

CVCtip Blood Hub Skin Nasal

mucosa TypeCVC Technical cultiveCVC tip Lengthofuse (days) S.epidermidis – – – – Umbilical SQ 2 S.epidermidis – – – – Phlebotomy QT 3 S.epidermidis – – – – Phlebotomy SQ/QT 3 S.epidermidis – – – – Umbilical SQ 4 S.epidermidis – – – – Phlebotomy SQ 4

S.epidermidis S.agalactiae – – – Umbilical SQ/QT 4

S.epidermidis – – – – Umbilical SQ/QT 5

S.epidermidis – – S.epidermidis – Umbilical SQ/QT 5

S.epidermidis – – S.epidermidis S.epidermidis Umbilical SQ/QT 5

S.epidermidis S.aureus S.aureus – S.aureus PICC SQ/QT 5

S.epidermidis – – – – PICC SQ/QT 6

S.epidermidis S.aureus/S. haemolyticus

– S.aureus S.epidermidis Umbilical SQ 6

S.epidermidis S.aureus – – – Umbilical SQ/QT 6

*_S._epidermidis *_S._epidermidis _– _– _S._epidermidis _Phlebotomy _SQ ₆

S.epidermidis S.epidermidis – – – Umbilical SQ/QT 6

*_S._epidermidis *_S._epidermidis _– _– _– _Flebotomia _SQ ₇

*_S._epidermidis *_S._epidermidis _– _– _– _Umbilical _SQ ₈

S.epidermidis S.bovis – S.lugdunensis – Umbilical SQ 8

S.epidermidis – – – S.epidermidis PICC SQ 9

S.epidermidis C.albicans – – – Phlebotomy SQ/QT 9

*_S._epidermidis *_S._epidermidis _– _– _– _PICC _SQ ₁₀

S.epidermidis – S.aureus – S.aureus PICC SQ 10

*_S._epidermidis *_S._epidermidis _– _– _– _Phlebotomy _SQ/QT ₁₁

S.epidermidis – – – – Umbilical SQ 12

S.epidermidis – – – S.aureus Phlebotomy SQ/QT 15

S.epidermidis – – – – PICC SQ 16

S.epidermidis S.liquefaciens/E. agglomerans

– S.epidermidis – Phlebotomy SQ/QT 17

S.epidermidis – – S.epidermidis – PICC SQ/QT 19

S.epidermidis – S.aureus S.epidermidis S.epidermidis Phlebotomy SQ/QT 19

S.epidermidis – – S.epidermidis – Phlebotomy SQ 20

*_S._epidermidis *_S._epidermidis _S._epidermidis _– *_S._epidermidis _PICC _SQ ₂₃

S.epidermidis A.baumannii/E.coli – – – PICC SQ/QT 24

S.epidermidis – – – – PICC QT 25

S.epidermidis – – – S.aureus Phlebotomy SQ 28

S.epidermidis – – – – Phlebotomy SQ 32

S.epidermidis – – S.epidermidis – Phlebotomy SQ 33

S.epidermidis – – – – PICC SQ 39

SQ,semi-quantitativetechnique;QT,quantitativetechnique;CVC,centralvenouscatheter;PICC,peripherallyinsertedcentralcatheter.

∗ _Samples_were_analyzed_by_PFGE.

Another importantﬁnding that reinforcesthe problems

ofhandling catheterswasthesubsequent detectionof

col-onization by S. aureus contaminating 75% of CVCs hubs

analyzed.Thisaspect wasalsoreported byMahieuet al.30

whoshowedanassociationbetweenhandlingthehubandan

increasedriskofCR-BSIandnosocomialsepsisinneonates.

The reduction in the incidence of BSI in our NICU was

gradualandsigniﬁcant,andappearedtooccurprominently

over two speciﬁc periods, namely from 2006 to 2008 and

from 2010 to 2012. During the second period we adopted

standard procedures for insertion of PICC and continuous

care. This strategy was based on our ﬁndings as well as

other studies conducted in the unit.31 _Although_improved

asepticproceduresandcarefulinsertionofCVCswere

docu-mentedtoreducetheincidenceofCR-BSIratesinourunit,

these procedures were not adhered to resulting again in

an increase in infection rates, thus reinforcing the

impor-tance ofmeasures to controland prevent infection at the

unit.

S.epidermidiswasresponsiblefor43%ofallCA-BSIs.While

S. epidermidis has long been considered non-pathogenic, it

is now recognizedas a relevantopportunistic pathogen in

hospitals.32 _Phenotypic _methods _demonstrated _that _66.7%

and 50%ofblood samplesandCVC tips,respectively,were

producingbioﬁlms,withdetectionoftheicaADgenein33.3%

ofthebloodsamplesand50%ofCVCtips.Thepresenceofthe

mecAgenewasalsocommonamongtheseisolates(50%).

BasedonourﬁndingsaboutthepathogenesisCR-BSIthe

pathogenesis oftheseinfections seem tobedifferentthan

(6)

Table3–Resultsofculturesfrom6casesofBSI,withpositivebloodandCVCtipcultures,andclonalproﬁlingofthemecA

andicaADgenesinsamplesofS.epidermidisisolatedfromhigh-riskneonates.

Infected patient

Sites BSIclassiﬁcation Routeof acquisition

CVCtype Catheterization time(days)

PFGE mecAgene icaADgene

1 Blood Deﬁnite Deﬁniteextraluminal PICC 10 A Positive Positive

CVCtip CR-BSI A Positive Positive

2 Blood Notrelated Indeterminate Umbilical 8 H Negative Negative

CVCtip C Negative Negative

3 Blood Notrelated Indeterminate Phlebotomy 11 F Positive Positive

CVCtip A Positive Positive

4 Blood Probable Probableextraluminal Phlebotomy 7 H Negative Negative

CVCtip CR-BSI ND Negative Negative

5 Blood NotRelated Indeterminate Phlebotomy 6 G Negative Negative

CVCtip A Positive Positive

Nasalmucosa D Negative Negative

6 Blood Probable Probableintraluminal PICC 23 E Positive Negative

CVCtip CR-BSI B Negative Negative

Hub ND Positive Positive

ND,notdone;BSI,bloodstreaminfection;PFGE,pulsedﬁeldgelelectrophoresis;PICC,peripherallyinsertedcentralcatheter.

Table4–Strainrelatednessof12isolatescategorizedaccordingtoPFGEtypeandgenotypebyPCRin6casesofBSIdue

toS.epidermidis.

PCRproﬁle Genotype PFGE PFGEpatterns Predominantclone(%) Samepatient Blood CVCtip

All 12

1 mecA+/icaAD+ 5 A,F A(80.0) Yes Yes

2 mecA−/icaAD− 6 H,C,G,D,B H(33.3) No No

3 mecA+/icaAD− 1 E E(100.0) No No PFGE,pulsedﬁeldgelelectrophoresis.

Sample Clone A 12 11 10 9 8 7 6 5 4 3 2 1 A A A B C D E F G H H 100 80 60

Fig.1–DendogramofgenotypicstrainsofStaphylococcusepidermidisafterfragmentationwithrestrictionenzymeSmaIand computeranalysisofPFGE.

Samples11and12:bloodandCVCtip,respectively,frompatient1.Samples2and7:bloodandCVCtip,respectively,from patient2.Samples4and9:bloodandCVCtip,respectively,frompatient3.Sample1:bloodsamplefrompatient4.Samples: 3,6and10:blood,nasalmucosaandCVCtip,respectively,frompatient5.Samples:8and5:bloodsampleandCVCtip, respectively,frompatient6.

(7)

Conclusions

Inconclusion,thereseemstobeevidencethatpathogenesis

ofCR-BSI inneonates differsfrom that reportedin adults,

as the majority of these infections that occurred in high

riskneonatespresentedroutesofindeterminateacquisition

andonly16.7%wereacquiredextraluminally.Findingsofthis

studyreinforcetheneedforcombinedstrategiestoprevent

extraluminallyand intraluminallyacquired BSIinhigh-risk

neonatesindevelopingcountries.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

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