brazjinfectdis2017;21(4):477–480
w w w . e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Brief
communication
Antimicrobial
resistance
and
plasmid
replicons
in
Yersinia
enterocolitica
strains
isolated
in
Brazil
in
30
years
Miliane
R.
Frazão
♦,
Leonardo
N.
Andrade
♦,
Ana
L.C.
Darini,
Juliana
P.
Falcão
∗UniversidadedeSãoPaulo(USP),FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto(FCFRP),DepartamentodeAnálisesClínicas, ToxicológicaseBromatológicas(DACTB),RibeirãoPreto,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received21December2016 Accepted17April2017 Availableonline27May2017
Keywords:
Yersiniaenterocolitica
Resistanceprofile Plasmidreplicon
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b
s
t
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c
t
SomestudiesevaluatedtheresistanceprofileoftheY.enterocoliticastrainsisolatedindiverse countries.However,inBraziltheisolationandthestudyofY.enterocolitica arenot com-monandthereforeinformationabouttheantimicrobialresistanceprofileofthisspeciesin thiscountryisscarce.Therefore,theaimofthisstudywastoevaluatetheantimicrobial resistanceofY.enterocoliticaofbiotypes1A,2and4isolatedfromclinicalandnon-clinical sourcesbetween1979and2012,inBrazil.ThisstudyshowedthatsomeYersiniaenterocolitica
ofdifferentbiotypesremainsusceptibletoantimicrobialsusedforgastroenteritistreatment. Moreover,neitheracquiredresistancegenesnordiversityofplasmidsrepliconswerefound; however,variationintheinvitrointrinsicresistantpatternwasdetected,exceptthe non-resistancetocefoxitininallstrains.Notwithstanding,duetoepidemiologicallinkbetween
Y.enterocoliticaandtheporkproductionchain,monitoringplasmidacquiredresistancein
Y.enterocoliticacouldalsobeconsideredforantimicrobialresistancecontrolpurposesand foodsafetymeasures.
©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).
YersiniaenterocoliticaisthemostprevalentYersiniaspeciesthat causesillnessinhumansandanimals.Y.enterocoliticastrains canbeclassifiedintosixbiotypes,beingbiotypes1B,2,3,4,and 5associatedwithillnessinhumansandanimals,while bio-type1Acomprisesstrainsthatareconsideredtobeprimarily nonpathogenic.1
Y.enterocoliticaintrinsicresistancetoampicillin,ticarcillin, amoxycillin-clavulanate,cefazolin,andcephalothinhasbeen assigned by the Clinical & Laboratory Standards Institute (CLSI).2 Inaddition,intrinsicresistancetocefamandoleand
∗ Correspondingauthor.
E-mailaddress:jufalcao@fcfrp.usp.br(J.P.Falcão).
♦ Theseauthorscontributedequallytothiswork.
cefoxitinhasalsobeenrecognizedbyEUCAST.3Y.enterocolitica
haveshownsusceptibilityinvitrotoaminoglycosides, tetracy-cline, chloramphenicol,extended-spectrum cephalosporins, and trimethoprim-sulfamethoxazole. Resistance to fluoro-quinolones has been observed in some countries due to chromosomalmutationmechanism.4InBrazil,theisolation
and the study ofY.enterocoliticaare notcommon and thus informationabouttheantimicrobialresistanceofisolatesof thisspeciesinthiscountryisscarce.5–8
Therefore, theaimsofthis studywere todeterminethe antimicrobial susceptibility profile and asses the intrinsic resistancepattern,tosearchforplasmidacquiredresistance genes,andtoinvestigateplasmidrepliconsinY.enterocolitica
isolatedinBrazil.
http://dx.doi.org/10.1016/j.bjid.2017.04.006
1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
478
braz j infect dis.2017;21(4):477–480A totalof 34 Y. enterocoliticastrains biotype 1A(n=2), 2 (n=12),and4(n=20)werestudied.Thesestrainswereselected from the collection of the “Brazilian Reference Center on
Yersiniaspp.otherthanY.pestis”isolatedfrom1979to2012, basedontheresistanceprofilesfoundforsomeotherstrains ofthebiotypesmentionedaboveinpreviousstudiesofour group.5–7
The antimicrobial susceptibility profilewas determined usingthe disk diffusionmethod and interpretedaccording tothebreakpointsforEnterobacteriaceae.2,3,9Moreover,
double-disksynergytest(DDST)wasperformedtodetect extended-spectrumbeta-lactamase(ESBL)production,10enzymesable
tohydrolyzethird-andfourth-generationcephalosporins. Plasmid acquired genes coding for resistance to extended-spectrum cephalosporins (blaCTX-M, blaTEM and
blaSVH), tetracyclines (tet), aminoglicosydes (aac(6)-Ib), and
fluoroquinolones (qnr, aac(6)-Ib-cr, qepA and oqxAB) were searched.11–14 Moreover, plasmids were searched
fol-lowing the PCR-based replicon typing (PBRT) scheme targeting replicons of the major incompatibility groups (Inc)harboring/disseminating antibioticresistancegenesin
Enterobacteriaceae.15
Y.enterocoliticasamplesshowedrecognizedintrinsic resis-tance to cefazolin (CFZ) (34/34), cephalotin (CF) (34/34), ampicillin(AMP)andticarcillin(TIC)(32/34),and amoxicillin-clavulanicacid(AMC)(19/34).Thisinherentnon-susceptibility patternhasbeendisplayedbyalloralmostallstrainsfrom dif-ferentbiotypesandisolationsources.However,cefoxitin(FOX) intrinsicresistance,additionallyalsorecognizedbyEUCAST,16
wasnotdetected(Table1).
MostY.enterocoliticaisolatesharboredchromosomalgenes
blaAandblaBencodingfortwobeta-lactamases,respectively,
Table1–Generaldataofthe34Yersiniaenterocoliticastrainsstudied.
Strains Biotype Source Year Resistanceprofile
IP7382a 4 Animalfeces 1979 AMP,TIC,CFZ,CF,NIT
IP7884a 4 Animalfeces 1979 AMP,TIC,CFZ,CF
FCF57 1A Milk 1980 AMP,TIC,AMC,CFZ,CF,NIT
FCF86 2 Freshwater 1982 AMP,TIC,AMC,CFZ,CF FCF88 2 Freshwater 1983 AMP,TIC,AMC,CFZ,CF FCF268 1A Chickenmeat 1984 AMP,TIC,AMC,CFZ,CF FCF90 2 Freshwater 1984 AMP,TIC,AMC,CFZ,CF FCF376a 4 Humanfeces 1984 AMP,TIC,CFZ,CF
FCF93 2 Freshwater 1985 AMP,TIC,AMC,CFZ,CF FCF94 2 Freshwater 1986 AMP,TIC,CFZ,CF FCF96 2 Freshwater 1987 AMP,TIC,CFZ,CF
FCF100 2 Freshwater 1988 AMP,TIC,AMC,CFZ,CF,PTZ
FCF418a 4 Humandiarrheicfeces 1988 AMP,TIC,CFZ,CF
FCF103 2 Freshwater 1989 AMP,TIC,CFZ,CF FCF105 2 Freshwater 1990 AMP,TIC,CFZ,CF FCF110 2 Freshwater 1991 AMP,TIC,AMC,CFZ,CF FCF113 2 Freshwater 1992 AMP,TIC,CFZ,CF FCF115 2 Freshwater 1993 AMC,CFZ,CF FCF600a 4 Humandiarrheicfeces 1998 AMP,TIC,CFZ,CF,CPM
FCF601 4 Animalfeces 1998 AMP,TIC,AMC,CFZ,CF FCF605a 4 Humandiarrheicfeces 1999 AMP,TIC,CFZ,CF,NIT
FCF606a 4 Humandiarrheicfeces 1999 AMP,TIC,AMC,CFZ,CF,NAL,NIT
FCF607a 4 Humandiarrheicfeces 1999 AMP,TIC,AMC,CFZ,CF,CPM,NIT
FCF609a 4 Humandiarrheicfeces 2000 AMP,TIC,CFZ,CF,NAL
FCF612a 4 Humandiarrheicfeces 2000 AMP,TIC,AMC,CFZ,CF
FCF613a 4 Humandiarrheicfeces 2003 AMP,TIC,CFZ,CF,NAL
FCF614a 4 Humandiarrheicfeces 2003 AMP,TIC,CFZ,CF,NAL,SXT
FCF615a 4 Humanblood 2004 AMP,TIC,AMC,CFZ,CF
FCF618a 4 Humandiarrheicfeces 2008 AMP,TIC,CFZ,CF,NAL,KAN
FCF619a 4 Humandiarrheicfeces 2008 AMP,TIC,AMC,CFZ,CF,SXT
FCF620a 4 Humanblood 2008 AMC,CFZ,CF
FCF624a 4 Lymphnodeswab 2010 AMP,TIC,AMC,CFZ,CF,CFX,NAL,
NOR,CIP,LEV,TET,DOX,SXT, NIT,FOS
FCF625a 4 Lymphnodeswab 2010 AMP,TIC,CFZ,CF
FCF626a 4 Humanblood 2012 AMP,TIC,AMC,CFZ,CF,NAL
Boldandunderlinetypemeansacquiredresistanceotherthanplasmidacquiredresistanceinvestigatedhere.
AMP,ampicillin;TIC,ticarcillin;AMC,amoxicillin-clavulanicacid;PTZ,piperacillin-tazobactam;CF,cephalotin;CFZ,cefazolin;CFX,cefuroxime; FOX,cefoxitin;CTX,cefotaxime;CAZ,ceftazidime;CPM,cefepime;ATM,aztreonam;ERT,ertapenem;GEN,gentamicin;AMI,amikacin;KAN, kanamycin;SXT,trimethoprim-sulfamethoxazole;NAL,nalidixicacid;NOR,norfloxacin;CIP,ciprofloxacin;LEV,levofloxacin;C, chlorampheni-col;NIT,nitrofurantoin;TET,tetracycline;DOX,doxycyclin;TGC,tigecycline;PB,polymyxinB;FOS,fosfomycin.AllmanufacturedbyOxoid (Basingstoke,Hampshire,UK).
a PresenceofIncFII
brazj infect dis.2017;21(4):477–480
479
BlaA(anon-induciblebroad-spectrum carbenicillinase)and BlaB(anAmpC-typeinduciblecephalosporinase).17The
dif-ferential expression and activities of these two enzymes determine the differential beta-lactam intrinsic resistance amongbiotypes ofY. enterocolitica.4 Moreover, a small
per-centageofstrainsmayappearsusceptibleduetolaboratory methodvariation,mutationorresistanceexpression. There-fore,invitrosusceptibleresultsshouldbeviewedwithcaution because in vivo non-susceptibility could lead to therapeu-ticfailure.Approximatelyhalfofthestrains(14/34)showed alsoacquiredresistance(Table1).Beta-lactamresistanceto piperacillin-tazobactam (PTZ) and cefuroxime (CFX) could havebeenduetoBlaB(AmpC)overproduction.However,BlaB overproductiondoesnotexplaincefepime(CPM)resistance, onceAmpCbeta-lactamasesarenotabletohydrolyze fourth-generation cephalosporins (like CPM). Thereby, once ESBL productionwasnotdetectedandtherecognizedenzymatic intrinsicresistanceisnotabletoconferthisphenotype,other mechanismofresistancelikeporinlossand/orover expres-sionofeffluxsystemcouldhavebeenresponsibletoCPMas wellasPTZandCFXresistanceintheisolatesstudiedhere.17,18
Nitrofurantoin(NIT)resistancewasdetectedin17.6%(6/34) ofstrains; otherstudiesreported36%resistant19aswell as
100%susceptible strains.20 Trimethoprim-sulfamethoxazole
(SXT)resistancewas foundedin8.8%(3/34)ofstrains, and 4%20 to 10%21 of resistance were seen in other studies.
Nalidixicacid(NAL)resistancewasobservedin20.5%(7/34)of strainsandotherstudieshavedescribedanincreasing num-ber of strains showing NAL resistance over recent years22
reaching23%.23Inaddition,fluoroquinoloneslikenorfloxacin
(NOR),ciprofloxacin (CIP), andlevofloxacin (LEV)resistance was foundin justone strain namedFCF624, corroborating thefindingsofotherstudieswhereratesoffluoroquinolone resistanceweresignificantlylower than those observedfor NALalone.22Tetracycline(TET),doxycyclin(DOX),kanamycin
(KAN),andfosfomycin(FOS)resistancewerealsodetectedin theFCF624strain(Table1),showingmultipleresistance phen-otypes,rarelyreportedinY.enterocolitica.4
No plasmid acquired genes coding for resistance to extended-spectrumcephalosporins,tetracyclines, aminogli-cosydes, and fluoroquinolones were found. Thereby, resistance to these antimicrobial classes could have chromosomalorigin.
Inthisstudy,justIncFIIY plasmidrepliconweredetected
mostlyinstrainsofthe pathogenicbiotype4isolatedfrom human diarrheic feces; however, it did not relate to any acquiredresistancegenes(Table1).IncFII-likeplasmidsare mostlyconsideredasvirulenceplasmids,suchaspYV harbor-ingtypeIIIsecretionsystem(yops),thatcanbepresentalone orco-resident andcompatible withotherFII-positive resis-tanceplasmids(typablebytheFII,FIA,FIB,FICloci)withinthe samebacterialcell24 (http://pubmlst.org/plasmid/).Plasmids
carrying transferable antibioticresistance geneshave been detectedfromavarietyofEnterobacteriaceae25;howeverreports
inY.enterocoliticaarerare,4 suchastheconjugativeplasmid
(30–40kb) transferring chloramphenicol, streptomycin, and sulfonamideresistancephenotypesfromasporadicY. ente-rocolitica4/O:3strain.26
Y. enterocolitica is a non-hospital pathogen and the absenceofacquiredresistancegenescouldbeexplainedby
littleinteraction withhospitalpathogens andconsequently a reduced possibility to genetic exchanges, such as hori-zontal gene transfer mediated by plasmids. Nevertheless, resistancegenesandresistantbacteriahavebeenalarmingly andincreasinglyfoundinfoodandfood-producinganimals,27
suchasmcr-1geneinESBL-producingEscherichiacolistrains isolatedfrompig.28Y.enterocoliticaisazoonoticpathogenthat
causesgastrointestinaldiseaseandporcineanimalseemsto bethemaincarrierofthisbacterialspecies.29Likewise,the
foodchaincouldboostY.enterocoliticaantimicrobialresistance. In summary, our study showed that some Brazilian Y. enterocoliticastrainsofdifferentbiotypesremainsusceptible to drugsusedfor treatinggastroenteritis,as well as extra-intestinalandhospitalinfections.Inaddition,variationinthe
invitrointrinsicresistantpatternwasdetected,except non-resistancetoFOXinall strains. Moreover,neitheracquired resistancegenesnordiversityofplasmidsrepliconssearched were found. Notwithstanding, due to epidemiological link betweenY.enterocoliticaandporkproductionchain, monitor-ingplasmidacquiredresistanceinY.enterocoliticacouldalsobe consideredforantimicrobialresistancecontrolpurposesand foodsafetymeasures.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WethankSaoPauloResearchFoundation (FAPESP-2012/19132-1)forfinancialsupport.Duringthecourseofthiswork,Frazão, M.R. was supportedby ascholarship granted by Coordina-tionfortheImprovementoftheHigherEducationPersonnel (CAPES).Andrade,L.N.wassupportedbyapostdoctoral fellow-shipfromProgramaNacionaldePósDoutorado(PNPD)/CAPES 2015.
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