w w w . e l s e v ie r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Microbial
investigation
of
biofilms
recovered
from
endotracheal
tubes
using
sonication
in
intensive
care
unit
pediatric
patients
Thiago
de
Oliveira
Ferreira
a,
Rafael
Yoshio
Koto
a,
Gabriel
Fialkovitz
da
Costa
Leite
a,
Giselle
Burlamaqui
Klautau
a,
Stanley
Nigro
b,
Cely
Barreto
da
Silva
b,
Ana
Paula
Idalgo
da
Fonseca
Souza
b,
Marcelo
Jenne
Mimica
c,
Regina
Grigolli
Cesar
c,
Mauro
José
Costa
Salles
a,∗aSantaCasadeSãoPaulo,EscoladeCiênciasMédicas,DepartamentodeMedicinaInterna,SãoPaulo,SP,Brazil
bSantaCasadeSãoPaulo,EscoladeCiênciasMédicas,DepartamentodeMedicinaLaboratorialePatologia,SãoPaulo,SP,Brazil cSantaCasadeSãoPaulo,EscoladeCiênciasMédicas,DepartamentodePediatria,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received10February2016 Accepted3July2016
Availableonline8August2016
Keywords: Sonication Endotrachealtube Biofilm Trachealaspirate
a
b
s
t
r
a
c
t
Objectives:Tocompareculturedmicroorganismsidentifiedonendotrachealtubesbiofilms throughsonicationtechniquewithtraditionaltrachealaspiratecollectedatextubationof pediatricintensivecareunitpatients.
Methods:Demographicandepidemiologicaldatawereanalyzedtoidentifyfactorspossibly relatedwiththemicrobiologicalprofileofthetwocollectionmethods.Associationsbetween categoricalandcontinuousvariableswereanalyzedusingthechi-squareorFisher’sexact test,orStudent’sttest.p-Value<0.05wereconsideredsignificant.
Results:Thirtyendotrachealtubesandtrachealaspiratessamplesfrom27subjectswere analyzed.Onlyonepatientpresentedtheclinicaldiagnosisofventilator-associated pneu-monia. Overall, 50%of bacteria wereGram-negative bacilli, followedby Gram-positive bacteriain 37%, andfungi in 10%.No statistically significant differenceon the distri-butionofGram-positiveor Gram-negativebacteria (p=0.996),andfungi(p=0.985)were observedbetweenthecollectionmethods.Pseudomonasspp.wasthemostfrequent microor-ganismidentified(23.8%),followedbyStreptococcusspp.(18.5%),Acinetobacterspp.(15.9%), coagulase-negative staphylococci (11.2%), and Klebsiella spp.(8.6%). Concordant results betweenmethodsamountedto83.3%.PseudomonasaeruginosaandAcinetobacterbaumannii
showedcarbapenemresistancein50%and43.7%oftheisolates,respectively.Ingeneral, cul-turesafterendotrachealtubessonication(non-centrifugedsonicationfluidandcentrifuged sonicationfluid)yieldedbacteriawithhigherratesofantimicrobialresistancecompared totrachealaspiratescultures.Additionally,in12subjects(40%),weobserveddiscrepancies regardingmicrobiologicprofilesofculturesperformedusingthecollectionmethods.
∗ Correspondingauthor.
E-mailaddress:mcsalles@osite.com.br(M.J.Salles).
http://dx.doi.org/10.1016/j.bjid.2016.07.003
1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Conclusions:OurstudydemonstratedthatsonicationtechniquecanbeappliedtoETbiofilms toidentifymicroorganismsattachedtotheirsurfacewithagreatvarietyofspecies iden-tified.However,wedidnotfindsignificantdifferencesincomparisonwiththetraditional trachealaspiratecultureapproach.
©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Nosocomialpneumoniarepresentsapproximatelyone quar-ter of all nosocomial infections and tracheal intubation increasestheriskforinfectionbysixto20timeshigher.1,2
Among patients under mechanic ventilation on intensive careunit(ICU), ventilator-associatedpneumonia(VAP) rep-resents the most frequent type of nosocomial infection, whichextends the lengthofhospitalization, increase mor-tality and costs of ICU treatment.1 The clinical diagnosis
ofVAP hasbeen based uponlocal and systemicsigns and symptomsofinfectioninadditiontoradiologicalcriteria.1,2
Furthermore,bacteriologicaldiagnosisusingprotected speci-menbrush(PSB),bronchoalveolarlavage(BAL),andprotected endotrachealaspirateshavebeenfullystandardizedbutlack specificity,asitreliesupontheidentificationof microorgan-ismsgrowingintrachealsecretion.1–4 Nevertheless,bacteria
growinginbiofilmsattachedtotheendotrachealtubemayalso playaroleinthepathogenesisofVAP.5,6Indeed,the
knowl-edgeofbiofilm formationbymicroorganisms inaseriesof medicaldevicesanditsassociationtodifficult-to-treat infec-tionshas becomea concerninginthe nosocomialsetting.7
Therefore,the VAP representsaclearexampleof multifac-torialinfectioninwhichthe biofilmmaybeimplicatednot only on the pathogenesis, but equally on the high preva-lence of multiresistant bacteria.8–12 Theendotracheal tube
(ET)allowsdirectentryofmicroorganismsfromthe orophar-ynxenvironmentintothelowerrespiratorytract,whereasthe innateimmunesystemcomponentsareineffective.12–14Thus,
ETallowsmicroorganismspresentingnaturalabilitytoform biofilmtoattachonthetubesurfacethroughproductionofa matrixofpolysaccharidesandproteinsthatprovides protec-tionandconsequentsurvivalintheenvironment.10According
topreviouspublications,bacteriawithinthebiofilmcaninfect thelungsbyseveralways:throughdetachmentofbiofilm por-tions,thusreachingthelungsandbyaspirationintodeeper airwaysofaerosolizedplanktonicpathogensdetachedfrom thebiofilm.13,14
Recently,microbialdiagnosisofimplant-associated infec-tionshasbeenincrementedbyusingvortexingand sonica-tion technique applied to dislodge sessile microorganisms attachedtoavarietyofmedicalimplants,bydisruptingthe polymermatrixonthesurfaceofthebiofilmwhile maintain-ingtheintegrityofthepathogen.15–17Trampuzetal.,Inacio
etal.,andVandecandelaereetal.weresomeoftheauthors whosestudies appliedsonication foridentifyingpathogens from biofilmsattached todifferent humanimplants.15,17–20
Despitethelackofdata,ETsonicationmay bean interest-ingtooltoincreasetheyieldofdetectionofsessilepathogens anditspossibleeffectonthepathogenesisofVAP.
Therefore, we aimed to compare the yield of cultured microorganisms collected by ET centrifuged and non-centrifugedsonicationfluidwithmicroorganismscollectedby conventional endotrachealaspirationofintubatedpediatric ICUpatients.
Materials
and
methods
Studypopulation
We performeda pilotmicrobiological cross-sectionalstudy comparing the microbial colonization of ET submitted to sonicationwiththemicrobialyieldofculturesofcollected tra-chealaspiratesatthetimeofelectiveextubation.Atotalof 27pediatricICUpatientsundermechanicalventilation,from December2012toJune2014attheSantaCasadeSãoPaulo SchoolofMedicine(Brazil)wereevaluated.Thecollectionof trachealaspirateswasperformedpriortoextubationaspart oftheICUphysiotherapistroutineinordertoprevent bron-choaspirations. Subjects were excluded when clinical data wereunavailableforanalysis,whennotrachealaspirateswere collected during extubation,or when contamination ofET occurredduringextubation,transportation,orprocessingin themicrobiologylaboratory.Asacross-sectionalstudy,study participantswerenotfollowed-up.TheResearchEthics Com-mitteeatourinstitutionapprovedtheprojectandwaiverof obtaininganinformedconsent,astheinvestigatorshave nei-therinterferedinpatientmanagementnorintheindication ofextubation.
DiagnosisofVAP
ThediagnosiscriteriaforVAPwerebasedontheCDC guide-lines, in whichpatients onmechanical ventilationdevelop pneumonia 48h after intubation.21 The diagnostic triad
consistedofclinicalevidenceofpulmonaryinfection, includ-ing fever, purulent secretions, leukocytosis or leukopenia, signs of respiratory distress and worsening gas exchange; radiologicsuggestionofpulmonaryinfection,suchas persis-tence oflunginfiltrate,consolidationsand cavitations;and microbialevidenceofpulmonaryinfection.21
Collectionofsamplesandsonicationmethod
Prior to extubation, the ICU physiotherapy team routinely collects trachealsecretionstopreventbronchoaspirationin pediatric patients. This collected secretion, that otherwise wouldbediscarded,wasthenusedforthemicrobialanalyses ofthestudy.Afterextubation,thedistal10cmoftheETwere
choppedoffandsubmittedtoasepticcleaningonitssurface witheitheralcohol70% orchlorhexidine. Thecleaned por-tionsoftheETwerestoredina50mLFalcontubeswiththe additionof20mLofsterileNaCl salinesolution(0.9%). The sampleswereprocessedandcultivatedwithin6hfrom col-lection;onthelaboratorytheFalcontubesweresubjectedto twocyclesofvortexingfor30susingaVortex-Genie2 (Sci-entific Industries Inc., Bohemia, NY, USA), interspersed by sonicationfor5minatafrequencyof40±2kHzwithpower density of 0.22W/cm2 ±0.04, using the ultrasonic washer
modelBactoSonic14.2(BandelinElectronicGmbH&Co.KG, Germany),accordingtopreviouspublications.15,17 Afterthe
secondvortexing,aliquotsof0.1mLofnon-centrifuged son-ication fluid were inoculated on aerobic sheep blood and chocolateagar plates.TheFalcon tubewasthensubmitted tocentrifugationfor5minat2500rpm,thesupernatant aspi-rated,leavingotheraliquotof0.1mLtobeplatedinthesame media.AlongtheET,trachealsecretionsamplesfromthesame patientswerecollectedbytheICUteam. Thetracheal aspi-ratesampleswerealsoinoculatedwithacalibratedloop,using aliquotsof0.1mLontobloodandchocolateagarplates,and theninterpretedafterovernightincubation inappropriated conditions.
Microbiologicalanalysis
Microbialanalysiswas performedforbothcentrifuged and non-centrifuged sonication fluid samples and for tracheal aspirates.Theculturemediawereincubatedfor48h,bothat atemperatureof35–37◦C.Thechocolateagarwasincubated
in a capnophilic atmosphere. The plates showing micro-bial growth underwent quantification and identification of bacteriaaccordingtotheroutineestablishedbythe labora-tory, considering the morphology and tinctorial properties displayed on Gram stain. In both methods, approximately 3–4 colony-forming units (CFU) were used as suspensions insaline until the inoculum reached0.5 MacFarland scale (1.5×108CFU/mL). The inoculum was plated in Mueller
HintonAgarorMuellerHintonBloodAgar.After24-h incu-bation,thediametersofinhibitionzonesweremeasuredand interpretedinaccordancetostandardsfortested antimicro-bial drugs.22 Catalase testswere performed on coloniesof
Gram-positive cocci for identifying Staphylococcus spp. and
Streptococcus spp. DNAse tests differentiated Staphylococcus aureusfromcoagulase-negativestaphylococci, whilegroups ofStreptococcusspp.andEnterococcusspp.wereidentifiedby theirhemolysis(alpha,betaorgamma),passingtohydrolysis ofbilesculinatestsandgrowthon6.5%NaClassociatedwith susceptibilitytestingtooptochinandbacitracinorCAMPTest, whennecessary.ThecoloniesofGram-negativebacteriawere identifiedbybiochemicalmethodstodeterminetheirgenus and species. The CLSI standardizeddisk diffusion method wasusedforall strainsin theidentification of susceptibil-ity profile,and forvancomycin, weapplied the Etest® (AB
Biodisk, France) for determination of minimum inhibitory concentration (MIC).23 Antimicrobials used for testing the
cultivated pathogens followed the recommendations of theCLSI. Tracheal aspirate (n=53) Sonication fluid (n=48) Centrifuged sonication fluid (n=50) Fungi 5 3 4 Gram - Bacteria 29 27 28 Gram + Bacteria 19 18 18 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Frequ en cy
Fig.1–FrequencyofGram-positive,Gram-negativeand fungigrowninthethreedifferenttechniquesperformed.
Statisticaldata
Subject and epidemiological variables and other microbi-ological findings were summarized by using frequencies, percentages, means and standard deviations. Associations between categoricalvariableswere analyzedusing the chi-square or Fisher’s exact test. Comparison of continuous variableswasperformedwiththeStudent’sttest.Differences wereconsideredsignificantatap-value<0.05.
Results
Studypopulationandempiricalantimicrobialtherapy
ThirtyETandtrachealaspiratessampleswereobtainedfrom 27patients.ThreeendotrachealtubesandTAsampleswere obtainedfromasinglesubjectatdifferentmomentsdueto reintubation. Thesubjects mean age was 3.25±3.93 years, in which 17 patients (56.6%) were younger than one-year-old; 63.33%were male. Medianlengthofhospitalstay and time on mechanical ventilationwere 16.2±12.76 days and 12.33±8.31days,respectively.Onlyonesubjectpresentedthe clinicaldiagnosis ofVAP.Table1summarizes epidemiologi-caldataandclinicalcharacteristicsofthestudypopulation, such as length ofhospital stay, days ofmechanical venti-lation, mainreasonforICU admission,empiricalantibiotic therapy.Inaddition,thistableshowsthefrequenciesof pos-itive culturesaccording to the collection methods, namely trachealaspirates(TA),sonicationfluid(SF),andcentrifuged sonicationfluidcultures(CSF).Therewerenosignificant dif-ferencesinthefrequencyofpositiveculturesaccordingtothe collectionmethods,afteradjustingforepidemiologicaldata, clinicalcharacteristics,andtypeofempiricalantibiotic ther-apy(Table1).
Microbiologyofcentrifuged,non-centrifugedsonication fluidsandtrachealaspiratecultures
In general, up to 50% of identified microorganisms were Gram-negative bacilli, followed by Gram-positive bacteria in 37%, whereas fungi represented less than 10% (Fig. 1). No statistically significant differences on the distribution of Gram-positive or negative bacteria (p=0.99), and fungi
Table1–Epidemiologicaldataandprescribedantibioticsofstudysubjectsandcomparisonwiththefrequencyof positiveculturesineachmicrobiologicaltechnique.
Hospitalizationdata Subjects
N=30
Positivecultures p-Valueb
TAa SFa CSFa Lengthofhospitalization 0–5days 3(10%) 1(33.3%) 2(66.6%) 1(33.3%) 0.78 6–10days 9(30%) 9(100%) 8(88.8%) 9(100%) 0.96 11–15days 7(23.3%) 7(100%) 6(85.7%) 4(57.14%) 0.66 16–20days 3(10%) 3(100%) 3(100%) 3(100%) – 20+days 8(26.6%) 8(100%) 8(100%) 8(100%) – Lengthofintubation 0–5days 8(26.6%) 6(75%) 7(87.5%) 6(75%) 0.95 6–10days 7(23.3%) 7(100%) 6(85.7%) 6(85.7%) 0.95 11–15days 6(20%) 6(100%) 5(83.3%) 3(50%) 0.6 16–20days 3(10%) 3(100%) 3(100%) 3(100%) – 20+days 6(20%) 6(100%) 6(100%) 6(100%) – Reasonsofhospitalization
Acuterespiratoryfailure
(bronchopneumonia,bronchiolitis)
12(40%) 10(83.3%) 11(91.6%) 9(75%) 0.9
Acuterespiratoryfailure(VAP) 1(3.3%) 1(100%) 0 0 0.36
Meningitis 2(6.6%) 2(100%) 2(100%) 2(100%) –
Septicshock 6(20%) 6(100%) 5(83.3%) 6(100%) 0.94
Non-infectiousdiseases
(Trauma,GIdiseases,congenitaldisorders)
9(30%) 8(88.8%) 8(88.8%) 7(77.7%) 0.95
Hospitalizationdata Subjects
N=27
Positivecultures p-Valueb
TAa SFa CSFa Antibioticsprescribed Ceftazidime 1(3.70%) 1(100%) 1(100%) 1(100%) – Amikacin 4(14.81%) 4(100%) 4(100%) 4(100%) – Metronidazole 3(11.11%) 3(100%) 2(66.6%) 3(100%) 0.88 Ceftriaxone 15(55.55%) 14(93.3%) 14(93.3%) 12(80%) 0.90 Clindamycin 4(14.81%) 4(100%) 3(75%) 4(100%) 0.91 Vancomycin 15(55.55%) 15(100%) 14(93.3%) 13(86.6%) 0.93 Meropenem 15(55.55%) 15(100%) 14(93.3%) 13(86.6%) 0.93 Fluconazole 8(29.62%) 8(100%) 7(87.5%) 7(87.5%) 0.95 Cefazolin 2(7.40%) 1(50%) 1(50%) 1(50%) – Micafungin 2(7.40%) 2(100%) 2(100%) 2(100%) – Cefepime 3(11.11%) 3(100%) 2(66.6%) 3(100%) 0.88 Oxacillin 13(48.14%) 12(92.3%) 11(84.6%) 10(77%) 0.91 Piperacillin/tazobactam 2(7.40%) 2(100%) 2(100%) 2(100%) – Crystallinepenicillin 3(11.11%) 3(100%) 3(100%) 3(100%) – Clarithromycin 2(7.40%) 2(100%) 2(100%) 2(100%) – AmphotericinB 1(3.70%) 1(100%) 1(100%) 1(100%) – Ampicillin/sulbactam 1(3.70%) 1(100%) 1(100%) 1(100%) – Azithromycin 1(3.70%) 1(100%) 1(100%) 1(100%) –
TA,trachealaspirate;SC,non-centrifugedsonicationfluid;CSF,centrifugedsonicationfluid.
a Hospitalizationdataweresummarizedasfrequenciesandpercentagesormedianandcomparedusingthechi-squareorFisher’sexacttest
asappropriatetocategoricalvariablesandt-testasappropriatetocontinuousvariables.Alltestsweretwosidedandp-valueslessthan0.05
wereconsideredstatisticallysignificant.
b Medicationfrequencywascalculatedbasedon27samples,sinceinthreeofthemthedatawasunavailable.
(p=0.98) according to the collection three methods. Pseu-domonasspp.(23.8%)and Streptococcusspp.(18.5%)werethe most frequently identified pathogens, followed by Acineto-bacterspp.(15.9%),coagulase-negativestaphylococci(11.2%),
Klebsiellaspp. (8.6%) and Staphylococcus aureus (4.6%). Inter-estingly, therewasno statisticallysignificant differenceon the distribution of microorganisms isolated from sonica-tionfluid(centrifugedand non-centrifuged)comparedwith tracheal aspirate. Table 2 summarizes the frequencies of
microorganismsculturedaccordingtothecollectionmethod ETsubmittedtosonicationandthemethodofconventional trachealaspirate.
DiscordantanddiscrepantcultureresultstoTA,SFand CSFcollectionmethods
Inall butoneETand TAsamplesnomicrobialgrowth was detectedneitheronSF,CSFnoronTAcultures(3.3%,1/30).The
Table2–Distributionofspeciesofbacteriaandfungiaccordingtoitsfrequenciesinculturesoftrachealaspirates, sonicationfluid,andcentrifugedsonicationfluid.
Microorganisms (N=151) TA (N=53) SF (N=48) CSF (N=50) p-Valuea Pseudomonasspp. 23.8%(36/151) 24.52%(13/53) 22.91%(11/48) 24%(12/50) 0.98 Streptococcusspp. 18.5%(28/151) 20.75%(11/53) 18.75%(9/48) 16%(8/50) 0.90 Acinetobacterspp. 15.9%(24/151) 13.2%(7/53) 16.66%(8/48) 18%(9/50) 0.80 CNSb 11.2%(17/151) 9.43%(5/53) 10.41%(5/48) 14%(7/50) 0.75 Klebsielaspp. 8.6%(13/151) 9.43%(5/53) 8.33%(4/48) 8%(4/50) 0.98 Candidaspp. 7.3%(11/151) 7.54%(4/53) 6.25%(3/48) 8%(4/50) 0.94 S.aureusc 4.6%(7/151) 5.66%(3/53) 4.16%(2/48) 4%(2/50) 0.92 Chromobacteriumspp. 2%(3/151) 1.88%(1/53) 2.08%(1/48) 2%(1/50) 0.99 Enterococccusspp. 2%(3/151) 0 4.16%(2/48) 2%(1/50) 0.33 Enterobacterspp. 2%(3/151) 1.88%(1/53) 2.08%(1/48) 2%(1/50) 0.99 Neisseriaspp. 2%(3/151) 1.88%(1/53) 2.08%(1/48) 2%(1/50) 0.99 C.indologenesd 0.66%(1/151) 0 2.08%(1/48) 0 0.34 Escherichiacoli 0.66%(1/151) 1.88%(1/53) 0 0 0.41 Rhodotorolarubra 0.66%(1/151) 1.88%(1/53) 0 0 0.41
TA,trachealaspirate;SC,non-centrifugedsonicationfluid;CSF,centrifugedsonicationfluid.
a Microbiologicresultsweresummarizedasfrequenciesandpercentagesandalsocomparedusingthechi-squareorFisher’sexacttestas
appropriatetocategoricalvariables.Alltestsweretwosidedandp-valueslessthan0.05wereconsideredstatisticallysignificant.
b Coagulase-negativestaphylococci.
c Staphylococcusaureus. dChryseobacteriumindologenes.
globalconcordance(positiveandnegativeresults)betweenTA, SF,andCSFwas83.3%.Therewerefivediscordantresults,in whichonlyonesubjecthadsonicationfluidculture-positive andTAculture-negativeresult.Ontheotherhand,theonly subject showing clinical diagnosis of VAP had TA culture-positive(Escherichiacoli)andSF/CSFculture-negativeresults. Surprisingly, subject #11 was notclinically diagnosed with VAP,butTAculturesyieldedPseudomonasspp.,Klebsiellaspp.,
S.aureus,andonlyKlebsiellaspp.onSFcultures.Twosubjects presentedCSFculture-negativeresults,butculturesofSFand TAwerepositiveanddiscrepancywasdetectedinoneofthem (Table3).Additionally,wefoundmicrobiologicaldiscrepancies (positiveculturesonTA,SF,andCSF,butdifferent microbio-logicalprofiles)among12(40%)subjects.Table3summarizes thediscrepantanddiscordantresultsbetweenTA,SF,andCSF cultures.
Regarding antimicrobial resistance, there were different patternsofsusceptibilitytoantimicrobialsamongthesame microorganisms identified on TA, SF,and SCF cultures. Of interest,Pseudomonasaeruginosaand Acinetobacterbaumannii
showedcarbapenemresistancein50%and43.7%ofthe iso-lates, respectively. In general, cultures after ET sonication (SF and CSF)yielded bacteria with higher rates of antimi-crobial resistance compared to TA cultures. For example, carbapenem-resistant and cefepime-resistant P. aeruginosa
isolatedfrom SF,CSF,andTAcultureswere 35%,35%,29%, and37.5%,37.5%,25%,respectively.Carbapenem-resistantA. baumanniiisolatedfromSF,CSF,andTAcultureswere28.5%, 42.8%, 28.5%, respectively. Methicillin-resistant S. aureus
(MRSA)wasidentifiedin28.5%oftheisolates,andKlebsiella pneumoniaeCarbapenemase (KPC) was isolated inonly one subject,identifiedinTA,SF,andSCFcultures.
Discussion
MicrobiologicaldatashowingETbiofilmfloraandupper air-way colonization remains insufficient, even though it has been frequently recognized as potential risk factors for nosocomialpneumonia.Therefore,weaimedtoprovide infor-mationregardingthemicrobiotaattachedtoETinnersurfaces biofilmsbyuseofsonicationandcomparedtoconventional tracheal aspirates cultures, among colonized pediatric ICU patients. Sonication has been frequently applied for dis-lodging biofilm sessile pathogens, and therefore validated asanadds-ontechniqueformicrobialdiagnosisofdifferent human device-associated infection.15–17,19 Vandecandelaere
et al.appliedsonication onETforbiofilmdetachment and compared with TA surveillance cultures among mechani-calventilatedadultpatients,andfoundcorrelationbetween antibiotic-resistantpathogensidentifiedeitheronETbiofilms andtrachealsecretioncultures.20Gil-Perotinetal.analyzed
the formation ofET biofilm and its implication to VAP by scrapingthebiofilmfromtheinnersurfaceofETfollowedby vortexing. Biofilmwasfoundalmost universallyonET and 50% ofbacteria associatedto VAPwere also cultured from biofilms.11
Toourknowledge,nopreviousstudieshaveapplied son-ication and vortexing on ET for biofilm microbial analysis in the ICU pediatric population. Unlike others authors,we investigatedmicrobialbiofilmformationonETmainlyamong subjectswithnoclinical diagnosisofVAP(withone excep-tion),even thoughtheywere all on systemicantimicrobial therapyforvariedreasons.Wefoundthatevenoncolonized mechanicallyventilatedpediatricpatients,therewereahigh prevalence(96.7%)ofETbiofilmformationafterintubation.
Table3–Subjectswithdiscordantanddiscrepantacultureresultsoftrachealaspirates,sonicationfluid,andcentrifuged
sonicationfluid.
Samples VAP TA SF CSF
1 N Klebsiellaspp. Klebsiellaspp.,CNSb,Acinetobacterspp. Klebsiellaspp.,Enterococcusspp.
2 N Streptococcusspp.
Rhodotorularubra,Candidaspp.
Streptococcusspp.,CNSc Negative 3 N Acinetobacterspp. Pseudomonasspp. Acinetobacterspp. Acinetobacterspp., Pseudomonasspp. CNSb 4 N Streptococcusspp., Candidaspp. Acinetobacterspp. Streptococcusspp., S.aureusc Streptococcusspp., Candidaspp.,CNSc 6 N Streptococcusspp., CNSb Streptococcusspp. Streptococcusspp., CNSb 7 N Pseudomonasspp. Streptococcusspp., Pseudomonasspp. CNSb 8 N Pseudomonasspp., Candidaspp. Streptococcusspp., Pseudomonasspp., Candidaspp. Streptococcusspp., Pseudomonasspp., Candidaspp.,S.aureusc 9 N Streptococcusspp., S.aureusc Klebsiellaspp. Klebsiellaspp.
10 N Negative Streptococcusspp. Negative
11 N Peusdomonasspp., Klebsiellaspp., S.aureusc Klebsiellaspp. Negative 14 N Acinetobacterspp. Klebsiellaspp. Acinetobacterspp., Candidaspp. Acinetobacterspp., Candidaspp.
17 N Streptococcusspp. Pseudomonasspp. Pseudomonasspp.
18 N Pseudomonasspp., CNSb Pseudomonasspp. CNSb,C.indologenesd Pseudomonasspp. 19 Y Streptococcusspp., CNSb,E.coli Negative Negative 20 N Streptococcusspp., Candidaspp. Streptococcusspp., Candidaspp. Acinetobacterspp., Enterococcusspp. Streptococcusspp., Candidaspp. Acinetobacterspp. 23 N Streptococcusspp., Klebsiellaspp. Klebsiellaspp., Enterococcusspp. Acinetobacterspp., Klebsiellaspp., Streptococcusspp.
28 N Pseudomonasspp. Pseudomonasspp. Negative
VAP,ventilator-associatedpneumonia;TA,trachealaspirate;SC,non-centrifugedsonicationfluid;CSF,centrifugedsonicationfluid.
a Discordance:theabsenceofpathogensinanytechniquesusedtoevaluateonepatientsamples.Discrepancy:thepresenceofdifferent
pathogensamongonepatient’ssamplewhencollectedbydifferenttechniques.
b Coagulase-negativestaphylococci.
c Staphylococcusaureus. d Chryseobacteriumindologenes.
Previousauthorshavealreadyacknowledgedtheassociation betweenupperrespiratorytractcolonization,microaspiration, andbiofilmformationonthesurfaceofETasriskfactorfor nosocomialpneumonia.5,6,8,9,11Anotherimportantaspectof
ourstudywasthespecialattentiontoinvestigatethe micro-bialresultsofSFusingaconcentrationstep.Indeed,many authorshaveadded aSFconcentration stepusing samples centrifugationtoidentifyhighernumberofmicroorganisms onthesample.15–19,24AtleastforbiofilmsattachedtoETinner
surface,centrifugationofsonicationfluidwasnotstatistically superiortonon-centrifugedSFtoincreasemicrobial diagno-sis.
Alongwithotherresults,5,11 inthisstudyTAandSF
cul-turesyieldedmostlyGram-negativebacilli,mainlyP.aeruginosa
andA. baumannii(40%).These pathogens havebeen highly
associatedwithsevereacuteandchronicnosocomial respi-ratory infections as they are easily capable of expressing biofilm-forming and antibiotic-resistant genes.1,5–11,20,25,26
Previousstudiesanalyzedthemicroorganismspresentin den-tal plaque of ICU patients and showed that more isolates ofP. aeruginosawereidentifiedwhencomparedtothe gen-eralpopulation.27–28Besides,Pseudomonasspp.,althoughnota
commoninhabitantoforalmicrofloraisanimportant respira-torypathogenfrequentlyassociatedwithlate-onsetVAP.7,11,27
The remarkableabilityofP. aeruginosa toadhereand form complexesbiofilmstructuresinsputum,lungtissues,andET andthereforeproducingdifficult-to-treatchronicinfections, such ascystic fibrosis and VAP,was the very beginning of studiesconnectingbiofilmsciencewithmedicine.5,13,14
inourstudiedsample,whichmighthavecontributedtothe highprevalenceofA.baumanniiisolated.Indeed,late-onset VAPhavebeen frequentlycausedbymultidrug-resistantA. baumannii, an environmentalopportunistic biofilm-forming low-virulentpathogencapableofsurvivingtomoistanddry conditionsforaprolongedperiod,andassociatedtoprevious useofsystemicempiricalantibiotics.25,26,29,30 Ofnote,
com-monoralmicrobiotasuchasStreptococcusspp.,whichpresents awidevarietyofspecies,includingS.mutans,abiofilm pro-moter, were also frequently isolated.27 Coagulase-negative
staphylococci, which have a well-known genetic predispo-sition to form biofilms, were also frequently detected on cultures.Ourresultsseemedtobeinaccordancewitharecent studythatappliedtraditionalculturemethodsandmolecular techniquestoassess themicrobial diversityofETbiofilms, and eventually identified coagulase-negative staphylococci, mainly S. epidermidis as the most frequently encountered organismoncultures.18
Inourstudy,theoverallmicrobialcultureresultsobserved fromeachcollectiontechniquewereconcordant(83%),which suggeststhatcultureofallthreetechniques(TA,SF,andCSF) mayhaveisolatedbothsessileandplanktoniccells.Astudyby Vandecandelaereetal.alsosuggestsanagreementofresults betweensurveillanceculturesandETbiofilmculturesusing sonicationtechnique.20However,inasituationofVAP,
accord-ingtoESCMIDguidelineforthe diagnosisandtreatmentof biofilm infections,it isdifficult todetermine ifthe biofilm withintheETcanbeeithertheprimarysourceofinfectionor merelyaconcomitantcolonizedsite.6Also,respiratory
secret-ionscanaccumulateintheETleadingtoavastconcentration ofplanktonicpathogensinairwaysecretion,underestimating thepresenceofsessilepathogens.
Regardingthepresenceofdiscordantresultsamong rein-tubatedpatientsanddiscrepancyinantibioticssusceptibility, biological variationand change ofmicrobial flora dueto a longerstayinICUmightbethereasonbehindthe discord-ance,oncecommonin-hospitalpathogenswerefoundatthe secondmoment,likeAcinetobacterspp.,P.aeruginosaand Can-didaalbicans.Wealsohypothesizethatplanktonicformsare moresusceptibletocreatedrug-resistantstrainsandare eas-ilydetectablethroughaspiratecultures.Besides,thebiofilm itselfrepresentsaresistancemechanismtoantibiotics,and oncethebacteriaaredispersedtheyaredetectedandrapidly become susceptibleto antibiotics. As no significant differ-ences werefoundbetweenthe techniquesassessedinthis study,thetrachealaspiratecouldbeanoptionforthe iden-tificationofpathogensfromthebiofilm,inwhichwasfound asimilarmicrobialsessileflorawhencomparedtothe iso-lates of planktonic pathogens. This study,however, had a smallsamplesize,whichlimitedthemicrobiologicalfindings, andthe strengthofourstatisticalanalysis. Theabsenceof morepatientsdiagnosedwithVAPcouldalsounderestimate thefindingsbyshowinglesspathogens,asdifferent sensibil-itypatternsincomparisontotheresultsobtained.Although ourstudydemonstrated83.3%ofconcordancebetween tech-niqueswedonotrecommendusingsonicationasadiagnostic testwithoutfurtherstudies,whichshouldbeperformedto betterunderstandtherelationbetweenVAPpathogenesisand ETbiofilms.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
We thank Eliete Celestino, Lucia Hiromi Kawai, and Maria Aparecida Murc¸afortheir outstandingtechnicalassistance andReginaGrigolliCesarforhersupportinthecollectionof trachealaspirateamongtheICUpediatricsubjects.Thiswork wassupportedbyFAPESP(Fundac¸ãodeAmparoaPesquisado EstadodeSãoPaulo)undergrantnumbers2013/00129-3and 00130-1.Thesponsorhadnoinvolvementinthestudydesign, inthecollection,analysis,andinterpretationofdata,inthe writing ofthe manuscript,or inthe decisiontosubmitthe manuscriptforpublication.
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