Epidemiology of extended-spectrum β-lactamase-producing Enterobacteriaceae among healthcare students, at the Portuguese Red Cross Health School of Lisbon, Portugal

Short

Communication

Epidemiology

of

extended-spectrum

β-lactamase-producing

Enterobacteriaceae

among

healthcare

students,

at

the

Portuguese

Red

Cross

Health

School

of

Lisbon,

Portugal

Claudine

Fournier

,

Marta

Aires

de

Sousa

,

Begoña

Fuster

Escriva

,

Leila

Sales

,

Patrice

Nordmann

,

Laurent

Poirel

*

a

MedicalandMolecularMicrobiologyUnit,FacultyofScienceandMedicine,UniversityofFribourg,Fribourg,Switzerland

b

EscolaSuperiordeSaúdedaCruzVermelhaPortuguesa(ESSCVP),Lisbon,Portugal

c_{LaboratoryofMolecularGenetics,InstitutodeTecnologiaQuimicaeBiológica(ITQB)AntónioXavier,UniversidadeNovadeLisboa(UNL),Oeiras,Portugal} d

INSERMEuropeanUnit(IAME,France),UniversityofFribourg,Fribourg,Switzerland

e

SwissNationalReferenceCenterforEmergingAntibioticResistance(NARA),UniversityofFribourg,Fribourg,Switzerland

f

InstituteforMicrobiology,UniversityofLausanneandUniversityHospitalCentre,Lausanne,Switzerland

ARTICLE INFO Articlehistory: Received6April2020

Receivedinrevisedform18June2020 Accepted1July2020

Availableonline11July2020 Keywords: Portugal ESBL Enterobacteriaceae Carriage Healtcareworkers ABSTRACT

Objective:Theaimofthepresentstudywastoprospectivelyevaluatetheprevalenceofintestinalcarriageby

extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae among Portuguese students

attendingaBachelors’courseinhealthcare,andtodeterminethemolecularfeaturesofESBL-producing

isolates.

Methods:One-hundredandelevenfaecalsamplesrecoveredfromPortuguesehealthcarestudentswere

screenedforeitherESBL-producing,carbapenem-resistant,colistin-resistantor

pan-aminoglycoside-resistantEnterobacteriaceae,usingrespectivescreeningmedia.Allrecoveredisolatesweretestedfor

antimicrobialsusceptibilityandcharacterisedbypulsed-fieldgelelectrophoresis(PFGE)andmultilocus

sequencetyping(MLST).

Results: Atotalof 17ESBL-producingEnterobacteriaceae (16Escherichia coli andasingle Klebsiella

pneumoniae)wererecoveredfrom16students,representingaprevalenceof14.5%.TheE.coliisolateswere

distributedintothreesequencetypes(STs)andsevenPFGEtypes.ThemostcommonESBLidentifiedwas

CTX-M-1(n=13;76%),followedbyCTX-M-15(n=3;18%)andCTX-M-8(n=1;6%).Themajorityofthe

strainswereresistanttosulfonamides(88%)andfosfomycin(71%).Resistancetoaminoglycosideswas

observedatalowrate,thatis12%forbothtobramycinandkanamycin.Nocolistin-,carbapenem-or

pan-aminoglycoside-resistantisolateswererecovered.Amajorclone,ST10-blaCTX-M-1,included12E.coli

isolates.TheblaCTX-M-1genewasalwayslocatedonanIncFIA/FIBplasmidtype,co-harbouringgenes

encodingresistancetotetracycline,sulfonamides,trimethoprim–sulfamethoxazoleandfosfomycin.

Conclusion:ThemostcommonlyidentifiedESBLgeneinE.coliwasblaCTX-M-1,usuallyidentifiedamong

ESBL-producingisolatesrecoveredfromanimals.AhighprevalenceoffaecalcarriageofESBL-producingE.

coliwasfoundamonghealthyhealthcarestudents,underlyingthispopulationasanimportantreservoir.

©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobial

Chemotherapy.This is an open access article underthe CCBYlicense (http://creativecommons.org/licenses/

by/4.0/).

1.Introduction

Thespreadofextended-spectrumβ-lactamase(ESBL) produc-ing Enterobacteriaceae has been considered as a global threat worldwideforthelast20years[1].ESBLsconferresistancetomost

β-lactam antibiotics, including penicillins and broad-spectrum cephalosporins, with only carbapenems being spared by those enzymes[2].

ESBL-producingEscherichiacoliareamajorcauseofcommunity andnosocomialacquiredinfections[3],whereasESBL-producing Klebsiellapneumoniaearemainlyhospital-acquiredpathogens[4]. Whereasuptothe1990s,ESBLsweremainlyencodedbytheblaTEM

and blaSHV genes, currently the most prevalent ESBL enzymes

recoveredfromhumansbelongtotheCTX-Mfamily,with CTX-M-15beingthemostfrequentESBLdeterminantidentifiedworldwide [5–7].

* Correspondingauthorat:MedicalandMolecularMicrobiologyUnit, Depart-mentofMedicine,FacultyofScience,UniversityofFribourg,CheminduMusée18, CH-1700Fribourg,Switzerland.

E-mailaddress:laurent.poirel@unifr.ch(L.Poirel). http://dx.doi.org/10.1016/j.jgar.2020.07.004

2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCCBY license(http://creativecommons.org/licenses/by/4.0/).

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

In Portugal, theproportion of ESBL-producing E.coli and K. pneumoniaeisolatescausinginvasiveinfectionswasestimatedin 2017at15.7%and44.9%,respectively[8].Althoughseveralstudies onESBL-producingEnterobacteriaceaehavebeendoneinPortugal, most have only focussed on clinical isolates (recovered from hospitalsornursinghomes).Theseactuallyshowed,similarlyto manyplaces worldwide,a predominanceof CTX-M-15and the occurrence of CTX-M-1 at a low rate [3,9–12]. A single study performedmorethan10yearsagoevaluatedthecarriageofESBL producersinthecommunity.TheprevalenceofESBL-producingE. coliamong healthychildren was 2.7% (threeisolates producing CTX-M-1, TEM-52and SHV-12only, respectively) [13]. Another studyconductedin2014amonghealthyadultsidentifiedfourE. coli ESBL-positive isolates, producing CTX-M-14 and CTX-M-27 [14]. However, no study had been designed to prospectively evaluatetheprevalenceofthecarriageofESBLproducersinthe community.

The aim ofthe presentstudy wastoprospectivelyevaluate theprevalence of intestinal carriage by ESBL-producing Enter-obacteriaceae as well as other multidrug-resistant isolates amonga group of Portuguese students attending a Bachelors’ courseinhealthcare,andtodeterminetheepidemiologicaland antimicrobial resistance features of those ESBL-producing iso-lates.

2.Methods 2.1.Sampling

BetweenMarchandMay2018,111rectalswabswerecollected from111studentsatEscolaSuperiordeSaúdedaCruzVermelha Portuguesa(ESSCVP)inLisbon,Portugal.Studentswereattending eitherthefirst,second,third orfourthyearoftheirdegrees.Of note, half of the students (n=55) degrees included a clinical internship, and subsequently had contact withhospitals, long-termcarefacilitiesorcommunitypatients.

Thestudywasexplainedorallyintheclassroomandasterile swabwithtransportmediumandaquestionnaireweredistributed toallthestudents.Thequestionnairesincludeddemographicdata, andriskfactorsforcolonisationwithmultidrug-resistantbacteria suchashospitalisationduringthelast6months,antibiotherapyat thetimeofsamplingorduringtheprevious2months,attendance attheclinicalinternship,contactwithisolationpatients,contact withanimalsandtravellingabroad.Allindividualswhoaccepted toparticipatereturnedtherectalswabsampleandthecompleted questionnaire. The protocol was approved by the Direction of ESSCVPandawritteninformedconsentwasobtainedfromeach participant(allwereagedover18years).

2.2.Bacterialisolates

Rectal swabswere enrichedovernight in 5mL Luria–Bertani (LB)broth.Tenmicrolitresofeachsamplewereplatedontofive differentselectivemedia:ChromIDESBL1_{(bioMérieux,}

Balme-La-Grotte, France), SuperPolymyxin, SuperAminoglycoside, Super-Carba and CarbaSmart1 (bioMérieux) in order to respectively selectforESBLproducers,colistin-resistant, pan-aminoglycoside-resistant and carbapenem-resistantEnterobacteriaceae. Species identification was performed onall selected isolates using the API20Esystem(bioMérieux).

2.3.Susceptibilitytesting

Antimicrobialsusceptibilitytestingwas performedusingthe discdiffusion methodon Mueller–Hinton agar plates (Bio-Rad,

Cressier,Switzerland)foramoxicillin,amoxicillin/clavulanicacid, piperacillin, piperacillin/tazobactam, ticarcillin, ticarcillin/clavu-lanic acid, temocillin, cephalotin, ceftazidime,cefotaxime, cefe-pime, cefoxitin,aztreonam, ertapenem, imipenem, meropenem, amikacin, gentamicin, tobramycin, kanamycin, fosfomycin, nali-dixicacid, chloramphenicol,tetracycline, colistin,sulfonamides, trimethoprim–sulfamethoxazole(SXT),ciprofloxacinand tigecy-cline,followingEuropeanCommitteeonAntimicrobial Suscepti-bility Testing (EUCAST) guidelines. Colistin resistance was evaluatedbybrothmicrodilutionaccordingtoEUCAST.Identi fica-tionofESBLproducerswasconfirmedwiththeRapidESBLNPtest [15].

2.4.Molecularcharacterisation

Identification of ESBL encoding genes was performed by polymerase chain reaction (PCR), as previously described [16], followed bysequencingof theamplicons (Microsynth,Balgach, Switzerland).

Theclonalrelationshipoftheisolateswasevaluatedby pulsed-field gel electrophoresis (PFGE) [17], and multilocus sequence typing (MLST), as previously described [18,19]. Sequence types (STs)wereassignedusingtheMLSTdatabasesforK.pneumoniae andE.coli(https://cge.cbs.dtu.dk/services/MLST-2.0/).

2.5.Transformationexperimentsandplasmidcharacterisation Plasmids carrying blaCTX-M and blaTEM geneswere extracted

fromtheoriginalisolatesusingtheKiesermethod[20]followedby electrotransformation. Escherichia coli TOP10 was used as the recipient and transformants were selected on LB agar plates supplemented withcefotaxime (1

m

transformantsastemplatestofurtherassessthepresenceofthe expectedgene.Plasmidsizewasevaluatedfromthetransformants using the Kieser method followed by electrophoresis. An incompatibilitygroupof theplasmids was determinedby PCR-basedreplicontyping(PBRT)oneachtransformant[21]. 2.6.Statisticalanalysis

Riskfactorswereassessed usingSPSSsoftware,version 21.0 (IBMPortugal,Lisbon,Portugal).Theχ2_{andFishertestswereused}

toidentifyvariablesassociatedwiththecarriageofESBLproducers andcolistin-resistantisolates(gender,age,schoolingyear, previ-ous hospital admission, current or previous antibiotherapy, attendance at the clinical internship, contact with isolation patients, contact with animals and travelling abroad). P-values of0.05wereconsideredstatisticallysignificant.

3.Results

3.1.Studypopulation

Amongthe111studentsscreened,85attendedtheBachelor'sin nursingand26werestudyingphysiotherapy.Theywere distrib-utedoverthe4yearsofschooling(Table1).Mostofthestudents werefemale(n=97;87%),andthemeanagewas25.7years.Onlya fewparticipants(n=3)hadbeenadmittedtoahospitalduringthe previous 6-monthperiod, and 11 (10%) wereunder current or previous(last6months)antibiotherapy.Halfofthestudentshad alreadyattendedtheclinicalinternship,and44%hadhadcontact withpatients.Regularcontactwithanimals(cat,dog,rabbit,sheep, goat)wasobservedfor17%oftheindividualsandtravellingabroad wasrecordedin30%.

3.2.PrevalenceofESBLproducersandriskfactors

Ofthe111samplescollectedfromthehealthcarestudents,17 ESBL-producingenterobacterialisolateswererecovered,ofwhich 16wereE.coliand onewas K.pneumoniae,correspondingtoa prevalence rate of 14.5%. Nocarbapenem-resistant, pan-amino-glycoside-resistantor colistin-resistantEnterobacteriaceae were isolated.

Amongthedifferentvariablesanalysed(Table1),attendingthe firstyearofschoolingofnursingorphysiotherapywassignificantly associated with intestinal carriage of ESBL producing Enter-obacteriaceae(P=0.009).

3.3.ESBLdeterminants

ThemostcommonlyidentifiedESBLamongE.coliwasCTX-M-1 (n=13, 76%),followed by CTX-M-15(n=2)and CTX-M-8 (n=1) (Table 2).The singleK.pneumoniae isolaterecovered produced CTX-M-15. This latter isolate, as well as the only CTX-M-15-producingE.coli,co-producedthenarrow-spectrumβ-lactamase, TEM-1.

PBRT analysis showed that the blaCTX-M-1 gene was either

located on IncFIA/FIB (n=11), IncFIC (n=1) and IncI1 (n=1) plasmidtypes,theblaCTX-M-8geneontheFIA/FIBplasmidtypeand

theblaCTX-M-15geneeitheronFIA/FIB,FICandPplasmidtypes.In

most cases (59%), plasmids carrying the blaCTX-M-1 gene

co-harboured genes encoding resistance to tetracycline, sulfona-mides,fosfomycinandSXT.

3.4.Susceptibilitytesting

Antimicrobialsusceptibilitytestingshowedthatamajorityof theESBL-producingisolateswereresistanttocefotaxime(94%), ceftazidime(94%), cefepime (100%), tetracycline (76%), sulfona-mides (88%), SXT (82%) and fosfomycin (71%). Resistance to

aminoglycosideswasobservedatalowrate,thatis12%forboth tobramycinand kanamycin.Allisolatesremainedsusceptibleto carbapenems,colistinandchloramphenicol.

3.5.Clonalrelationship

PFGEanalysisdistributedthe16ESBL-positiveE.coliisolates intosevenPFGE types(Fig.1)and threeSTs wereidentifiedby MLST(Table2),namelyST10(n=13),ST130(n=2)andST38(n=1). The unique ESBL-producing K. pneumoniae isolate belonged to ST348.

4.Discussion

The present study identified a prevalence of 15% ESBL-producing Enterobacteriaceaeamongthehealthcarestudents at a teaching university in Lisbon, Portugal. This rate may be consideredveryhigh,owingtothescreenedpopulation.Indeed, theseindividualswereforthemostpartyoung,healthypeople, whodidnotpresent(forthemajority)significantwell-established risk factors (travelling in endemic countries, hospitalisation, antibiotic selective pressure). This rate is twice as high when comparedwitharecentstudyperformedinSpain,aneighbouring country) which focussed on hospitalised patients screened at admission(7.7%),despitethefactthatthoseindividualspresented additionalriskfactors,beingunhealthyandolder(meanageof69 yearsold)[22].Thishighratemightbeinpartrelatedtothespread of asinglecloneamongthose students,possiblyresultingfrom cross-contaminations or contamination from a unique and unknownsource.Thisprevalenceratewasmuchlowerthanthat observedin Indiawith34%of thehealthypopulationattending hospitalforregularcheck-upsbeingcolonisedbyESBL-producing Enterobacteriaceae[23].

Overthe17ESBL-producingisolates,allbutonecorresponded toE.coli,withonlyasingleK.pneumoniaebeingidentified.

CTX-M-Table1

CharacteristicsofstudentsenrolledintheprospectivestudyandriskfactorsforintestinalcarriageofESBL-producingEnterobacteriaceae.

Nursing(n=85) Physiotherapy(n=26) Total(n=111) CarriageofESBLproducers P-value

Yes(n=17) No(n=94) Gender Male 13 1 14 3 11 0.422 Female 72 25 97(87%) 14(82%) 83(88%) Age(years) Mean 26.5 23.2 25.7 Schoolingyear First 40 7 47(42%) 14(82%) 33(35%) 0.009 Second 18 0 18(16%) 1(6%) 17(18%) Third 11 19 30(27%) 1(6%) 29(31%) Fourth 16 0 16(14%) 1(6%) 15(16%)

Previoushospitaladmission

Yes 2 1 3(3%) 0 3(3%) 0.455

No 83 25 108 17 91

Currentorpreviousantibiotherapy

Yes 9 2 11(10%) 2(12%) 9(10%) 0.781

No 76 24 100 15 85

Clinicalinternshipattendance

Yes 37 18 55(50%) 4(25%) 44(46%) 0.034

No 48 8 56 12 51

Contactwithisolatedpatients

Yes 45 4 49(44%) 4(25%) 45(47%) 0.096

No 40 22 62 12 50

Contactwithanimals

Yes 11 8 19(17%) 3(19%) 16(17%) 0.851

No 74 18 92 13 79

Travelabroad

Yes 23 10 33(30%) 3(19%) 30(32%) 0.299

No 62 16 78 13 65

1wasthemostcommonlyidentifiedESBL(76%)whereas CTX-M-15(18%)wasmuchlessprevalent.Thissituationissurprisingas what is observed in most European countries is actually the opposite, with CTX-M-15 producers over-predominating [24]. Conversely,CTX-M-1 producersarecommonlyobserved among animalsinmanyEuropeancountries,beingthemostcommonly identifiedESBLinmanydifferentfood-producinganimals(poultry, cattle,swine)[25–28].

Sequence typing identified three different STs among the recoveredE.coliisolates,namelyST10,ST131andST38.ThoseSTs arecommonlyidentifiedamonghumanisolates,withST10beinga commensallineagethatisalsooftenrecoveredfromanimals.By contrast,ST131ismainlyfoundinhumansandrelatedto extra-intestinalinfectionsbutalsoincarriageinhealthyindividuals[29]. AmongthetwostudentscolonisedwithanST131E.coli,there were different particularities that could be noted; one of the students colonised with the E. coli blaCTX-M-1-ST131 was a

vegetarian,hadtravelledinIndia,ItalyandIrelandduringthe 6-monthperiodpriortothestudy,wasattendingtheirsecondyearof schooling,andhadcontactwithhospitalisedpatientsduringtheir clinicalinternship.ThesecondindividualcolonisedwithanST131 E.coli(beingaCTX-M-8producer)wasafourth-yearstudentwho alsohadcontactwithpatients.

RegardingtheCTX-M-15-producingE.coliisolates,theywereof theST38type,whichisnotsurprisingconsideringthatthisclonal backgroundhasbeenreportedtobethesecondmostprevalentone producingCTX-M-15afterST131[30].TheCTX-M-15-producingK. pneumoniaewasanST348,anSTthatwasshowntobethesourceof urinarytractinfectionsinbothhumansandcompanionanimalsin Portugal[31].

The most commonly identified clone was ST10-blaCTX-M-1,

correspondingto12E.coliisolates.Surprisingly,11outofthose 12isolateswererecoveredfrom11studentsintheirfirstyearof study,withonlyasingleisolatefromastudentattendingthethird year.Thisresultwasverysurprising,asourprimaryhypotheses werethateithernodifferencewouldbeobservedamongallthe studentsduringallthestudentyears,oranincreasedESBLrate mightbeobservedalongtheyearsincaseadifferencewouldbe noticed.Thislatterhypothesiswouldhavecorrelatedwithsome previousstudiesshowingthathealthcareworkersincontactwith patientsbeingcolonisedwithESBLproducersareathigherriskof beingcolonisedwithESBLproducers[32].Heretheoppositewas observed, with most students in contact with patients (ESBL colonisationstatusunknown)beingnegativeforESBLproducers. Thereasonwhysuchahighproportionoffirst-yearstudentsare significantlycolonisedremainsintriguing.Suchahighproportion ofcolonisationwithESBLproducersmaybealsoidentifiedinthe generalandthehealthypopulationinPortugal.Itisimportantto highlight that those first-yearstudents werefor themost part colonisedwithasingleclone,belongingtoST10,whichisavery commonbackground,E.colibeingfoundnotonlyinhumans,but alsoinanimalsandinfood.Therefore,wemightspeculatethatthe sourceofcolonisationofstudentsbytheST10-blaCTX-M-1couldbe

food-related,consideringtheyactuallysharesomecommonmeals wheneatingattheuniversitycafeteria.

TheblaCTX-M-1geneofthismajorclonewaslocatedona56-kb

IncFIA/FIBplasmidthatco-harbouredgenesencodingresistanceto tetracycline, sulfonamides, SXT and fosfomycin. Besides the worldwide dissemination of E. coli CTX-M-1 in animals, an increasing prevalence of blaCTX-M-1-positive E. coli in human

communitieshaslatelybeendescribedinMediterraneancountries includingSpain(2%in2008[33])andPortugal(1%in2009[34]). Wemightthereforehypothesisethatatleastpartofwhatwehave shownisrepresentativeofwhatishappeninginthecommunity.Of note, data collected about antibiotic consumption among the subjectsstudied showed that littleantibioticselective pressure

Table2

GeneticandphenotypicfeaturesassociatedwiththeESBL-producingisolates.

Isolate Species ST Resistancedeterminants PFGE Plasmidincompatibility Plasmidsize(kb) Resistancephenotypetransformantsa

AA1 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA2 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA3 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA5 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA6 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA8 E.coli ST10 blaCTX-M-1 B IncFIA/FIB 84 TET,KMN,TMN,SUL,SXT,FOS

AA10 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA11 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AA13 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

AB3 E.coli ST10 blaCTX-M-1 C IncI1 84 TET,SUL,SXT,FOS

AB14 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

BB8 E.coli ST131 blaCTX-M-1 D IncFIC 76 Noco-resistance

CA1 E.coli ST10 blaCTX-M-1 A1 IncFIA/FIB 56 TET,SUL,SXT,FOS

DA11 E.coli ST131 blaCTX-M-8 A2 IncFIA/FIB 56 SUL,SXT

AB13 E.coli ST10 blaCTX-M-15, blaTEM-1 E IncFIA/FIB 61 TET,KMN,TMN

KA1 E.coli ST38 blaCTX-M-15 F IncFIC 23 SUL,SXT

KA7 K.pneumoniae ST348 blaCTX-M-15, blaTEM-1 Kp IncP 319 SUL,FOS

ESBL,extended-spectrumβ-lactamase;PFGE,pulsed-fieldgelelectrophoresis;ST,sequencetype.

Underlinedtermsindicatethoseresistancedeterminantsbeingco-localizedtogetherwithESBLencodinggeneonasameplasmid.

a_{Antibioticabbreviations:FOS,fosfomycin;KMN,kanamycin;SUL,sulfamide;SXT,trimethoprim–sulfamethoxazole;TET,-tetracycline;TMN,tobramycin.}

Fig.1.Pulsed-fieldgelelectrophoresisoftheESBL-positiveE.coliisolates.Sal, Salmonellabraenderupasmolecularsizestandard;AXX,first-yearnursestudent; BXX,second-yearnursestudent;CXX,third-yearnursestudent;DXX,fourth-year nursestudent;KA,first-yearphysiotherapystudent.

existedrulingoutsuchanexplanationforaputativecolonisation byresistantisolates.

In conclusion, our study revealed a very high rate of colonisation by ESBL-producing E. coli (15%) within a young healthypopulation.Asurprisingfindingwasthehighrateofthe detectionofCTX-M-1producersidentified,contrastingwiththe lowrateofCTX-M-15producersusuallyidentifiedamonghealthy populationsworldwide.

Funding

ThisworkhasbeenfundedbytheUniversityofFribourgandthe SwissNationalScienceFoundation(projectFNS-407240_177381). Itwas alsopartlysupportedbytheprojectPTDC/DTP-EPI/0842/ 2014fromFundaçãoparaaCiênciaeaTecnologia(FCT),Portugal. Competinginterests

Nonetodeclare. Ethicalapproval

Aconsentformwascompletedbyeachparticipant. References

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