Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period

(1)

w w w . e l s e v i e r . e s / e i m c

Original

article

Virulence

and

resistance

determinants

of

Klebsiella

pneumoniae

isolated

from

a

Portuguese

tertiary

university

hospital

centre

over

a

31-year

period

Cátia

Caneiras

a,b,∗

_,

_Luís

_Lito

c

_,

_Sagrario

_{Mayoralas-Alises}

d,e

_,

_Salvador

_Díaz-Lobato

f,g

_,

José

Melo-Cristino

c,h,i

_,

_Aida

_Duarte

a

a_Microbiology_and_Immunology_Department,_{Interdisciplinary}_Research_Centre_Egas_Moniz_(CiiEM),_Faculty_of_Pharmacy,_University_of_Lisbon,_1649-003,_Lisbon,_Portugal b_Institute_of_{Environmental}_Health_(ISAMB),_Faculty_of_Medicine,_University_of_Lisbon,_1649-028,_Lisbon,_Portugal

c_Laboratory_of_{Microbiology,}_Centro_Hospitalar_Lisboa_Norte,_1649-035,_Lisbon,_Portugal d_{Pneumological}_Department,_Moncloa_University_Hospital,_28008,_Madrid,_Spain e_European_University,_28108,_Alcobendas,_Madrid,_Spain

f_{Pneumological}_Department,_Ramón_y_Cajal_University_Hospital,_Madrid,_Spain

g_Institute_Ramón_y_Cajal_for_Health_Research_(IRYCIS),_Alcalá_de_Henares_University,_28034,_Madrid,_Spain h_Institute_of_{Microbiology,}_Faculty_of_Medicine,_University_of_Lisbon,_1649-028,_Lisbon,_Portugal

i_Institute_of_{Microbiology,}_Institute_of_Molecular_Medicine,_Faculty_of_Medicine,_University_of_Lisbon,_Lisbon,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received20June2018 Accepted2November2018 Availableonline12December2018 Keywords: Klebsiellapneumoniae Virulence Beta-lactamases Bacterialinfections Cross-infection

a

b

s

t

r

a

c

t

Introduction: The rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniaeproducingextended-spectrum␤-lactamasesandcarbapenemasesinKlebsiellapneumoniae isoneofthemostsigniﬁcantthreatstopublichealth.However,questionsandcontroversiesregarding theinteractionsbetweenresistanceandvirulenceinmultidrug-resistantK.pneumoniaeisolatesremain unclear.

Methods:Aretrospectivecohortstudywasperformedwith100K.pneumoniaeisolatesrecoveredfroma tertiarycareuniversityhospitalcentreinLisbonovera31-yearperiod.Resistanceandvirulence deter-minantswerescreenedusingmolecularmethods(PCR,M13-PCRandMLST).

Results:Thepredominantvirulenceproﬁle(ﬁmH,mrkDv1,khe)wassharedbyallisolates,indicativeofan

importantroleoftype1and3fimbrialadhesinsandhaemolysin,regardlessofthetypeof␤-lactamase produced.However,accumulationofvirulencefactorswasidentifiedinKPC-3-producers,withahigher frequency(p<0.05)ofcapsularserotypeK2andiucCaerobactinwhencomparedwithnon-KPC-3 ␤-lactamasesorcarbapenemases.Additionally,9differentvirulenceprofileswerefound,indicatingthat theKPC-3carbapenemaseproducersseemtoadaptsuccessfullytothehostenvironmentandmaintain virulenceviaseveralpathways.

Conclusion:Thisstudydescribesanoverlappingofmultidrug-resistanceandvirulencedeterminantsin ST-14K2_KPC-3_K._pneumoniae_clinical_isolates_that_may_impose_an_additional_challenge_in_the_treatment

ofinfectionscausedbythispathogen.

Determinantes

de

virulencia

y

resistencia

en

Klebsiella

pneumoniae

aisladas

en

un

centro

hospitalario

universitario

en

Portugal

durante

31 a ˜

nos

Palabrasclave: Klebsiellapneumoniae Virulencia

Beta-lactamasas

r

e

s

u

m

e

n

Introducción:LarápidaycomplejaevolucióndelosmecanismosderesistenciadeKlebsiellapneumoniae productoradebeta-lactamasasdeespectroextendidoycarbapenemasasenKlebsiellapneumoniaees unadelasamenazasmásimportantesparalasaludpública.Sinembargo,aunexistecontroversiasobre lainteracciónentrelaresistenciaylavirulenciaenaisladosdeK.pneumoniaeresistentesamúltiples antimicrobianos.

∗ Correspondingauthor.

E-mailaddress:ccaneiras@gmail.com(C.Caneiras).

https://doi.org/10.1016/j.eimc.2018.11.001

(2)

Infeccionesbacterianas Infeccióncruzada

Métodos: Serealizóunestudiodecohorteretrospectivocon100aisladosdeKlebsiellapneumoniaede uncentrohospitalariouniversitarioenLisboadurante31a ˜nos.Losdeterminantesdelaresistenciay virulenciaserastrearonutilizandométodosmoleculares(PCR,M13-PCRyMLST).

Resultados: Todoslosaisladoscompartíanunperﬁldevirulenciapredominante(ﬁmH,mrkDv1,khe),lo

queindicaunpapelimportantedelasadhesinasfimbrialesdetipo1y3,ydelahemolisina, indepen-dientementedeltipode␤-lactamasaproducida.Sinembargo,laacumulacióndefactoresdevirulencia delserotipocapsularK2ylaaerobactinaiucCseidentificóconunamayorfrecuenciaenlascepas produc-torasdeKPC-3(p<0,05)encomparaciónconlasproductorasdeotras␤-lactamasasocarbapenemasas. Además,seencontraron9perfilesdevirulenciadiferentes,indicativosdequelascepasproductorasde carbapenemasaKPC-3parecenadaptarseconéxitoalentornoymantenerlavirulenciaporvariasvías. Conclusión:Esteestudiodescribelauniónderesistenciaamúltiplesantimicrobianosjuntocon determi-nantesdevirulenciaenaisladosclínicosdeK.pneumoniaeST-14K2_KPC-3_lo_que_puede_suponer_un_desafío

adicionaleneltratamientodeinfeccionescausadasporestepatógeno.

Introduction

Klebsiellapneumoniaeisaleadingcauseofhealthcare-associated infections(HAIs),mainlyresponsibleforurinary,respiratoryand bloodstreaminfectionsandisnowrecognizedasanurgentthreat topublichealth.1_Since_the_ﬁrst_{broad-spectrum}_{␤-lactamase}

TEM-1,therapidevolutionofextended-spectrum␤-lactamases(ESBL) andcarbapenemases,areoneofthemostsigniﬁcantepidemiologic changes ininfectious diseases.2 _In _Portugal, _the _ﬁrst

carbapen-emasewas reported in 20093 _and _an _important _{hospital-wide}

disseminationhasbeenreportedpreviously.4_However,_the

molec-ulardeterminantsofvirulenceandresistancewerenotdescribed todate.

FactorsthatareimplicatedinthevirulenceofK.pneumoniae iso-latescanincludethecapsularserotype,iron-scavengingsystems, ﬁmbrialandnon-ﬁmbrialadhesins,5_which_have_an_important_role

inthedevelopmentoftheinfection.6_This_knowledge_can_be

fun-damentaltosupporteffortstocontrolthethreattohumanhealth posedbythisbacteriumwiththepurposeofrecognizeor under-standthe emergence of clinically important clones within this highlygeneticallydiversespecies.7 _Despite_this,_there_remains_a

lackofdataregardingthevirulencegenescarriedbyK.pneumoniae producingbeta-lactamases8_and,_particularly_little_is_known_about

thevirulencepotentialofKPC-producers.9

Theaimofthisstudywasevaluatethecorrelationofbacterial resistanceandvirulencedeterminantsamongK.pneumoniae iso-latescollectedoveraperiodof31yearsinasingletertiary-care universityhospitalcentre.

Methods

Hospitalcentresettingandbacterialisolates

TheK.pneumoniae isolatesselected forthis study were col-lectedbetween1980and 2011frompatientshospitalizaedat a tertiary care university hospital centre located in Lisbon, with approximately 1100 beds that provides direct care to a pop-ulation of 370,000 people. All isolates were recovered from inpatients using standard operating procedures and identiﬁed as␤-lactamase-producersbythelaboratory,usingconventional methodsorautomatedsystemslikeVitek2®(BioMérieux,Marcy, l’Étoile,France)orMicroScan® (Snap-on,Kenosha,WI,USA).The isolateswerealsosenttotheMicrobiologylaboratoryforadditional studies.AllisolatesweremaintainedfrozeninBHIbrothplus15% glycerolat−80◦_C._For_analysis,_the_strains_were_grown_in_BHI_Broth

for18hat37◦CandseededinnutrientagarorLBagar.

TheK.pneumoniaestrainswereselectedaccordingtothe iso-lationperiodandthebeta-lactamasetypeproduced(determined bypreliminaryantibioticsusceptibilityproﬁle)andwithinthis,by randomselection.TheselectedK.pneumoniaeisolateswerethen classiﬁedin4groups:groupA(1980–1989,15strainssusceptible to ␤-lactams, excluding aminopenicillins, indicative of broad-spectrum␤-lactamases–BSBLproduction);groupB(1990–2001, 12 strainswithresistancetoceftazidimeand reduced suscepti-bilitytocefotaxime, indicative of TEM-typeextended spectrum ␤-lactamases–ESBLproduction),groupC(2002–2008,48strains withresistancetoceftazidimeandcefotaxime, indicativeofthe production of CTX-M-type extended spectrum ␤-lactamases – ESBL); groupD (2009–2011),27 strainswithresistanceto car-bapenems,indicativeofcarbapenemaseproduction.

Amongthe100isolatesthatwerecharacterizedinthisstudy, 88wereclinicalisolates:urine(n=31);blood(n=21),pus(n=17), sputum(n=17),catheter(n=1)andcerebrospinalﬂuid(n=1).The remaining12isolates,includedingroupA,werefromonecolonized healthcareprofessional(n=1),colonizedpatients(n=7)and hos-pitalenvironmentalisolateslikesurfacesandmedicalequipment (n=4).Theisolateswererecoveredin26wardsorICUs,mainlyin medicalandcardiothoracicsurgerywards.

Antimicrobialsusceptibilitytesting

Antimicrobialsusceptibilitytesting wasperformedfollowing theEuropeanCommitteeonAntimicrobialSusceptibilityTesting (EUCAST) guidelinesbythe standardizeddiskdiffusionmethod in Mueller-Hinton agar medium. Quality controls were carried outinaccordancewithEUCAST(version6.0,2016), andClinical andLaboratoryStandardsInstitute(CLSI)guidelines(M100-S20), namelyEscherichiacoliATCC25922andEscherichiacoliATCC35218. Allisolatesweretestedwiththesamepanelofantibioticsas fol-lows:amoxicillin/clavulanicacid(20␮g/10␮g),cefoxitin(30␮g), cefotaxime(5␮g),ceftazidime(10␮g),imipenem(10␮g), gentam-icin(10␮g),andciproﬂoxacin(5␮g).Isolatesbelongingtogroup B,CandDwerealsotestedfortigecycline(15␮g)andfosfomycin (200_␮g)inordertoevaluatetheroleoftheseantibioticsas alter-nativetherapeuticoptions.

Theinhibition zones were interpretedaccording toEUCAST, withtheexceptionoffosfomycin,thatwasinterpretedfollowing theCLSIguidelines.Theisolateswerecategorizedassusceptible (S),intermediate(I)andresistant(R)byapplyingthebreakpoints inthephenotypictestresults.Theimipenemminimuminhibitory concentration(MICs)wasdetermined bytheagargradient test (E-test®,Biomérieux,France)followingtheEUCASTguidelines.

(3)

Table1

Primersusedinthisstudyforthedetectionofbeta-lactamasesandvirulencegenes.

Gene DNAsequence(5to3) Ampliconsize(bp) EMBLaccessionnumber(Genbank)

blaTEM F:GAAAGGGCCTCGTGATAC R:TTACCAATGCTTAATCAGTGA 1058 HM749966 blaNDM F:TATCGCCGTCTAGTTCTGCTG R:ACTGCCCGTTGACGCCCAAT 871 AB604954 K2A F:CAACCATGGTGGTCGATTAG R:TGGTAGCCATATCCCTTTGG 531 EF221827 ﬁmH F:TGTTCACCACCCTGCTGCTG R:CACCACGTCGTTCTTGGCGT 512 NC012731.1 mrkDV1 F:CGGTGATGCTGGACATGGT R:CCTCTAGCGAATAGTTGGTG 300 EU682505.2 mrkDV2-4 F:CTTAATGGCGMTGGGCACCA R:TCATATGCGACTCCACCTCG 950 AY225463.1AY225464.1AY225465.1 khe F:TGATTGCATTCGCCACTGG R:GGTCAACCCAACGATCCTGG 428 NC012731.1 iucC F:GTGCTGTCGATGAGCGATGC R:GTGAGCCAGGTTTCAGCGTC 944 NC005249.1 rmpA F:ACTGGGCTACCTCTGCTTCA R:CTTGCATGAGCCATCTTTCA 516 NC012731.1 magA F:TCTGTCATGGCTTAGACCGAT R:GCAATCGAAGTGAAGAGTGC 1137 NC012731.1

F:forwardprimer;R:reverseprimer.

Resistanceandvirulencedeterminants

PCRbasedscreeningforthemostcommonlyfound␤-lactamase families was performed with speciﬁc primers (blaSHV, blaDHA, blaCMY,blaCTX-M)includingcarbapenemasegenes(blaKPC,blaIMP, blaVIMandblaOXA).10ThevirulencefactorswereassessedbyPCR

withspecificprimersforK2serotype(K2A),fimbrialadhesinstype 1(fimH)andtype3(mrkDv1andmrKDV2-4),haemolysin(khe),

aer-obactin(iucC),mucoid(rmpA)andhypermucoviscosityphenotype (magA).Theprimersweredesignedinourstudy(Table1).

Polymerase chain reactionswere performedusing the com-mercial kit puReTaq Ready-To-Go PCR Beads (GE Healthcare®) according to the manufacturer’s instructions. Subsequently the PCRproductswereresolvedinagarosegel1%,inTBE(1_×),and stainedwithGelRed(Biotium).Positiveandnegativecontrolswere includedinallPCRassays.ResultingPCRproductswere submit-tedtopuriﬁcationusingtheJETquickSpinColumnTechniquePCR PuriﬁcationKit(Genomed®),accordingtoproducer’sinstructions and were sequenced at Macrogen Korea and STABVida Portu-gal.Searchesfornucleotidesequenceswereperformedwiththe BLASTprogram,availableattheNationalCenterforBiotechnology InformationWeb site (http://www.ncbi.nim.nih.gov/). Multiple-sequencealignmentswereperformedwiththeClustalXprogram, available at the European Bioinformatics Institute Web site (http://www.ebi.ac.uk/Tools/msa/clustalw2).

MoleculartypingbyM13-PCRﬁngerprinting

ClonalrelatednesswasestablishedbyM13-PCRﬁngerprinting, basedonrandomlyampliﬁedpolymorphicDNAanalysis.11_Strains

classiﬁedasgeneticallyrelatedandassignedtothesamelineage wereidentiﬁedwithletters(A–Z).Differentnumberswereassigned tomorecloselyrelatedstrains(≤2bands)anddifferentiate mem-ber’stypesassubtypes(e.g.,A1,A2).

Multilocussequencetyping(MLST)

MLSTwasperformedaspreviouslydescribed12_on₂₅_selected

isolates representing each of the ␤-lactamases currently with more clinical relevance: CTX-M-15 (n=8) and KPC-3 (n=17). ThesequenceswereperformedatMacrogenKorea and submit-ted to the MLST database for allele attribution. The Klebsiella

pneumoniae database is available at the Pasteur MLST site (http://www.pasteur.fr/mlst/).LastaccessedatMay2,2018.

Statisticalanalysis

The statistical signiﬁcance of comparisons made through-out this study was carried out by Fisher Exact Test, for whichwe usedthecomputerprogramavailableinhttp://www. graphpad.com/quickcalcs/index.Ap-value<0.05wasconsidered statisticallysigniﬁcant.

Ethicalapproval

Isolateswereobtainedaspartofroutinediagnostictestingand wereanalyzedanonymously.Alldatawerecollectedinaccordance withtheEuropeanParliamentandCouncildecision forthe epi-demiologicalsurveillance and control of communicable disease intheEuropeanCommunity.Epidemiologicaldatawerecollected fromclinicalrecords.Thestudyproposalwasalsoapprovedby ResearchEthicsCommitteeofFacultyofMedicine,Universityof Lisbon,Portugal.

Results

Antimicrobialsusceptibilitypatterns

ThisstudyincludedthesusceptibilityprofileofK.pneumoniae strainsisolatedfrom1980to2011againstauniformpanelof antibi-otics(Fig.1).AllisolatesfromgroupAexceptone(93.3%,14/15) weresusceptibletoallcephalosporinsand13isolatesshowed resis-tancetogentamicin(86.7%,13/15).Allthestrainsincludedingroup B(n=12)wereresistanttoceftazidime(100.0%,12/12).Moreover, 66.7%(8/12)and50.0%(6/12)showedresistancetogentamicinand ciprofloxacin.AmongtheisolatesofgroupCitwasobservedthat 84.8%(39/46)and82.6%(38/46)oftheisolateswereresistantto ceftazidimeandcefotaxime,respectively.Additionally,resistance togentamicin(82.6%,38/46)andciprofloxacin(76.1%,35/42)was alsofound.

Amongthe27strainsbelongingtogroupD,96.3%(26/27)were resistanttocefotaximeandceftazidime,74.1%ofisolates(20/27) wereresistanttoimipenemwhile81.5%(22/27)and40.7%(11/27) wereresistanttogentamicinandciproﬂoxacin,respectively.

(4)

Group A Group C Group D Group B AMC FOX CAZ CTX GM CIP CIP IMP AMC FOX CAZ CTX GM IMP CIP AMC FOX CAZ CTX GM IMP AMC FOX CAZ CTX GM CIP IMP 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100

Resistant Intermediate Susceptible Resistant Intermediate Susceptible

Fig.1.Antimicrobialsusceptibilitypatternofthe100representativeKlebsiellapneumoniaestrainsisolatedfrompatientswithhealthcare-associatedinfections.AMC: amoxicillin/clavulanicacid;FOX:cefoxitin:CAZ:ceftazidime;CTX:cefotaxime;GM:gentamicin;CIP:ciproﬂoxacin;IMP:imipenem.

Fig.2.ActivityoftigecyclineandfosfomycinagainstKlebsiellapneumoniaeisolates.

TheisolatesofgroupsB–Dwerealsotestedfortigecyclineand fosfomycinin order evaluatetheroleof theseantibiotics asan alternativetherapeuticoption aswelltounderstandthe evolu-tionof their resistancepatterns over thestudy period (Fig.2).

Fig.2 shows the phenotypiccharacterization of K.pneumoniae isolates.Theisolates resistanttotigecyclinefoundrangedfrom 8.3% (1/12) among broad-spectrum ␤-lactamase producers, to 29.6%(8/27)incarbapenemaseproducers.Ahighfrequencyof iso-lateswithclinicallyintermediatesusceptibilitytotigecyclinewas found(rangingfrom 48.1%to83.3%) whencompared with fos-fomycin(0%).Thesusceptibilitytofosfomycinwas100%(broadand extended-spectrum␤-lactamases producers) and 88.9% (period 2009–2011). Fosfomycin showed the highest activity to all ␤-lactamase-producingisolates(88.9%)(Fig.1),incomparisonwith gentamicin(3.7%)orciproﬂoxacin(33.3%).

Identiﬁcationoftheˇ-lactamases

Resultsoftheidentiﬁcationof␤-lactamasesinK.pneumoniae areshowninTable2.The15 K.pneumoniaeisolatesingroupA

(1980–1989)presented bothTEM-1and SHV-1 broad-spectrum ␤-lactamases. Among the 12 isolates in group B(1990–2001), seven(58.3%,7/12)presentedtheTEM-10andﬁve(41.7%,5/12) theTEM-24extended-spectrum␤-lactamases(ESBL).All46 iso-lates(100.0%,46/46) ingroup C(2002–2008) werepositive for cefotaxime-hydrolyzingCTX-M-15ESBL.The27isolatesingroup D(2009–2011)presentedtheKPC-3carbapenemasealone(59.3%, 16/27)or in combination withother ␤-lactamases,namelythe broad-spectrum␤-lactamaseSHV-11(14.8%,4/27)andtheESBL SHV-35(3.7%,1/27)andCTX-M-15(22.2%,6/27).ThegenesblaDHA,

blaCMY,blaIMP,blaVIM,blaNDMandblaOXAwerenotdetectedinour

collection.

Detectionofvirulencegenes

Virulencegenes as ﬁmHand mrkD adhesins, khe toxin, iucC siderophore,K2capsulartypeaswellthemagAandrmpAgenethat confersahypermucoviscosityandmucoidphenotype,were inves-tigatedinallK.pneumoniaeisolates.AprevalenceofﬁmH(96.0%), mrkDv1(90.0%), khe(63.0%)and K2(23.0%)virulence geneswas

(5)

Table2

ResistancedeterminantsfoundinKlebsiellapneumoniaeisolates(n=100)recoveredovera31-yearperiodinatertiarycarehospitalcentreinLisbon,Portugal.

Group Resistancedeterminants

Totalofisolates ␤-Lactamases No.ofisolates(%)

N=100

A BSBL n=15 TEM-1,SHV-1 15(100.0)

B TEM-typeESBL n=12 TEM-10 7(58.3)

TEM-24 5(41.7) C CTX-M-typeESBL n=46 CTX-M-15 46(100.0) D Carbapenemases n=27 KPC-3 16(59.3) KPC-3,SHV-11 4(14.8) KPC-3,SHV-35 1(3.7) KPC-3,CTX-M-15 6(22.2)

BSBL:broad-spectrum␤-lactamase;ESBL:extended-spectrum␤-lactamase.

Table3

DistributionofvirulencefactorsandtheirrespectivegenesamongKlebsiellapneumoniaeisolatesproducingdifferenttypesof␤-lactamases.

Virulencefactor Target

gene

␤-Lactamases Numberofisolates(%)

BSBL TEM-typeESBL CTX-M-typeESBL Carbapenemases

Total

TEM-1/SHV-1 TEM-10/TEM-24 CTX-M-15 KPC-3

n=15 n=12 n=46 n=27 N=100

Fimbrialadhesins Type1 ﬁmH 12(80.0) 12(100.0) 46(100.0) 26(96.3) 96(96.0)

Type3 Variant1 mrkDV1 10(66.7) 9(75.0) 45(97.8) 26(96.3) 90(90.0)

Variant2–4 mrkDV2–4 4(26.7) 0.0 1(2.2) 1(3.7) 6(6.0)

Toxin Haemolysin Khe 12(80.0) 11(91.7) 24(52.2) 16(59.0) 63(63.0)

Capsulartype K2serotype K2A 4(26.7) 3(25.0) 0.0 16(59.0)* ₂₃_(23.0)

Siderophore Aerobactin iucC 0.0 0.0 0.0 6(22.0)* _0.0

Protectinesorinvasins Mucoviscosityphenotype magA 0.0 0.0 0.0 0.0 0.0

Regulatorofmucoidphenotype rmpA 0.0 0.0 0.0 0.0 0.0

*_p_<_0.05_KPC-3_producers_vs_non-KPC-3_producers;_BSBL:_{broad-spectrum}_{␤-lactamase;}_ESBL:_{extended-spectrum}_{␤-lactamase.}

identiﬁed.Only6.0%oftheisolatesshowedthemrkDV2-4andiucC

gene.NomagAandrmpAgeneswereampliﬁed.Allisolatesanalyzed (n=100)presentedatleastoneofthevirulencegenesstudied.

Thedistribution ofvirulence genesby the␤-lactamase type produced is shown in Table 3. The ﬁmbrial adhesins ﬁmH (80.0–100.0%), mrkDV1 (66.7–97.8%) and hemolysin gene khe

(52.2–91.7%) were identified in all K. pneumoniae ␤-lactamase types,althoughtheiucCgenewasonlyidentifiedinKPC-3(22.0%). TheK2genewasidentifiedinTEM-1/SHV-1(26.7%), TEM-10/TEM-24(25.0%)andKPC-3producers(59.0%).ThemrkDV2–4 genewas

more frequent (p<0.05) among the TEM-BSBL isolates (26.7%) compared with TEM-ESBL (0.0%), CTX-M-15 (2.2%) and KPC-3 (3.7%).TheK2andiucCvirulencegenesweresigniﬁcantlyrelated withKPC-3clinicalisolatescomparedwithstrainswithother ␤-lactamases(p=0.0267andp=0.0002,respectively).

Thecharacterizationofthevirulenceprofile(VP)wasperformed inordertoevaluatethesimultaneouslyaccumulationofvirulence genesinthesameisolate,andnumberedaccordingthedecreasing numberofvirulencegenesfound(Table3).AccordingtoTable4, threevirulenceprofileswerepredominant,namelyVP10(35.0%), VP13(27.0%)andVP4(11.0%).Theprevalentprofilesfoundinthe ␤-lactamase-producingK.pneumoniaeisolateswerethosethat pre-sentedlessaccumulationofvirulencegenesperisolate,namelythe VP1–3(2genes),VP10(3genes)and,finally,VP4(4genes).The VP10(fimH,mrkDv1,khe)wasfoundin35isolates(35.0%)andwas sharedbyall␤-lactamaseproducers,namelybyTEM-1andSHV-1 (13.3%),TEM-10andTEM-24(50.0%),CTX-M-15(50.0%)and KPC-3(14.8%).TheVP13(fimH,mrkDv1)wasfoundamong27(27.0%) strainsandwasmainlydetectedinCTX-M-15producers.Instead, theVP4(fimH,mrkDv1,khe, K2)wasspecificallyfoundinKPC-3

Table4

VirulenceproﬁlesandnumberofvirulencegenesidentiﬁedinisolatesofKlebsiellapneumoniaeproducingdifferenttypesof␤-lactamasesandcarbapenemases.

Numberofvirulence

genes

Virulence

proﬁlea

BSBL TEM-typeESBL CTX-M-typeESBL Carbapenemases Total TEM-1/SHV-1 TEM-10/TEM-24 CTX-M-15 KPC-3 n=15 n=12 n=46 n=27 N=100 5 VP1 fimH,mrkDv2–4,khe,iucC,K2 0.0 0.0 0.0 1(3.7) 1(1.0) VP2 fimH,mrkDv1,khe,iucC,K2 0.0 0.0 0.0 1(3.7) 1(1.0) 4 VP3 fimH,mrkDv1,khe,iucC 0.0 0.0 0.0 2(7.4) 2(2.0) VP4 fimH,mrkDv1,khe,K2 2(13.3) 2(16.7) 0.0 7(25.9) 11(11.0) 3 VP5 fimH,khe,K2 1(6.7) 0.0 0.0 0.0 1(1.0) VP6 fimH,mrkDv1,iucC 0.0 0.0 0.0 1(3.7) 1(1.0) VP7 mrkDv1,khe,iucC 0.0 0.0 0.0 1(3.7) 1(1.0) VP8 fimH,mrkDv2–4,khe 4(26.7) 0.0 1(2.2) 0.0 5(5.0) VP9 fimH,mrkDv1,K2 1(6.7) 1(8.3) 0.0 7(25.9) 9(9.0) VP10 fimH,mrkDv1,khe 2(13.3) 6(50.0) 23(50.0) 4(14.8) 35(35.0) 2 VP11 fimH,khe 0.0 3(25.0) 0.0 0.0 3(3.0) VP12 mrkDv1,khe 3(20.0) 0.0 0.0 0.0 3(3.0) VP13 fimH,mrkDv1 2(13.3) 0.0 22(47.8) 3(11.1) 27(27.0)

VP:virulenceproﬁle;BSBL:broad-spectrum␤-lactamase;ESBL:extended-spectrum␤-lactamase.

(6)

producers.KPC-3isolatesshowed9virulenceproﬁles(allexcept VP5,-8,-11and-12)whereasinCTX-M-15producing-isolatesonly 3weredetected(VP10,-13and-8).

GenotypingbyM13-PCRﬁngerprintingandMLSTresults

TheclonalrelatednessoftheK.pneumoniaeisolateswas estab-lishedbyM13-PCRfingerprintingtoaddressiftherewasacommon clonebetweenthegroupofdifferent␤-lactamaseproducing iso-latesorwithinthesame␤-lactamasegroup.Apolyclonalsituation was found with 33 different genotypes. No similar genotypes wereidentified among thedifferent␤-lactamases groups, indi-catinggeneticvariability.Despitethis,geneticallyrelatedisolates wereidentified withinthesame␤-lactamase groupas follows: ingroupA–broad-spectrum ␤-lactamasesproducingstrains–, withsevencolonizedpatientsandfourenvironmentalisolates,all fromneonatologydepartment,sharedthesamegenotype;ingroup C,CTX-M-15␤-lactamaseproducers,presented4maingenotypes withisolatesfromdifferentwardsand,finally,ingroupD,66.7%of theKPC-3producerssharedthesamegenotype(genotypeA).The intensivecareunitofhematologicdiseases,thethoracicsurgery departmentand thesurgeryobservationroomwerethemainly affectedwards,allsharingtheKPC-3typeAgenotype.

MLSTwasperformedonCTX-M-15(n=8)andKPC-3(n= 17)-producingclinicalisolatesforinternationalcomparativepurposes. TheST15accountedfor62.5%(5/8)oftheCTX-M-15isolates ana-lyzedbyMLST.Additionally,5distinctallelicprofileswerefound: ST133(n=1); ST147(n=1),ST276(n=1)forCTX-M-15 produc-ingisolates,whereastheKPC-3carbapenemase-producingisolates wereST11(n=2)andST14(n=15).ThefirsttwoKPC-3isolates, identifiedin2009,showedtheST11andtheremainingKPC-3 iso-lates(2009–2011)exhibitedthesamecombinationofallelesacross thesevensequencedloci,correspondingtotheST14(88.2%ofthe KPC-3isolates).

Discussion

Multidrugresistantand virulentpopulationswere,foralong time,non-overlapping13 _but_the_virulence _of_K._pneumoniae_and

the interplay between resistance and virulence is still poorly understood.14_Available_reports_do_not_include_the_KPC-3_isolates

orST14clones15_and_mainly_report_the_{hypermucoviscosity}

pheno-type,associatedwithpyogenicliverabscess.16_To_our_knowledge,

this is the ﬁrst study that characterizes the evolution of viru-lencefeaturesofK.pneumoniae␤-lactamase-producers,including carbapenemaseproducersrecoveredfrompatientswith hospital-acquiredinfections.

Acomparisonofthepopulationstructureandvirulencefactors betweengroupsofstrainswithESBLandKPCresistance mecha-nismsversusbroad-spectrum␤-lactamaseproducers(BSBL)was performed.TheBSBLstrainsshowedasusceptibilityproﬁleto ␤-lactams and quinolones (>90%), that decreased to <60% among TEM-typeESBLproducersandto<20%amongCTX-M-typeESBL producers.ItisofconcernthatKPC-3carbapenemaseproducers wereresistantto␤-lactams,quinolonesandaminoglycosides, lim-itingthetherapeuticoptionstotreatinfectionscausedbythese bacteria.AccordingtotheEuropeanCentreforDiseasePrevention andControl,therateofresistancetocarbapenemsinPortugalhas increasedsix-fold(0.3–1.8%)overtheperiodfrom2011to2014,17

highlightingthatspecialattentionshouldbepaidtocarbapenem resistancetrends.

The rateof resistanceto tigecycline among TEM- and CTX-M-type extended-spectrum _{␤-lactamase} producers was 8.5%, in line with previous reports that mentioned <10% resistance ratesinwide-scalesurveillancestudies18_but_surprisingly_among

carbapenem-resistantstrainsthisresistanceratereached30%, fol-lowing thetigecyclinecommercializationin Portugal(2009).Of relevance,also48%oftheKPC-3isolatespresentedintermediate susceptibilitytotigecycline,aphenotypeassociatedwithuncertain clinicaltherapeuticeffect.

Ofnote,89%ofKPC-3isolatesweresusceptibletofosfomycin andtheresistanceratesfoundinallgroupsofisolatesrangedfrom 8to11%,supportingfosfomycinasatherapeuticoptionfor difﬁcult-to-treatK.pneumoniaeinfections.Thepotentialuseoffosfomycin hasbeenreportedbyotherauthorsand itsability topenetrate throughbioﬁlmlayers.19_However,_the_report_of_FosA3_(resistance

tofosfomycin)andKPC-2onasingleplasmidanditslikelyclonal spreadareworryingandshouldbemonitored.20

Type1 and type 3 ﬁmbrial adhesins aremainly involved in adherencetoseveralcelltypesandinbioﬁlmformation21_which

protectsbacteriaagainstantibioticsandhostdefenses19_and_can

actas a startingpoint of infectionfrommedical devices.22 _The

highprevalence(>90%) of type 1 and type 3 fimbrial adhesins observedinourstudy,togetherwiththeantimicrobial multiresis-tanceobserved,canexplainthepersistenceofthismultiresistant isolatesforlongtimeinthehospitalenvironmentand the diffi-cultyoftheireradication.Outof100strainsstudied,92showedthe fimHandmrkDgenesinthesameisolate,demonstratingarelation betweenthesetwogenes.Additionally,thevirulenceprofileVP10 (fimH,mrkDv1,khe)withthegenesencodingtype1andtype3 fim-brialadhesinsandhaemolysin,apore-formingtoxinthatmakes somenutrientsavailablesuchas theferrousionin hemoglobin andoftenassociatedwithpathogenicmicroorganisms,23_were

pre-dominantintheK.pneumoniaecollectionandweresharedbyall ␤-lactamaseproducers,conclusiveofacommonpathogenicorigin. TheKPC-producingstrainswerepreviouslydescribedashighly resistantandlowvirulentstrainsandnospeciﬁcvirulencefactors havebeenidentiﬁed.24_{Nevertheless,}_this_conclusion_is_sustained

intheabsenceofaerobactinandcapsularK2andmainlyincluded KPC-2isolates.24,25_In_addition,_Siu_et_al._reported_that_known

viru-lencefactorssuchasK1,K2,andK5capsularpolysaccharides,rmpA andtheaerobactingene wereabsentin KPC-producingisolates andthatthesestrainspresentlowvirulencein murinelethality model.26

Ourdatacontrastwithpreviousstudiesconsideringahigher detection rate (p<0.05) of K2 capsular antigen (59%) and aer-obactin (22%) in KPC-3 K. pneumoniae isolates than in the KPC-negativegroups.TheK2antigenconfersresistanceto phago-cytosisandserumresistance,27_being_frequently_associated_with

severeinfections28_and_aerobactin_is_one_of_the_siderophores

(extra-cellularferricchelatingagents)secretedbybacterialcellsandplay acriticalroleinbacterialvirulencegiventhatironbioavailabilityin thehostisextremelylowanditisessentialformicrobialgrowth.29

Moreover,DeCassiaAndradeMeloetal.reportedthepresenceof type1andtype3ﬁmbrialadhesinsandyersiniabactinsiderophore inKPC-2-producingK.pneumoniaeisolatesfromBrazil,2 _a

coun-trymaintainingacloserelationshipwithPortugal.However,the bacterialsequencetypesoftheisolateswerenotanalyzed.

Inconclusion,theST14KPC-3K.pneumoniaeclonehasrevealed ahighervirulenceproﬁlediversityandabilitytoaccumulate vir-ulencegenes in thesame isolate,when compared with strains producingother␤-lactamases.Adeeperunderstandingofthe viru-lenceandresistancetraitsofhigh-riskclonesistheﬁrststeptoward thedevelopmentofmore effectiveapproaches tominimizethe impactofhospitalacquiredinfectionsbymultiresistantbacteria.

Funding

ThisworkwassupportedbytheResearchInstituteforMedicines (iMed.ULisboa),FacultyofPharmacy,UniversityofLisbon.

(7)

Conﬂictsofinterest

J.Melo-Cristinoresearchgrantsadministeredthroughhis uni-versityandhonorariaforservingonspeaker’sbureausofPfizer, GileadandNovartis.Theotherauthorsdeclarethattheresearch wasconductedintheabsenceofanycommercialorfinancial rela-tionshipsthatcouldbeconstruedasapotentialconflictofinterest.

Acknowledgments

The authorswould like to thank toall the membersof the Microbiologylaboratoryforthecollaborationinisolationand iden-tiﬁcationofbacteria.

References

1.TacconelliE,MagriniN.Globalprioritylistofantibiotic-resistantbacteriato guideresearch,discovery,anddevelopmentofnewantibiotics.WorldHealth Organization;2017.

2.deCassiaAndradeMeloR,deBarrosEM,LoureiroNG,deMeloHR,MacielMA, SouzaLopesAC.PresenceofﬁmH,mrkD,andirp2virulencegenesin KPC-2-producingKlebsiellapneumoniaeisolatesinRecife-PE,Brazil.CurrMicrobiol. 2014;69:824–31.

3.MachadoP,SilvaA,LitoL,Melo-CristinoJ,DuarteA.EmergenceofKlebsiella pneumoniaeST11-producingKPC-3carbapenemaseataLisbonhospital.Clin MicrobiolInfect.2010;16Suppl.2:S28.

4.PiresD,ZagaloA,SantosC,CotadeMedeirosF,DuarteA,LitoL,etal.Evolving epi-demiologyofcarbapenemase-producingEnterobacteriaceaeinPortugal:2012 retrospectivecohortatatertiaryhospitalinLisbon.JHospInfect.2016;92:82–5. 5.YuVL,HansenDS,KoWC,SagnimeniA,KlugmanKP,vonGottbergA,etal. Vir-ulencecharacteristicsofKlebsiellaandclinicalmanifestationsofK.pneumoniae bloodstreaminfections.EmergInfectDis.2007;13:986–93.

6.StruveC,BojerM,KrogfeltKA.Identificationofaconservedchromosomalregion encodingKlebsiellapneumoniaetype1andtype3fimbriaeandassessmentof theroleoffimbriaeinpathogenicity.InfectImmun.2009;77:5016–24. 7.HoltKE,WertheimH,ZadoksRN,BakerS,WhitehouseCA,DanceD,etal.

Genomicanalysisofdiversity,populationstructure,virulence,and antimicro-bialresistanceinKlebsiellapneumoniae,anurgentthreattopublichealth.Proc NatlAcadSciUSA.2015;112:E3574–81.

8.BeceiroA,BouG.Resistenciaalosantimicrobianosyvirulencia,¿una aso-ciaciónbeneﬁciosaparaelmundomicrobiano?EnfermInfeccMicrobiolClin. 2012;30:492–9.

9.BouG.Relaciónentreresistenciayvirulenciaenbacteriasdeinterésclínico. EnfermInfeccMicrobiolClin.2014;32:1–3.

10.CalistoF.EmergênciadecarbapenemasesemKlebsiellapneumoniae:odesaﬁo debactériasmultirresistentesevirulentas[TesedeMestradoem Microbiolo-giaAplicada].RepositóriodaUniversidadedeLisboa.UniversidadedeLisboa, FaculdadedeCiências,DepartamentodeBiologiaVegetal;2011.

11.GrundmannHJ,TownerKJ,DijkshoornL,Gerner-SmidtP,MaherM,SeifertH, etal.Multicenterstudyusingstandardizedprotocolsandreagentsforevaluation

ofreproducibilityofPCR-basedﬁngerprintingofAcinetobacterspp.JClin Micro-biol.1997;35:3071–7.

12.DiancourtL,PassetV,VerhoefJ,GrimontPA,BrisseS.Multilocussequencetyping ofKlebsiellapneumoniaenosocomialisolates.JClinMicrobiol.2005;43:4178–82. 13.Bialek-DavenetS,CriscuoloA,AilloudF,PassetV,JonesL,Delannoy-Vieillard AS,etal.Genomicdeﬁnitionofhypervirulentandmultidrug-resistantKlebsiella pneumoniaeclonalgroups.EmergInfectDis.2014;20:1812–20.

14.HennequinC,RobinF.Correlationbetweenantimicrobialresistanceand vir-ulence in Klebsiella pneumoniae.EurJ Clin Microbiol InfectDis. 2016;35: 333–41.

15.LevAI,AstashkinEI,KislichkinaAA,SolovievaEV,KombarovaTI,Korobova OV,etal.ComparativeanalysisofKlebsiella pneumoniaestrainsisolatedin 2012–2016thatdifferbyantibioticresistancegenesandvirulencegenes pro-ﬁles.PathogensGlobalHealth.2018:1–36.

16.Catalán-NájeraJC,Garza-RamosU,Barrios-CamachoH.Hypervirulenceand hypermucoviscosity:twodifferentbutcomplementaryKlebsiellaspp. pheno-types?Virulence.2017;8:1111–23.

17.AntimicrobialresistancesurveillanceinEurope2014.Stockholm:European AntimicrobialResistanceSurveillanceNetwork(EARS-Net),EuropeanCentrefor DiseasePreventionandControl(ECDC);2015.

18.PournarasS,KoumakiV,SpanakisN,GennimataV,TsakrisA.Current perspec-tivesontigecyclineresistanceinEnterobacteriaceae:susceptibilitytestingissues andmechanismsofresistance.IntJAntimicrobAgents.2016;48:11–8. 19.BandeiraM,CarvalhoPA,DuarteA,JordaoL.Exploringdangerousconnections

betweenKlebsiellapneumoniaebioﬁlmsandhealthcare-associatedinfections. Pathogens.2014;3:720–31.

20.JiangY,ShenP,WeiZ,LiuL,HeF,ShiK,etal.Disseminationofaclone car-ryingafosA3-harbouringplasmidmediateshighfosfomycinresistancerate ofKPC-producingKlebsiella pneumoniae inChina.IntJ AntimicrobAgents. 2015;45:66–70.

21.SchrollC,BarkenKB,KrogfeltKA,StruveC.Roleoftype1andtype3ﬁmbriaein Klebsiellapneumoniaebioﬁlmformation.BMCMicrobiol.2010;10:179. 22.SanchezCJJr,MendeK,BeckiusML,AkersKS,RomanoDR,WenkeJC,etal.

Bioﬁlmformationbyclinicalisolatesandtheimplicationsinchronicinfections. BMCInfectDis.2013;13:47.

23.KoczuraR,KaznowskiA.OccurrenceoftheYersiniahigh-pathogenicityisland andironuptakesystemsinclinicalisolatesofKlebsiellapneumoniae.Microb Pathog.2003;35:197–202.

24.Nordmann P, CuzonG, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producingbacteria.LancetInfectDis.2009;9:228–36. 25.LavigneJP,CuzonG,CombescureC,BourgG,SottoA,NordmannP.Virulence

ofKlebsiellapneumoniaeisolatesharboringblaKPC-2carbapenemasegeneina Caenorhabditiselegansmodel.PLOSONE.2013;8:e67847.

26.SiuLK,LinJC,GomezE,EngR,ChiangT.Virulenceandplasmidtransferability ofKPCKlebsiellapneumoniaeattheVeteransAffairsHealthcareSystemofNew Jersey.MicrobDrugResist.2012;18:380–4.

27.PanYJ,LinTL,ChenYH,HsuCR,HsiehPF,WuMC,etal.CapsulartypesofKlebsiella pneumoniaerevisitedbywzcsequencing.PLOSONE.2013;8:e80670. 28.TurtonJF,PerryC,ElgohariS,HamptonCV.PCRcharacterizationandtyping

ofKlebsiellapneumoniaeusingcapsulartype-speciﬁc,variablenumbertandem repeatandvirulencegenetargets.JMedMicrobiol.2010;59:541–7.

29.ElFertas-AissaniR, MessaiY,AlouacheS,BakourR. Virulenceproﬁlesand antibioticsusceptibilitypatternsofKlebsiellapneumoniaestrainsisolatedfrom differentclinicalspecimens.PatholBiol.2013;61:209–16.

View publication stats View publication stats