Lorena Salazar-Ospina, Judy Natalia Jiménez year old with 1 skin and soft tissue infections Staphylococcus aureus in childrenunder frequency High of methicillin-susceptible andmethicillin-resistant ORIGINAL ARTICLE

(1)

www.jped.com.br

ORIGINAL ARTICLE

High frequency of methicillin-susceptible and

methicillin-resistant Staphylococcus aureus in children under 1 year old with skin and soft tissue infections ^夽

Lorena Salazar-Ospina, Judy Natalia Jiménez

^∗

UniversidaddeAntioquia,EscueladeMicrobiología,GrupodeInvestigaciónenMicrobiologíaBásicayAplicada(MICROBA), Medellín,Colombia

Received14March2017;accepted31May2017 Availableonline21September2017

KEYWORDS Staphylococcus aureus;

MSSA;

MRSA;

Pediatric;

Epidemiology;

Molecularbiology

Abstract

Objective: Staphylococcusaureusisresponsibleforalargenumberofinfectionsinpediatric population;however, informationaboutthebehaviorofsuchinfectionsinthispopulationis limited.Theaimofthestudywastodescribetheclinical,epidemiological,andmolecularchar- acteristicsofinfectionscausedbymethicillin-susceptibleandresistantS.aureus(MSSA---MRSA) inapediatricpopulation.

Method: Across-sectionaldescriptivestudyinpatientsfrombirthto14yearsofagefromthree high-complexityinstitutionswasconducted(2008---2010).Allpatientsinfectedwithmethicillin- resistantS.aureusandarepresentativesampleofpatientsinfectedwithmethicillin-susceptible S.aureuswereincluded.Clinicalandepidemiologicalinformationwasobtainedfrommedical recordsandmolecularcharacterization includedspatyping, pulsed-ﬁeldgel electrophoresis (PFGE),andmultilocussequencetyping(MLST).Inaddition,staphylococcalcassettechromo- somemec(SCCmec)andvirulencefactorgenesweredetected.

Results: Atotalof182patients,65withmethicillin-susceptibleS.aureusinfectionsand117 withmethicillin-resistantS.aureusinfections,wereincludedinthestudy;41.4%ofthepatients beingunder1year.Themostfrequentinfectionswereoftheskinandsofttissues.Backgrounds suchashavingstayedindaycarecentersandprevioususeofantibioticsweremorecommon inpatientswithmethicillin-resistantS.aureusinfections(p≤0.05).Sixteenclonalcomplexes wereidentiﬁedandmethicillin-susceptibleS.aureusstrainsweremorediverse.Themostcom- moncassettewas staphylococcalcassette chromosomemec IVc(70.8%),whichwaslinkedto Panton---Valentineleukocidin(pvl).

Conclusions: Incontrastwithotherlocations,aprevalenceofinfectionsinchildren under1 yearofageinthecitycouldbeobserved;thisemphasizestheimportanceofepidemiological knowledgeatthelocallevel.

4.0/).

夽 Pleasecitethisarticleas:Salazar-OspinaL,JiménezJN.Highfrequencyofmethicillin-susceptibleandmethicillin-resistantStaphylo- coccusaureusinchildrenunder1yearoldwithskinandsofttissueinfections.JPediatr(RioJ).2018;94:380---9.

∗Correspondingauthor.

E-mails:nataliajiudea@gmail.com,jnatalia.jimenez@udea.edu.co(J.N.Jiménez).

https://doi.org/10.1016/j.jped.2017.06.020

(2)

S.aureusinfectionsinchildrenunder1yearold 381

PALAVRAS-CHAVE Staphylococcus aureus;

MSSA;

MRSA;

Pediatria;

Epidemiologia;

Biologiamolecular

AltafrequênciadeS.aureus(MSSA-MRSA)emcrianc¸ascommenosdeumanode idadecominfecc¸õesdepeleedotecidomole

Resumo

Objetivo: O Staphylococcus aureus é responsável por um grande número de infecções na populaçãopediátrica;contudo,asinformaçõessobreocomportamentodessasinfecçõesnessa população sãolimitadas.Oobjetivodo estudofoidescrever ascaracterísticas clínicas,epi- demiológicas e moleculares deinfecções causadas porStaphylococcus aureus suscetíveis e resistentesàmeticilina(MSSA-MSRA)emumapopulaçãopediátrica.

Método: Umestudotransversaldescritivofoirealizadoempacientesentre0e14anosdeidade detrêsinstituic¸õesdealtacomplexidade(2008-2010).TodosospacientesinfectadoscomS.

aureusresistentesàmeticilinaeumaamostrarepresentativadepacientesinfectadoscomS.

aureussuscetíveisàmeticilinaforamincluídos.Asinformaçõesclínicaseepidemiológicasforam obtidasdeprontuáriosmédicos,eacaracterizaçãomolecularincluiutipagemspa,Eletroforese em GeldeCampoPulsado(PFGE)eTipagemdesequênciasmultilocus(MLST).Alémdisso, o Cassete Cromossômico Estafilocócicomec (SCCmec) egenes de fatores devirulência foram detectados.

Resultados: 182pacientes,65cominfecçõesporS.aureussuscetíveisàmeticilinae117com infecçõesporS.aureusresistentesàmeticilina,foramincluídosnoestudo;41,4%dospacientes commenosdeumanodeidade.Asinfecçõesmaisfrequentesforamdapeleedostecidosmoles.

Oshistóricoscomointernac¸õesemcentrosdeatendimentoeousopréviodeantibióticosforam maiscomunsem pacientescominfecc¸õesporS.aureusresistentesàmeticilina(p≤0,05).

Dezesseiscomplexosclonaisforamidentificados,eascepasdeS.aureussuscetíveisàmeticilina forammaisdiversificadas.OcassetemaiscomumfoioCasseteCromossômicoEstafilocócicomec IVc(70,8%),relacionadoàleucocidinadepanton-valentine(pvl).

Conclusões: Em comparação aoutros locais, observamos uma prevalência deinfecções em criançascommenosdeumanodeidadenacidade;oqueenfatizaaimportânciadeconhecer aepidemiologiaemnívellocal.

0/).

Introduction

ThecomplexsituationofworldwideStaphylococcusaureus resistancetomethicillinhasledtoitscurrentmanagement, representing a priority for the World Health Organization (WHO)andachallengeforhumanpublichealthindifferent regions;however,bothmethicillin-susceptibleandresistant S.aureus (MSSAandMRSA)strainspossessavirulenceand pathogeniccapacitythatallowsthemtoreachhighinfection rates.

SomepopulationgroupsaremoresusceptibletoS.aureus infections; in particular, the pediatric population hasless effective immunologicalfunction,persistentlyhigh bacte- rial colonization rates, poor hygiene habits, and constant exposuretoschoolenvironmentsthatfavortheacquisition ofinfectionandthedisseminationofthemicroorganism.^1,2 The epidemiology of S. aureus infections in this popula- tionisdiverse,andthefrequenciesofMRSAinfectiondiffer betweengeographicregions,rangingfrom6%³to69.9%⁴;in Colombia,frequenciesupto47.4%havebeenfound.⁵

InMedellin,althoughS.aureusisoneofthemainagents responsibleforinfectionsinthepediatricpopulation,both inhospitals andin thecommunity,thereis littleinforma- tiononthecharacteristicsoftheinfectionscausedbythis microorganisminthispopulation.Thisstudyaimstodescribe theclinical,epidemiological,andmolecularcharacteristics ofS.aureusinfections(MSSA---MRSA)inapediatricpopula- tionofthecity.

Methods

Studypopulation

Anobservationalcross-sectionalstudywasconductedfrom February 2008 to June 2010, at three tertiary care hos- pitalsfromMedellin,the second largest city inColombia.

Patientsbetweenbirthand14yearsinfectedwithS.aureus (MSSA---MRSA) were recruited prospectively; only the ﬁrst isolatefromeachindividualwasevaluated.Allpatientswith MRSAisolatesobtainedduringthe timeofthe studywere included, and considering that the prevalence of MSSA is higher,asample wasdeﬁned.The sample size wascalcu- latedbasedontheMSSAprevalenceduring2007withineach institution,whichnumbered65isolates.TheMSSA isolates includedwererandomlyselectedeachmonth,fromFebru- ary2008 toJune 2010, using a table of random numbers accordingtorecordsofeachparticipatinginstitution.

The research protocol and informed consent (signed by parents or guardians) were approved by the Bioethics CommitteeforHumanResearchoftheUniversityResearch CenteratUniversidaddeAntioquia(approvalno.0841150) andbythebioethicscommitteeofeachhospital.

Clinicalandepidemiologicaldata

Clinical and epidemiological data for each patient were obtained from medical records. Information included

(3)

clinicalanddemographiccharacteristics,antimicrobialuse, risk factors,co-morbidities, typeof infection, treatment, lengthofhospitalstay,andoutcome.AccordingtotheCen- tersfor Disease Control andPrevention (CDC) criteria, an infectionwasconsideredpresentonadmission(POA)ifthe date of the event occurred on the day of admission to an inpatient location, twodays before admission, or the calendardayafteradmission.An infectionwasconsidered healthcare-associated(HAI) ifthe dateofeventoccurred onorafterthethirdcalendardayofadmissiontoaninpa- tient location. Patients with surgical site infections and ventilator-associatedeventwereexcluded.⁶

Identiﬁcationandantibioticsusceptibility

IdentiﬁcationofS.aureuswasconductedbystandardlabo- ratorymethodsbasedoncolonymorphologyinsheepblood agar and positive catalase and coagulase tests. Antibi- otic susceptibilities of S. aureus isolates were assessed in accordance with Clinical Laboratory Standards Insti- tute guidelines (CLSI, 2009) using the VITEK^® 2 system (BioMérieux,Inc.,NC,EUA).Theantibioticstestedincluded clindamycin, erythromycin, gentamycin, linezolid, mox- iﬂoxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim-sulfamethoxazole, and vancomycin. The S.

aureusstrainATCC29213wasusedforqualitycontrol.

Polymerasechainreaction(PCR)conﬁrmationofS.

aureusandmethicillinresistance

Thepresenceofthespecies-speciﬁcnucandfemAgenesas wellasthemecAgenewereveriﬁedbyPCR,aspreviously described.^7,8

Moleculartyping:spatyping(spa),multilocus sequencetyping(MLST)andpulsedﬁeldgel electrophoresis(PFGE)

In all isolates, the polymorphic X region of the protein A gene (spa) was ampliﬁed and sequenced as previously described.⁹ Corresponding spa types were assigned using eGenomics software^9,10 and Ridom spa- types were subsequently assigned using the spa typing website (http://www.spaserver.ridom.de/) developed by Ridom GmbH and curated by SeqNet.org (http://www.

SeqNet.org/).¹¹MLSTwasperformedonasubsetofteniso- latesrepresentingthe morefrequentspa types,usingthe methodologydescribed by Enright et al.¹² Allele numbers andsequencetypes(ST)wereassignedusingthedatabase maintained at http://saureus.mlst.net/while clonal complexes (CC)were inferredusing eBURSTanalysis.¹³ Clonal complexes for all remaining strains were inferred by spa repeatpatternanalysis,¹⁰orbyreferringtotheRidomspa serverwebsite.

PFGE,followingSmaIdigestion,wasperformedaccording to a protocol described elsewhere.¹⁴ DNA fragment pat- ternswere normalized using S. aureus strain NCTC 8325.

ClusteranalysiswasperformedusingtheDicecoefﬁcientin BioNumericssoftware(BioNumerics^®,softwareversion6.0, Belgium).Dendrogramsweregeneratedbytheunweighted

pairgroupmethodusingaveragelinkages(UPGMA).Similar- itycut-offsof80% and95%wereusedtodeﬁne typesand subtypes,respectively.¹⁴

SCCmectyping

ForMRSAisolates,SCCmectypesandsubtypesweredeter- minedusingsetsofmultiplexPCR reactions,aspreviously described.^15,16 MRSAstrainswereincludedaspositivecon- trolsforSCCmectypesandsubtypes.

Detectionofstaphylococcalvirulencefactors Allisolateswerescreenedforthegenesencodingstaphylo- coccalenterotoxins(sea,seb,sec,sed,see),toxicshocksyn- drometoxin1(tst),andexfoliativetoxinsaandb(eta,etb), usingtheprotocolsandprimersdescribedbyMehrotraetal.⁸ ThelukS/F-PVgenes,encodingPanton-Valentineleukocidin (PVL); and the arcA gene, associated arginine catabolic mobileelement(ACME),werealsoassessedbyPCR.^17,18 Statisticalanalyses

Comparisons of clinical, epidemiological, and molecular characteristics were conducted between MSSA and MRSA infectedpatients,andamongthedifferentgroupsofinfec- tionsobtainedafterapplyingCDCcriteria.

Categorical variables were compared using the chi- squared test or Fisher’s exact test; Student’s t- and Mann---WhitneyUtestswereusedforcontinuousvariables.p values≤0.05wereconsideredstatisticallysigniﬁcant.Sta- tisticalanalyseswerecarriedoutusingthesoftwarepackage inSPSS^® (IBMSPSSStatisticsforWindows,version21.0,NY, USA).

Results

Clinicalandepidemiologicaldata

Therewere182pediatricpatientswithS.aureusinfections included;ofthese,65hadMSSAinfectionsand117hadMRSA infections;119patientscamefromhospitalA,51fromhos- pitalB,and12fromhospitalC.

Intheselectedstudy population,theratioof boysand girlswas2:1andthemedianagewas2years;however,41.2%

(n=75)oftheinfectionsoccurredinpatientsbetweenbirth and1yearof age,inwhichahigh frequencyofMRSAand MSSAinfectionswasobserved(Fig.1).

AccordingtotheCDCcriteria,62.7%(n=101)oftheinfec- tions were POA, and 37.3% (n=60) corresponded to HAI (p=0.391). Infection type showed statistically significant differences by hospital. In hospitals A and B therewas a predominanceofPOAinfections;inhospitalCtherewasa predominanceofHAI(p=0.029).Twenty-one(11.5%)surgi- calsiteinfectionswereidentified,whichwerenotincluded inthisclassification.

The clinical---epidemiological characteristics of the patientsevaluatedinthestudyaredescribedinTable1.In general,aprevioushistoryofhospitalizationwasthemost frequentantecedentamongpatients,with51.65%(n=94);

(4)

S.aureusinfectionsinchildrenunder1yearold 383

50

45

30

20

MRSA MSSA

% of isolates

Age in years 10

0

<1 2-4 5-8 9-12 13-14

35

25

15

5 40

Figure 1 Frequency of infection by age group. MSSA, methicillin-susceptible Staphylococcus aureus (n=65); MRSA, methicillin-resistant Staphylococcusaureus (n=117). The ﬁg- ureshowstheagedistributionofpatientsinfectedbyMSSAand MRSAstrains. A higherfrequencyofinfectionsis observedin childrenunder1year.

furthermore,havingstayedindaycarecenters(MRSA11.1%, n=13 vs. MSSA 1.5%, n=1; p=0.020) and previous use of antibiotics (MRSA 55%, n=60 vs. MSSA 32.8%, n=20;

p=0.005)wasthemostfrequentantecedentinpatientswith MRSAinfections.

InHAI,aprevioushistoryofintensivecareunit(ICU)stay (MRSA21.4%,n=9vs.MSSA0%;p=0.047)andpriorantibiotic useinthelastsixmonths(MRSA64.1%,n=25vs.MSSA29.4%, n=5;p=0.017)weremorefrequentinpatientswithMRSA infections.

However, thehospital stay waslongerin patients with HAI,withasigniﬁcant differenceinfavor ofpatientswith MRSAinfections (MRSA 0---434days, Me=14 vs.MSSA 2---43 days,Me=5.5;p=0.038).

The mostfrequentsites ofinfectionwereskin andsoft tissue,with41.21%(n=75),andthemostcommonmedical servicewasinternalmedicine,with64.84%(n=110).

The frequency of co-morbiditiesin the population was 74.1%(n=135),morefrequentinpatients withPOAinfec- tions causedby MSSA(MSSA 81.1%, n=30vs.MRSA 54.7%, n=35;p=0.008).

Ofthepopulation,57.6%requiredsurgicaltreatmentand healingwasthemostfrequent outcome;especiallyinPOA infections caused by MSSA (MSSA 56.8%, n=21 vs. MRSA 34.4%,n=22;p=0.028).Sixofthepatientsincludedinthe studydied;however,thiswasthecrudemortalityandnot attributedtoS.aureusinfection.

Resistanceproﬁle

TheMSSAisolateshadsixresistanceproﬁles;61.5%(n=40) weresusceptibletoallantibioticsevaluated,16.9%(n=11)

Figure2 ResistanceproﬁlesofthemainMRSAclones.MRSA, methicillin-resistantStaphylococcusaureus. Figureshows the proﬁle of antimicrobial resistance of the main MRSA clones foundinthestudy.Antibioticsassessed:oxacillin(Oxa),tetracycline(Tet),clindamycin(Clin),erythromycin(Eri),gentamycin (Gen). Dark column: isolates belonging to CC5-SCCmec-I (n=22); clearcolumn: isolates belonging to CC8-SCCmec-IVc (n=75).

wereresistanttotetracycline,and9.2%(n=6)toerythromy- cinandclindamycin.TheMRSAisolateshadeightresistance proﬁles;44.4%(n=52)wereresistantonlytooxacillin,fol- lowed by 28.2% (n=33) with resistance to oxacillin and tetracycline. All of the isolates were susceptible to vancomycin,linezolid,andtigecycline.

Moleculartyping

Amongalltheisolates,16clonalcomplexes(CC)wereiden- tiﬁed.MSSA strainswere the morediverse, with ahigher numberofCCs;themostcommonwereCC8(29.2%,n=19), CC45(16.9%,n=11),andCC1(10.8%,n=7),whereasinthe MRSAstrainsthemostcommonCCswereCC8(70.9%,n=83) andCC5(22.2%,n=26).

The presence of mecA gene wasconfirmed in the 117 selectedMRSAstrains.Ofthese,itwaspossibletoidentify the SCCmec type in 113 strains; four isolates were non- typeable.OftheMSRAisolates,70.8%harboredSCCmecIVc (n=80), followedbySCCmecI(20.3%,n=23),SCCmecIVa (5.3%,n=6),andSCCmecV(2.7%,n=3).Anisolatewasiden- tifiedwithSCCmecIV,butitssubtypewasnotidentifiable (0.9%;n=1).

Among patients with POA infections, 87.1% (n=54) of MRSAisolatesharboredSCCmecIVc,followedbySCCmecI (8.1%,n=5)andSCCmecIVa(4.8%,n=3).Meanwhile,MRSA isolates from HAI patients harbored SCCmec type IVc in 56.1%(n=23),followedbySCCmectypeI(26.8%;n=11),IVa (7.3%;n=3),V(7.3%,n=3),andIV(2.43%,n=1).SCCmec IVcwasthemostfrequenttypeinbothtypesofinfection.

TheresistanceproﬁlesofthemostimportantMRSAclones areshowninFig.2.

Forty different spa types were identiﬁed in MSSA and MRSAstrains.Themost commontypes weret1610(17.6%, n=32),t008(17%,n=31),t149(11%,n=20),andt024(9.9%;

n=18).InMSSAisolatesbelongingtoCC8,typest1635and

(5)

Salazar-OspinaL,JiménezJN Table1 ClinicalandepidemiologicalcharacteristicsofpatientswithStaphylococcusaureus(MSSA---MRSA)infections.

POA-I(n=101)n(%) HAI(n=60)n(%) Total(n=182)n(%)

MSSA MRSA p-Value MSSA MRSA p-Value MSSA MRSA p-Value

n=37(36.6) n=64(63.4) n=18(30.0) n=42(70.0) n=65 n=117

Gender

Male 25(67.6) 41(64.1)

0.721 11(61.1) 32(76.2)

0.235 43(66.2) 78(66.7)

0.944

Female 12(32.4) 23(35.9) 7(38.9) 10(23.8) 22(33.8) 39(33.3)

Age(years)

Median 4 4

0.534^b 3.5 1 0.141^b 3 2

0.330^b

Range 0---14 0---14 0---12 0---14 0---14 0---14

≤1years 10(27.0) 21(32.8)

0.601

5(27.8) 22(52.4) 0.130 23(35.4) 52(44.5)

0.826

2---4years 12(32.4) 16(25.0) 5(27.8) 10(23.8) 18(27.7) 27(23.1)

5---8years 3(8.1) 8(12.5) 3(16.7) 2(4.8) 7(10.8) 11(9.5)

9---12years 7(18.9) 15(23.4) 5(27.8) 5(11.9) 12(18.5) 20(17.1)

13---14years 5(13.5) 4(6.3) 0(0) 3(7.1) 5(7.7) 7(6.0)

Previoushistory

Hospitalizationinthepastyear 21(56.8) 29(45.3) 0.268 5(27.8) 23(54.8) 0.055 33(50.8) 61(52.1) 0.860

StayinICUinthepastyear 3(8.1) 1(1.6) 0.138^a 0(0) 9(21.4) 0.047^a 5(7.7) 14(12.0) 0.366

Surgeryinthepastyear 11(29.7) 11(17.2) 0.141 4(22.2) 14(33.3) 0.389 25(38.5) 36(30.8) 0.292

Dialysisinthepastyear 4(10.8) 1(1.6) 0.059^a 1(5.6) 4(9.5) 1.000 5(7.7) 5(4.3) 0.333^a

MRSAisolationinthepastyear 1(2.7) 2(3.1) 1.000â 0(0) 4(9.5) 0.306â 1(1.5) 7(6.0) 0.262â

Antimicrobialsuseinpastsix months

11(31.4) 27(45.8) 0.171 5(29.4) 25(64.1) 0.017 20(32.8) 60(55.0) 0.005

Traumainpastsixmonths 9(24.3) 10(15.6) 0.281 7(38.9) 8(19.0) 0.118^a 16(24.6) 19(16.2) 0.169

Stayinachildren’sdaycare centersinthepastyear

1(2.7) 6(9.4) 0.418^a 0(0) 5(11.9) 0.309^a 1(1.5) 13(11.1) 0.020

Familyinfectioninthepastyear 5(21.7) 10(21.7) 1.000 2(15.4) 7(19.4) 1.000^a 9(21.4%) 17(18.3) 0.668 Sportsparticipationinthepast

year

8(27.6) 16(32.7) 0.639 2(14.3) 3(7.9) 0.602^a 10(19.6) 20(20.4) 0.908

Hospitalstay(days)

Median 0 0 0.098

b

5.50 14 0.038

b

0 0 0.309

Range 0---1 0---1 (2---43) (0---434) (0---50) (0---434) b

(6)

S.aureusinfectionsinchildrenunder1yearold385 Table1 (Continued)

POA-I(n=101)n(%) HAI(n=60)n(%) Total(n=182)n(%)

MSSA MRSA p-Value MSSA MRSA p-Value MSSA MRSA p-Value

n=37(36.6) n=64(63.4) n=18(30.0) n=42(70.0) n=65 n=117

Service

Internalmedicine 28(75.7) 42(65.6)

0.264

10(55.6) 27(64.3)

0.369

43(66.1) 75(64.1)

0.388

PediatricICU 2(5.4) 5(7.8) 2(11.1) 9(21.4) 5(7.7) 15(12.8)

Orthopedics 3(8.1) 14(21.9) 1(5.6) 0(0) 4(6.2) 15(12.8)

Surgery 1(2.7) 0(0) 3(16.7) 5(11.9) 7(10.8) 8(6.8)

Emergency 1(2.7) 2(3.1) 1(5.6) 0(0) 3(4.6) 2(1.7)

Other 2(5.4) 1(1.6) 1(5.6) 1(2.4) 3(4.6) 2(1.7)

Infectiontype

Skinandsofttissue 18(48.6) 32(50.0)

0.011

9(50) 16(38.1)

0.329

27(41.5) 48(41.0)

0.155

Pneumonia 1(2.7) 13(20.3) 2(11.1) 7(16.7) 3(4.6) 20(17.1)

Bloodstream 4(10.8) 5(7.8) 4(22.2) 3(7.1) 8(12.3) 8(6.8)

Catheter-relatedbloodstream 4(10.8) 0(0) 3(16.7) 11(26.2) 7(10.8) 11(9.4)

Osteomyelitis 1(2.7) 6(9.4) 0(0) 0(0) 1(1.5) 6(5.1)

Arthritis 1(2.7) 3(4.7) 0(0) 1(2.4) 1(1.5) 4(3.4)

Intra-abdominal 1(2.7) 0(0) 0(0) 0(0) 1(1.5) 0(0)

Surgicalsite – – – – 10(15.4) 11(9.4)

Other 7(18.9) 5(7.8) 0(0) 4(9.5) 7(10.8) 9(7.7)

Comorbidities

Co-morbidities 30(81.1) 35(54.7) 0.008 15(83.3) 38(90.5) 0.419 53(81.5) 82(70.1) 0.091

Atopy 7(18.9) 8(12.5) 0.382 1(5.6) 8(19.0) 0.255^a 9(13.8) 16(13.71) 0.974

Immunosuppression 6(16.2) 5(7.8) 0.204^a 3(16.7) 3(7.1) 0.352^a 9(13.8) 9(7.7) 0.183

Chronicrenaldisease 3(8.1) 2(3.1) 0.353â 1(5.6) 3(7.1) 1.000â 4(6.2) 5(4.3) 0.493â

Neoplasia 3(8.1) 1(1.6) 0.138â 0(0) 2(4.8) 1.000â 3(4.6) 4(3.4) 0.702â

Otherco-morbidity^c 12(32.4) 13(20.3) 0.174 6(33.3) 24(57.1) 0.091 26(40.0) 45(38.5) 0.838

Cardiovasculardisease 3(8.1) 1(1.6) 0.138^a 1(5.6) 5(11.9) 0.658^a 11(16.9) 11(9.4) 0.136

Lungdisease 1(2.7) 1(1.6) 1.000â 0(0) 8(19.0) 0.091â 1(1.5) 9(7.7) 0.099â

Treatment

Surgicaltreatment 22(59.5) 43(67.2) 0.435 8(44.4) 20(47.6) 0.821 34(52.3) 71(60.7) 0.273

Outcome

Healing 21(56.8) 22(34.4) 0.028 14(77.8) 35(83.3) 0.719^a 41(63.1) 62(53.0) 0.188

Improvement 16(43.2) 40(62.5) 0.061 4(22.2) 5(11.9) 0.431^a 24(36.9) 49(41.9) 0.513

Death 0(0) 2(3.1)â 0.531â 0(0) 2(4.8) 1.000â 0(0) 6(5.1) 0.090â

POA-I,presentonadmissioninfection;HAI,healthcare-associatedinfection;MSSA,methicillin-susceptibleStaphylococcusaureus;MRSA,methicillin-resistantStaphylococcusaureus;ICU, intensivecareunit.

Signiﬁcantdifferences(p-values≤0.05)areshowninbold.

a Fisher’sexacttest.

b Mann---WhitneyU-test.

c Otherco-morbidities:cardiovasculardisease,chroniclungdisease,nervoussystemdiseases,skindiseases,malnutrition,cholestaticsyndrome,subglotticstenosis,snakebite,Rh-ABO incompatibility,shortbowelsyndrome,nesidioblastosis,laryngealandlungpapillomatosis,hemophiliaB,chronicosteomyelitis,congenitalmalformations,nephropathy,andothers.

(7)

t008, each with 9.2% (n=6) were the most frequent and t922,with6.2%(n=4),belongingtoCC1,werehighlighted.

MostimportantstrainsofMRSAwerethosebelongingtoCC8, andthemostfrequentwereCC8-SCCmecIVc-t1610(27.43%, n=31),CC8-SCCmecIVc-t008(19.47%,n=22),CC5-SCCmec I-t149(16.81%,n=19), andCC8-SCCmecIVc-t024(15.04%, n=17).

Duringthestudy period,achange wasobserved inthe mostimportantclonal complexes,both in MSSAandMRSA isolates. CC5 almost completely disappeared, while CC8 andCC45 remainedconstant. Inaddition, theincrease of otherCCswasevident,althoughnoneofthemwerespeciﬁ- callypredominant.Duringthethreeyearsofthestudy,the clonebelongingtoCC8-SCCmecIVcofMRSAwasincreasing, representingthe most prevalent,while the CC5-SCCmec I disappearedinthethirdyear.

SixteenisolatesofMSSAwereanalyzedusingPFGE,anda greatdiversitywasfoundintheresults(Fig.3A).Analysisof MRSAbyPFGEwasperformedon26isolatesandfourrelated groupswere identiﬁed.Thelargest grouphad15 isolates, which belonged to CC8, and harbored SCCmec IVc; 14 of themwerepositiveforpvlandhaddifferentspatypes(t008, 7/15;t1610,4/15;t024,3/15;t2031,1/15;Fig.3B).

Virulencefactors

Eight virulencefactor geneswere detected,both in MSSA and MRSA strains; however, higher gene prevalence was observedin MSSAcomparedtoMRSAisolates.Statistically signiﬁcant differences were observed between MSSA and MRSAstrains regarding the pvl genes (MRSA 76.9%, n=90 vs.MSSA32.3%,n=21;p=0.000),sed(MSSA33.8%,n=8vs.

MRSA10.3%,n=12;p=0.000),andsee(MSSA12.3%,n=8vs.

MRSA2.6%,n=3;p=0.018).

The presenceofpvl showedstatisticallysigniﬁcantdif- ferences between isolates with SCCmec IVc and other typesofSCCmec(SCCmecIVc95%,n=76vs.otherSCCmec 30.3%, n=10; p=0.000); It suggests that the pvl gene is morefrequentlyassociated withisolates withSCCmecIVc than SCCmec I. Genes such as sed and tst were associ- atedwithSCCmecIVa(sed,p=0.001;tst,p=0.012),while the eta gene was associated with SCCmec V (p=0.027).

No etb and arcA (ACME) genes were found in any of the isolates.

Discussion

InfectionscausedbyS.aureus(MSSA---MRSA)inchildpopula- tionscontinuetobeamajorconcern,bothinthecommunity and the hospital environment. In general, studies in the pediatric population are few, and some have limitations:

many of them focus on certain infection types and most donot present informationon infections caused by MSSA strains,whichremainrelevantandhavenotbeendisplaced byMRSAstrains.

Inthisstudy,asigniﬁcantnumberofinfectionsoccurred onpatients betweenbirthand1 yearofage (41.2%),who mainlyhadskinandsofttissueinfectionscausedbyMRSA.

Interestingly,fewstudiesonS.aureusinfectionsinchildren populationagreewiththeseﬁndings,asreportedin China byWuetal.in2010;theyfoundthat37.6%ofskinandsoft

tissueinfectionsoccurredinchildrenyoungerthan1year.¹⁹ Likewise,morbidityandmortalityreportsbytheCDCinthe United States have described an important prevalence of skinandsofttissueinfectionsinneonatalpatients.²⁰ How- ever, other publications differon infectiontypes; authors suchasIlczyszynetal.inPoland²¹andQiaoetal.inChina²² have shown that S.aureus infections in children between birthand1yeararemainlyoftheinvasivetype(bacteremia andpneumonia).²²

Likewise, results of other studies contrast with the present study regarding age; studies in Brazil²³ and Argentina,²⁴forexample,haveshownahighprevalenceofS.

aureusinfectionsinpatientsbetween2and5yearsofage, andotherstudiescarriedoutinColombia,incitiessuchas Bucaramanga²⁵ andCartagena,⁵describeahighprevalence of S. aureus infections in children agedbetween 4 and 5 yearsandbetween10and17years,respectively.

Infectionsindifferentagegroupsaredeterminedbythe particularitiesofeachpopulationandbytheriskfactorsto whichinfantsareexposed;furthermore,theinfantpopula- tionhasalessadaptedimmunesystem,whichallowsgreater colonization,morereadilydevelopedclinicalpresentations, andmorecomplications.^1,2

Inthisregard,apreviousstudy,carriedoutinMedellin during2011,revealedcolonizationfrequenciesbyS.aureus (MSSAandMRSA)of45.9%inchildrenunder2years.²⁶This is an important aspect taking into account the relation- shipbetweencolonizationandthedevelopmentofS.aureus infections,sinceitispossiblethatthefrequenciesofinfec- tion found in pediatric patientsare related,amongother aspects,tothefrequenciesofcolonizationreportedinthe city.

In particular, in this study a relationship between the antecedentofhavingstayedindaycarecentersandMRSA infectionswasfound,whichcouldbeduetothefrequency ofcolonizationpreviouslyreportedinchildrenfromMedel- líndaycarecenters.Rodríguezetal.wereabletoestablish thatthecolonizingstrainswerecloselyrelatedtothestrains causinginfectioninthecity.²⁶ Daycarecentershavebeen describedasanimportantreservoir ofthemicroorganism, becomingfavorableplacesforitsdissemination.Theseenvi- ronmentsallowtheswappingobjectsbetweenchildrenwho have poorhygienehabits, whichthereforemakesdissemi- nationmorelikely.²

According to a previous study published on S. aureus infectionsintheadultpopulationfromMedellín,²⁷thisstudy foundthatS.aureusstrainsharboringSCCmecIVc,usually associatedwiththecommunityenvironment,predominated as etiological agents of HAIs, displacing the traditional strains with the SCCmec type I that dominated in hospitals.Further,frequenciesofSCCmecIVcwerehigherinthe infantpopulationcomparedtothosereportedintheadult population(58.7%adultvs.70.8%pediatric).²⁷

This discovery shows the success of SCCmec IVc in the child population, which is a cause of great concern becauseit facilitates themaintaining of virulencefactors like Panton-Valentine leukocidin (PVL) and its dissemina- tion may be greater in this population. As described, SCCmecIVissmaller;therefore,thebiologicalcostofresis- tancedecreases,favoringitspropagationoverotherstrains.

Spreadoccursmainlyinstrainsbelongingtospeciﬁcclonal

(8)

S.aureusinfectionsinchildrenunder1yearold 387 PFGE

Strain ID Strain ID MSSA H339

MRSA P80 MRSA H135

MRSA P171

MRSA H229 MRSA H384

MRSA HP54 MRSA H324 MRSA C25 MRSA C66 MRSA P96 MRSA H287 MRSA H276 MRSA P244 MRSA H286 MRSA P219 MRSA H422 MRSA P78 MRSA P221 MRSA H271 MRSA C47 MRSA C23 MRSA H474 MRSA H426 MRSA H417 MRSA H347 MRSA H314

t008 ; YHGFMB.

t008 ; YHGFMBQBLO

t024 ; YGFMBQBLO t024 ; YGFMBQBLO t008 ; YHGFMBQBLO

8 8 8 8 8 8 8

8 8 8 8 8 8/72

5 I

IVc IVc

V V V NT NT

I IVc IVc IVc IVc IVc IVc IVc IVc IVc IVc IVc IVc

+ + + +

+ + + + + + + + +

+ + +

IVc IVc IVc

IVc

I 5 5 30 9 59 8 8 8 8 8 8 8

t1610 ; YHGFMBQBBLO t1610 ; YHGFMBQBBLO t008 ; YHGFMBQBLO

t008 ; YHGFMBQBLO

t008 ; YHGFMBQBLO t008 ; YHGFMBQBLO t008 ; YHGFMBQBLO

442 (t149) 1273 (t7279) 442 (t149) 43 (t021) 37 (t209) 17 (t216) 451 (t324) 366 (t068) 1037 (t1635) t024 ; YGFMBQBLO t008 ; YHGFMBQBLO 873 (t2031)

8 1 12 97 1/188 15

45 121

unk unk unk 45 45 45 45 152 105 (t2:67)

122 (t1:89)

207 (t3:55) 263 (t2:143)

715 (t6:30) 715 (t6:30)

411 (t6:45) 1264 (t:1445)

400 (t7:463) 400 (t7:463) 263 (t2:143) 1260 (t:5211) 151 (t2:28) 110 (t2:13) 175 (19:22) MSSA H369

MSSA H326 MSSA H430 MSSA H445

MSSA P137 MSSA H402 MSSA P228

MSSA H289 MSSA P156 MSSA H475

MSSA H309 MSSA P152 MSSA H525 MSSA C19 MSSA H493

CC

spa type SCCmec pvl

spa type

40 50 60 70

70 75 80 90 10085 95

80 90 100

PFGE

A

B

Figure 3 Genetic relatedness in MSSA and MRSA isolates. UPGMA dendrogram showing genetic relatedness in a sample of methicillin-susceptibleStaphylococcusaureus(MSSA)(A)andmethicillin-resistantStaphylococcusaureus(MRSA)(B)isolates.Dotted lineindicatesthecut-offpoint,ortheDicecoefficientof80%;thisisusedtodefinePFGEclones.Clustersabovethispercentage areconsideredtobegeneticallyrelated.IsolatesidentifiedwiththeletterHbelongtohospitalA,isolatesidentifiedwiththeletter PbelongtohospitalB,andisolatesidentifiedwiththeletterCbelongtohospitalC.

complexes such asCC8, which could beevidenced in the PFGEresults,aswasreportedpreviously.²⁸

Nonetheless, the spa types predominant in this study;

t1610, t008, t149, and t024 have been previously identi- ﬁed,mainly in MRSAstrains infecting pediatric andadult

patients,notonlyinColombiaandLatinAmericanbutalso inmanycountriesaroundtheworld.^29,30Theseﬁndingsshow the wide circulation among the general populations, and suggest thepathogenic capacity and disseminationability ofthesestrains.

(9)

Withregardtotheresistanceprofiles, adifferencewas observedbetweenthisstudyandothers,whichmaybedue todifferencesinuseofantimicrobials,eitherintheclinical setting or elsewhere. Heterogeneity confirms the impor- tanceofknowing thebehaviorof theseinfectionsin each regionandinstitution.Further,thedifferencesbetweenthe resistanceprofilesofthemainMRSAclonesreportedinthe study allow a clinical approach tothe recognition of the predominantclones,withouttheneedformoleculartyping.

Inthe present studya predominance ofPOAinfections wereobserved;however,health-careassociatedinfections continuebeingveryimportantinthepediatricpopulation.

At the same time, MSSA strains constitute an important sourceof infections;in thiscase, theyharbored avariety ofvirulencefactorsgenesincontrastwiththeMRSAstrains, anaspectpreviouslydescribed.

Theresultsofthepresentstudyrepresentvaluableinfor- mation for the knowledge of local epidemiology and the controlofpediatricinfectionsinthecity.Inaddition,they demonstratethattheepidemiologyofthismicroorganismis diverseandthat,duetotheparticularitiesofeachregion, the data cannot be extrapolated. In general, the study demonstrateda higher prevalence of SCCmec IVc in chil- drenthanadults,whichcouldindicateagreaterspreadof thechromosomalcassetteinthispopulationgroupandmay requireadditionalstudies.

Althoughthestudygroupdoesnotconstituteacomplete cohort,thepatientsandtheevaluatedisolatesaretheresult ofcarefullydesignedsampling.

Funding

Sustainabilitystrategyconsolidation processCODI(Comitê de Desenvolvimento e Pesquisa). University of Antioquia, researchGrouponBasicandAppliedMicrobiology.MICROBA (Grupo de Pesquisa em Microbiologia Básica e Aplicada) 2016---2017.

Conﬂicts of interest

Theauthorsdeclarenoconﬂictsofinterest.

References

1.Rodríguez EA, Jiménez JN. Factores relacionados con la colonización por Staphylococcus aureus. IATREIA (Medellín).

2015;28:66---77.

2.Lamaro-Cardoso J, De Lencastre H, Kipnis A, Pimenta FC, OliveiraLS,OliveiraRM,etal.Molecularepidemiologyandrisk factorsfornasalcarriageofStaphylococcusaureusandmethi- cillinresistantS.aureusininfantsattendingdaycarecenterin Brazil.JClinMicrobiol.2009;47:3991---7.

3.VanderMeeMarquetN,PoissonDM,LavigneJP,FranciaT,Tris- tanA, Vandenesch F,etal. Theincidence ofStaphylococcus aureusST8-USA300amongFrenchpediatricinpatientsisrising.

EurJClinMicrobiolInfectDis.2015;34:935---42.

4.Jimenez-Truque N, Saye EJ, Thomsen I,Herrera ML, Creech CB. Molecularepidemiologyofmethicillin-resistantStaphylo- coccus aureus in Costa Rican children. Pediatr Infect Dis J.

2014;33:e180---2.

5.Correa-JiménezO,Pinzón-RedondoH,ReyesN.Highfrequency ofPanton-ValentineleukocidininStaphylococcusaureuscausing

pediatricinfectionsinthecityofCartagena-Colombia.JInfect PublicHealth.2016;9:415---20.

6.Centerfor DiseaseControl and Prevention(CDC).Identifying healthcare-associatedinfections(HAI)forNHSNSurveillance;

2016. Available from: https://www.cdc.gov/nhsn/pdfs/

pscmanual/2pscidentifyinghaisnhsncurrent.pdf [cited 05.04.16].

7.BrakstadOG,AasbakkK,MaelandJA.DetectionofStaphylococ- cusaureusbypolymerasechainreactionampliﬁcationofthe nucgene.JClinMicrobiol.1992;30:1654---60.

8.MehrotraM,WangG,JohnsonWM.MultiplexPCRfordetection ofgenes for Staphylococcus aureusenterotoxins, exfoliative toxins,toxicshocksyndrometoxin1,andmethicillinresistance.

JClinMicrobiol.2000;38:1032---5.

9.ShopsinB,GomezM,MontgomerySO,Smith DH,Waddington M, Dodge DE, et al. Evaluation of protein A gene polymor- phicregionDNAsequencingfortypingofStaphylococcusaureus strains.JClinMicrobiol.1999;37:3556---63.

10.MathemaB,MediavillaJ,KreiswirthBN.Sequenceanalysisof thevariablenumbertandemrepeatinStaphylococcusaureus proteinAgen:spatyping.MethodsMolBiol.2008;431:285---305.

11.HarmsenD,ClausH,WitteW,RothgängerJ,ClausH,Turnwald D,etal.Typingofmethicillin-resistantStaphylococcusaureus inauniversityhospitalsettingbyusingnovelsoftwareforspa repeatdeterminationanddatabasemanagement.JClinMicro- biol.2003;41:5443---8.

12.EnrightMC,DayNP,DaviesCE,PeacockSJ.MultilocusSequence typingforcharacterizationofmethicillin-resistantmethicillin- susceptibleclonesofStaphylococcusaureus.JClinMicrobiol.

2000;38:1008---15.

13.FeilEJ, LiBC,AanensenDM,Hanage WP,SprattBG.eBURST:

inferringpatterns ofevolutionary descentamong clustersof relatedbacterialgenotypes frommultilocus sequence typing data.JBacteriol.2004;186:1518---30.

14.MulveyMR,ChuiL,IsmailJ,LouieL,MurphyC,ChangN,etal.

DevelopmentofaCanadianstandardizedprotocolforsubtyping methicillin-resistant Staphylococcusaureususing pulsed-ﬁeld gelelectrophoresis.JClinMicrobiol.2001;39:3481---5.

15.KondoY,ItoT,MaXX,WatanabeS,KreiswirthBN,EtienneJ, etal.Combination ofmultiplexPCRsforstaphylococcalcas- sette chromosomemectype assignment:rapididentiﬁcation systemformec,ccr,andmajordifferencesinjunkyardregions.

AntimicrobAgentsChemother.2007;51:264---74.

16.MilheiricoC,OliveiraDC,deLencastreH.MultiplexPCRstrategy forsubtypingthestaphylococcalcassettechromosomemectype IVinmethicillin-resistantStaphylococcusaureus:‘‘SCCmecIV multiplex’’.JAntimicrobChemother.2007;60:42---8.

17.DiepBA,GillSR,ChangRF, PhanTH,ChenJH,DavidsonMG, et al. Complete genome sequence of USA300, an epidemic cloneofcommunity-acquiredmethicillin-resistantStaphylococ- cusaureus.Lancet.2006;367:731---9.

18.McclureJ,ConlyJM,LauV,ElsayedS,LouieT,HutchinsW,etal.

NovelmultiplexPCRassayfordetectionofthestaphylococcal virulencemarkerPanton-Velentineleukocidingenesandsimul- taneousdiscriminationofMethicillin-susceptiblefromresistant Staphylococci.JClinMicrobiol.2006;44:1141---4.

19.Wu D, Wang Q, Yang Y, Geng W, Wang Q, Yu S, et al.

Epidemiology and molecular characteristics of community- associatedmethicillin-resistant and methicillin --- susceptible Staphylococcusaureusfromskin/softtissueinfectionsinachil- dren’shospital in Beijing, China. DiagnMicrobiol Infect Dis.

2010;67:1---8.

20.CenterforDiseasesControlandPrevention(CDC).Community- associated methicillin-resistant and Staphylococcus aureus infection among healthy newborns. Morb Mortal Wkly Rep.

2006;55:329---32.

21.IlczyszynMW,SabatAJ,AkkerboomV,SzkarlatA,KlepackaJ, Sowa-SierantI,etal.Clonalstructureandcharacterizationof

(10)

S.aureusinfectionsinchildrenunder1yearold 389 Staphylococcusaureusstrainsfrominvasiveinfectionsinpedi-

atric patients from South Poland: association between age, spatypes,clonalcomplexes,andgeneticMarkers.PLOSONE.

2016;11:e0151937.

22.Qiao Y, Ning X, Chen Q, Zhao R, Song W, Zheng Y, et al.

Clinicalandmolecularcharacteristicsofinvasivecommunity- acquiredStaphylococcusaureusinfectionsinChinesechildren.

BMCInfectDis.2014;14:582.

23.Gomes RT, Lyra TG, Alvez NN, Caldas RM, Barberino MG, Nascimento-CarvalhoCM.Methicillin-resistantandmethicillin- susceptibleStaphylococcus aureus infection amongchildren.

BrazJInfectDis.2013;17:573---8.

24.PaganiniH,VerdaguerV,RodriguezAC,DellaLattaP,Hernán- dezC,BerberianG, etal. Aclinicaland riskfactor analysis of methicillin-resistant community-acquired infections. Arch ArgentPediatr.2006;104:295---300.

25.ForeroMJ.Infecciones porStaphylococcus aureusadquiridas en la comunidad. Diferenciación clínica según sensibilidad y resistencia a la meticilina en el servicio de infectología pediátricadelHospitalUniversitariodeSantander.2006---2008.

Santander:UniversidaddeSantander;2009.

26.RodríguezEA,CorreaMM,OspinaS,AtehortúaSL,JiménezJN.

Differencesinepidemiologicalandmolecularcharacteristicsof nasalcolonizationwithStaphylococcusaureus(MSSA---MRSA)in childrenfromauniversityhospitalanddaycarecenters.PLoS ONE.2014;9:e101417.

27.JiménezJN,OcampoAM,VanegasJM,RodríguezEA,Mediav- illa JR, Chen L, et al. CC8 MRSA strains harboring SCCmec typeIVc arepredominant inColombian hospitals.PLoSONE.

2012;7:e38576.

28.ChambersHF, DeLeo FR. Waves ofresistance: Staphylococ- cus aureus inthe antibiotic era. NatRev Microbiol. 2009;7:

629---41.

29.MachucaMA,SosaLM,GonzálezCI.Moleculartypingandvir- ulence characteristic of methicillin-resistant Staphylococcus aureusisolatesfrompediatricpatientsinBucaramanga,Colom- bia.PLoSONE.2013;8:e73434.

30.SolaC,PaganiniH,EgeaAL,MoyanoAJ,GarneroA,KevricI, etal.SpreadofepidemicMRSA-ST5-IVcloneencodingPVLas amajorcauseofcommunityonsetstaphylococcalinfectionsin Argentineanchildren.PLoSONE.2012;7:e30487.