Pressure ulcer as a reservoir of multiresistant Gram-negative bacilli: risk factors for colonization and development of bacteremia

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Pressure

ulcer

as

a

reservoir

of

multiresistant

Gram-negative

bacilli:

risk

factors

for

colonization

and

development

of

bacteremia

Iolanda

A.

Braga

_,

_Cristiane

_S.

_Brito

_,

_Augusto

_Diogo

_Filho

_,

_Paulo

_P.

_Gontijo

_Filho

_,

Rosineide

M.

Ribas

a_{UniversidadeFederaldeUberlândia,HospitaldeClínicas,Uberlândia,MG,Brazil}

b_{UniversidadeFederaldeUberlândia,InstitutodeCiênciasBiomédicas,Uberlândia,MG,Brazil}

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Articlehistory:

Received31March2016 Accepted16November2016 Availableonline6December2016

Keywords: Pressureulcer Gram-negativebacilli Colonization Bacteremia

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Thepurposeofthisstudywastoidentifytheriskfactorsthatpredisposepatientswhoare hospitalizedwithpressureulcers(PUs)colonizedbyGram-negativebacilli(GNB)todevelop bacteremia.Inaddition,wealsodetectedmainphenotypesofresistanceininfectedand uninfectedPUs.AprospectivecohortstudywasconductedattheClinicalHospitalofthe FederalUniversityofUberlândiaincludingpatientswithStageIIorgreaterPUs,colonized ornotwithGNB,fromAugust2009toJuly2010.Infectedulcersweredefinedbasedon clin-icalsignsandonpositiveevaluationofsmearsofwoundmaterialtranslatedbyaratioof polymorphonuclearcellstoepithelialcells≥2:1,afterGiemsastaining.Atotalof60patients withStageIIPUswereincluded.Ofthese83.3%hadPUscolonizedand/orinfected.The fre-quencyofpolymicrobialcolonizationwas74%.EnterobacteriaceaeandGNBnon-fermenting bacteriawerethemostfrequentisolatesofPUswith44.0%ofmultiresistantisolates.Among patientswhohadinfectedPUs,sixdevelopedbacteremiabythesamemicroorganismwith a100%mortalityrate.Inaddition,PUsinhospitalizedpatientsweremajorreservoirof mul-tiresistantGNB,alsoahigh-riskpopulationforthedevelopmentofbacteremiawithhigh mortalityrates.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

Pressure ulcer (PUs) is a common clinical problem associ-atedwithhighcostintermsofbothtreatmentandhuman

∗ _{Correspondingauthor.}

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

suffering.1_{Thisisparticularlycommoninelderlypatientsin}

generalhospitalsandhomecaresettings,mainlyincritical unitswhereitsincidencerangesfrom8%to40%.2

The microbiota of PUs is usually polymicrobial and complex and can be colonized with Gram-negative bacilli

http://dx.doi.org/10.1016/j.bjid.2016.11.007

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

(GNB) multidrug-resistant and methicillin-resistant Staphy-lococcus aureus (MRSA). PUs can be reservoirs for resistant microorganisms3 _{andmay evolveinto localinfections,also}

becomingasourceofbacteremiainhospitalizedpatients.4

This study aimed to identify the risk factors that pre-disposehospitalizedpatientswithPUscolonizedbyGNBto developbacteremiabythesemicroorganisms.Inaddition,we alsodetectedthemainphenotypesofbacterialresistancein infectedanduninfectedPUs.

Materials

and

methods

Settingandstudydesign

Theresearchwasconductedatthe ClinicalHospitalofthe Federal University of Uberlândia, a tertiary 510-bed teach-inghospital,fromAugust2009toJuly2010.Duringthestudy period,60patientswithStageIIorgreaterPUswereincludedin aprospectivecohortstudy,whosemedicalrecordswere iden-tifiedbyactivesurveillanceandreviewedfordemographicand riskfactordata.

Definitions

InfectedPU

Thepresenceofinfectionattheulcersitewasbasedonclinical signsandsymptoms(i.e.,erythema,edema,pain,foulodor, andpurulentexudates,fever,delayedhealing,discoloration ofgranulationtissue,friable granulationtissue,andwound breakdown).5,6 _{Inaddition,aPUwasdeemedinfectedwhen}

thesmearofitsmaterialyieldedaratioofpolymorphonuclear tosquamousepithelialcells≥2:1afterGiemsastaining.7

PUstaging

PUswereclassifiedaccordingtothecriteriaproposedby San-tosetal.8

Bacteremia

Definedasthepresenceofviablebacteriaintheblood docu-mentedbyapositivebloodculture.9_{OnlythefirstMRSAand}

GNBbacteremiaepisodewastakenintoconsideration. Multidrug-resistant(MDR)phenotype

The criteria used for defining MDR phenotype was: non-susceptibleto≥1agentof≥3antimicrobialclasses.10

Specimencollectionandmicrobiologicaltechniques

PUcleaningwasperformedwithsterileandwarmsaline solu-tion(around37.5◦C)andanaseptictechniqueunderirrigation pressurewithasyringe(20ml)andneedle(gauges25X8-21).11

Afterwards,asterileswabmoistenedwithsalinesolutionwas rotatedovera1cm2_{areaofgranulationtissuewithsufficient}

pressuretopressfluidoutofthewoundtissue.12

The clinical specimen was inoculated into MacConkey Agar, and the differentiation of Gram-negative microor-ganisms in Enterobacteriaceae family and non-fermenting bacilli was made through oxidation-fermentation (OF) and oxidase tests. For Enterobacteriaceae it was used glucose

andlactosefermentation;indoleproduction;motility;citrate; urea hydrolysis; sulfidric gas production; phenylalanine deaminase;lysineand ornithinedecarboxylase;methyl red reaction;andVoges-Proskauertest.Fornon-fermentingGNB it was used nitrate reduction;gluconateuse; pigment pro-duction;lysinedecarboxylaseactivity;ureaseactivity;indole production;acetamideandesculinhydrolysis.Theswabwas inoculatedintoMannitolAgar,andStaphylococcusaureuswas identifiedascoagulase-positivebyclassictechniques.13

SelectedS.aureusandGNBisolateswereevaluatedusing thediskdiffusiontestforresistancetomethicillinand fluoro-quinolones,andtothirdandfourthgenerationcephalosporins and carbapenems, respectively. Quality-control protocols wereusedaccordingtotheguidelinesoftheClinicaland Lab-oratoryStandardsInstitute(CLSI,2014).14

Ethicalaspects

EthicalapprovalwasobtainedfromtheEthicsCommitteeof UberlandiaFederalUniversityaccordingtotherequirements oftheMinistryofHealth,underprotocolnumbers333/09and 118/05.

Statisticalanalysis

To assess the association of risk factors with bacteremia, patients with infected and uninfected PU were compared usingtheStudent’st-testforcontinuousdataandtheFisher exacttestorchi-squaretestforcategoricaldata,when appro-priate.Ap-value lessthan orequalto0.05was considered significant.Statisticalanalyseswereperformedwith Graph-PadPrismversion4(GraphPadSoftware,SanDiego,CA).

Results

A totalof 60patients were investigated.117 patients were excludedduetoStageIPUsorbecausethepatientsand/or familymembershavenotagreedtoparticipateinthisstudy. The patients were predominantly men (70.0%), mean age of 61 years with a wide range (20–88 years). The major-ityofpatientswerehospitalizedforclinicalreasons(53.3%) or trauma (26.7%).Onlysix (10.0%)patientswere admitted becauseofinfectedPUs.

The patients were hospitalized for an average of 103 days (SD±84.8days). Cardiomyopathy(78.3%)and diabetes mellitus(43.3%)werethemostcommoncomorbidities. Gas-trointestinalcatheter(85.0%),centralvenouscatheter(55.0%), mechanicalventilation(45.0%),urinarycatheter(40.0%),three ormoreinvasivedevices(55.0%),andthreeormoreclassesof antimicrobialagents(77.9%)wereusedbymostofthepatients. Thehospitalmortalityratewasalsohigh(41.7%)inthiscohort (datanotshown).

Enterobacteriaceae were the most commonly isolated (49.0%) group of bacteria in colonized/infected patients withPUs,followedbyStaphylococcusaureus(28.0%)and non-fermentingGNB(23.0%),mostlyPseudomonasaeruginosaand Acinetobacter spp. Colonization with multiresistant bacteria including Klebsiellapneumoniae(17/20, 85.0%), Escherichiacoli (6/24, 25.0%), Enterobacter (1/4, 25.0%), Pseudomonas

Table1–Microorganismsandresistancephenotypesinisolatedpressureulcers.

Microorganisms/phenotypes TotalisolatesofPUa _{IsolatedofinfectedPU IsolatedofnoinfectedPU}

N=100(%) N=35(%) N=65(%)

Enterobacteriaceae 49(49.0) 16(45.7) 33(50.8)

Escherichiacoli 24(49.0) 7(43.8) 17(51.5)

Resistantto3rdgenerationcephalosporins 10(41.7) 7(100.0) 3(17.6) Resistanttofluoroquinolones 7(29.2) 5(71.4) 2(11.8) Multiresistant 6(25.0) 2(28.6) 4(23.5)

Klebsiellapneumoniae 20(40.8) 8(50.0) 12(36.4)

Resistantto3rd/4thgenerationcephalosporins 17(85.0) 7(87.5) 10(83.3) Multiresistant 17(85.0) 7(87.5) 10(83.3)

Enterobacterspp. 4(8.2) 1(6.3) 3(9.1)

Resistantto3rdgenerationcephalosporins 4(100.0) 1(100.0) 3(100.0) Multiresistant 1(25.0) 1(100.0) 0

Proteusspp. 1(2.0) 0 1(3.0)

Resistantto3rdgenerationcephalosporins 1(100.0) 0 1(100.0) Multiresistant 1(100.0) 0 1(100.0)

Non-fermentingGNB 23(23.0) 14(40.0) 9(13.8)

Pseudomonasaeruginosa 18(78.3) 11(78.6) 7(77.8)

Resistantto3rdgenerationcephalosporins 18(100.0) 11(100.0) 7(100.0) Resistanttocarbapenems 7(38.9) 6(54.5) 1(14.3) Resistanttofluoroquinolones 8(44.4) 6(54.4) 2(28.6) Multiresistant 18(100.0) 11(100.0) 7(100.0)

Acinetobacterspp. 5(21.7) 3(21.4) 2(22.2)

Resistantto3rdgenerationcephalosporins 5(100.0) 3(100.0) 2(100.0) Resistanttocarbapenems 3(60.0) 3(100.0) 0 Multiresistant 3(60.0) 3(100.0) 0

Staphylococcusaureus 28(28.0) 5(14.3) 23(35.4)

MRSAb _{17(60.7) 5(100.0) 12(52.2)}

a _{Pressureulcer.}

b _{Methicillin-resistantStaphylococcusaureus.}

aeruginosa (18/18, 100.0%), Acinetobacter spp. (3/5, 60.0%), andmethicillin-resistantStaphylococcusaureus(17/28,60.7%) were the predominant bacteria in our patients. The fre-quency of epidemiologically relevant phenotypes such as fluoroquinolones- and carbapenems-resistant Pseudomonas aeruginosa, carbapenems-resistant Acinetobacter spp, third generation cephalosporins-resistant Klebsiella pneumoniae, andEscherichiacoliweresurpassed40%amongGNBisolates. Amongthe 16patientswithinfectedPUs,35sampleswere isolated of which 29 (82.9%) were multiresistant bacteria, whereasamong34patientswithnon-infectedPUs,65 sam-pleswere isolated,ofwhich35 (52.3%)were multiresistant (Table1).Byunivariateanalysis,noriskfactorswere statisti-callyassociatedwithGNBbacteremiainpatientswithStageII orgreaterPUscolonizedbythesepathogens(datanotshown). Ofthe 50patients(83.3%)withPUcolonized byGNB,16 (32.0%)developedlocalinfections.Out ofthese16patients with infected ulcer, 10 patients (62.5%) developed bac-teremia.Amongthese10patients,sixhadthesameisolates recoveredfrombothexudateandbloodwithsimilar pheno-type/antibiotic susceptibility pattern. Themortality in this groupofpatientswithPUinfectedwithGNBbacteremiawas higher(OR:7.43,95%CI,1.23–45.0,p=0.04)than inpatients withnon-infectedPUs(Table2).

Discussion

Pressure ulcers are a public health problem that results indistress and disability, and constitute a great challenge

to health administrators. They are particularly common amongelderlypatientsingeneralhospitalsand homecare settings.2

Ourstudyincluded60patientswithStageIIorgreaterPUs withcharacteristicsofcriticallyillpatients,themajoritywith threeormoreinvasiveprocedures (55.0%)andtwoormore comorbidities(75.0%).Inaddition,thesepatientshadarather longlengthofhospitalstay(mean103days)andahigh hospi-talmortalityrate(41.7%).StageIIIPUswerethemostcommon (32.0%)inourseries.

PUs are also frequently colonized by several species of bacteria and surface cultures yield a polymicrobial floraofbothGram-positiveand Gram-negativeaerobicand anaerobicspecies.15_{Themicroorganismsmostcommonly}

iso-lated in chronic wounds are usually Staphylococcus aureus, ␤-hemolytic Streptococci, Enterococcus spp., aerobic GNB EnterobacteriaceaeandPseudomonasspp., andinparticular, resistantbacteriasuchasmethicillin-resistantStaphylococcus aureus,vancomycin-resistantEnterococcusspp., ciprofloxacin-resistant Pseudomonas aeruginosa, and extended-spectrum beta-lactamase (Escherichia coli and Citrobacter).16,17 _{In our}

study,50outof60patients(83.3%)hadPUswithcultures posi-tiveforGNB,mostwithmixedflora(74.0%).Mostisolateswere identifiedasEnterobacteriaceae(49.0%),Escherichiacoli(49.0%) and Klebsiella pneumoniae (40.8%), and non-fermentingGNB (23.0%), mainly Pseudomonasaeruginosa (78.3%),and Staphy-lococcus aureus (28.0%). A total of 63% ofthe isolateswere multiresistanttodifferentantibiotics,includingPseudomonas aeruginosa(100.0%),Proteusspp.(100.0%),Klebsiellaspp(85.0%),

Table2–BacteremiainpatientswithpressureulcerscolonizedbyGram-negativebacilli.

Infectedpressureulcer Non-infectedpressureulcer p-valuea _ORb_(95%CI)

N=16(%) N=34(%)

Bacteremia 10(62.5) 20(58.8) 0.80 1.17(0.34–3.96)

Concordancec _{6(60.0) 5(25.0) 0.10 4.50(0.89–22.75)}

Death 8(80.0) 7(35.0) 0.04 7.43(1.23–45.0)

a _{Statisticalsignificanceat5%.} b _{Oddsratio(95%confidenceinterval).}

c _{Phenotypicconcordancebetweentheorganisminthebloodandulcer.}

MRSA(60.7%),Acinetobacterspp(60.0%),Escherichiacoli(25.0%), andEnterobacterspp.(25.0%).

Oneofthefewreportsintheliteratureabouttheroleof polymicrobialsynergyinlocalwoundinfection pointedout thattherecoveredbacteriafromPUsaresimilartothoseof acutenecrotizingsofttissueinfections,namelyanaerobicand aerobicbacteria,thuslikelycontributingtothedeterioration ofa lesion.18 _{Ourresultsconfirmingthat thepolymicrobial}

florawasobservedinthemajority(74.0%)ofinfectedwounds areinlinewiththefindingsofthatreport.

TheassociationofPUswithbacteremiaiswellestablished, butfewstudieshaveaddressedthisproblemsystematically. PUsmayserveasoccultfociforbloodstreaminfection,the mostcommon majorcomplicationofPUs.InfectedPUsare more likely to cause bacteremia than wounds only colo-nizedwithpolymicrobialmicroorganisms,17 _{alsoevidentin}

ourresults.

In hospitalized patients, the relationship between bac-teremiaandPUhasbeenassociatedwith50%mortalityrate.19

Among21patientswithsepsisattributedsolelytoPU, bac-teremiawasdocumentedin16(76%)cases,and47.6%ofthe patientsdied,eightofthesedespiteappropriateantibiotics treatment.20 _{Our datashowed thatout of16 patients with}

infectedPUs,10(62.5%)developedGNBbacteremia,sixhad thesamemicroorganismisolatedfromthePUandblood.Out ofthesesix,Pseudomonasaeruginosawasrecoveredfromfour patientsandKlebsiellapneumoniaefromtwo.

Inourstudy,the16patientswithbacteremiacausedbythe sameGNBthatwasrecoveredfromthePUhadahigherrisk ofdying(OR=7.43;p≤0.04)whencomparedwith34patients who didnothaveinfected PUsand werejust colonized by thesemicroorganisms.Thehospitalmortalityratewas80.0% inpatientswithinfectedPUswhodevelopedbacteremia, com-paredto35.0%inthosewhodevelopedbacteremia,buthadno infectedPUsinspiteofthepresenceofthesame microorgan-isminPUsandblood.

In conclusion, Stage II or greater PUs in hospitalized patients are reservoirs of multiresistant GNB, such as Escherichiacoli,Klebsiellapneumoniae,Pseudomonasaeruginosa, andAcinetobacterspp.ColonizationofPUswaspredominantly polymicrobialwithsignificant association withinfection in otheranatomicsites.Thisstudyalsoemphasizesthe impor-tanceofPUsasapotentialsourceofbacteremiainhospitalized patientsleadingtohighmortalityrates.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethankthepatientsofClinicalHospitalofFederal Univer-sityofUberlandiawhoconsentedtoparticipateinthestudy andthestaffmembersfortheircooperation.Wealsothankto DeividW.F.Batistãoforhelpinginstatisticalanalysisandthe BrazilianAgencyFAPEMIGandCNPqforfinancialsupport.

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