h tt p:/ / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Resistance
patterns,
ESBL
genes,
and
genetic
relatedness
of
Escherichia
coli
from
dogs
and
owners
A.C.
Carvalho
a,b,∗,
A.V.
Barbosa
b,
L.R.
Arais
b,
P.F.
Ribeiro
b,
V.C.
Carneiro
b,
A.M.F.
Cerqueira
baLaboratóriodeBiologiaMolecular,DepartamentoInterdisciplinardeRiodasOstras,InstitutodeHumanidadeseSaúde, PóloUniversitáriodeRiodasOstras,RiodasOstras,RJ,Brazil
bLaboratóriodeEnteropatógenos,MicrobiologiaVeterináriaedeAlimentos/(LEMA),DepartamentodeMicrobiologiaeParasitologia, InstitutoBiomédico,UniversidadeFederalFluminense,Niterói,RJ,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received20December2013
Accepted9October2014
AssociateEditor:RoxaneMaria
FontesPiazza Keywords: Escherichiacoli Antimicrobialresistance Dogs Geneticrelatedness
a
b
s
t
r
a
c
t
AntimicrobialresistanceinEscherichiacoliisolatedfrompetdogscanbeconsidereda
poten-tialthreatofinfectionforthehumanpopulation.Ourobjectivewastocharacterizethe
resistancepattern,extendedspectrumbeta-lactamaseproductionandgeneticrelatedness
ofmultiresistantE.colistrainsisolatedfromdogs(n=134),theirowners(n=134),andhumans
whoclaimtohavenocontactwithdogs(n=44,control),searchingforsharingofstrains.The
strainswereassessedfortheirgeneticrelatednessbyphylogeneticgroupingandpulsed-field
gelelectrophoresis.MultiresistantE.colistrainswereisolatedfrom42(31.3%)fecalsamples
frompairsofdogsandowners,totaling84isolates,andfrom19(43.1%)controlgroup
sub-jects.Thestrainsshowedhighlevelsofresistancetoampicillin,streptomycin,tetracycline,
trimethoprimandsulfamethoxazoleregardlessofhostspeciesorgroupoforigin.TheblaTEM,
blaCTX-M,andblaSHVgenesweredetectedinsimilarproportionsinallgroups.Allisolates
pos-itiveforblageneswereESBLproducers.ThephylogeneticgroupAwasthemostprevalent,
irrespectiveofthehostspecies.NoneofthestrainsbelongingtotheB2groupcontainedbla
genes.Similarresistancepatternswerefoundforstrainsfromdogs,ownersandcontrols;
furthermore,identicalPFGEprofilesweredetectedinfour(9.5%)isolatepairsfromdogsand
owners,denotingthesharingofstrains.Petdogswereshowntobeapotentialhousehold
sourceofmultiresistantE.colistrains.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:doutoracristiane@ig.com.br(A.C.Carvalho).
http://dx.doi.org/10.1016/j.bjm.2015.11.005
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
Introduction
Theimplicationsofmicrobialresistanceforpublichealthhave
increasedtheinterestofthescientificcommunityinthe
pres-enceandcirculationofresistantorganismsbetweenpetsand
thehumanpopulation.Amongpets,dogsshareaclose
prox-imitywithhumans,andproximityisknowntoincreasethe
possibilityoftransmitting resistant bacteriabetweenthese
hostspecies.Itisimportanttonotethatthebacterialabilityto
transfergeneticcassettes,whichconferresistancetoseveral
classesofdrugs,andobservationsofthespreadofresistance
haveincreasedinrecentyears.1,2 Themolecular
character-izationofantimicrobialresistancecouldbeveryuseful not
onlyinsurveillancestudiesandmonitoringandtrackingof
multidrug-resistantstrainsbutalsoinobtaininginformation
aboutcommonalityamonghumanandanimalbacterial
iso-lates.
Escherichia coli is characterized by a substantial genetic
diversity,broadhostrange,versatilityinpathogenicpotential,
anddistribution betweenhostsinthe environment.3 E. coli
isolates,commensal or pathogenic,obtainedfrom humans
andotheranimals,havebeenextensivelystudiedand
char-acterizedintermsoftheirdrugresistanceprofilesbasedon
their phenotypic sensitivity to various antimicrobialdrugs
as well as by their genetic resistance to major classes of
antibiotics detected by molecular assays of genetic
simi-larities amongisolates.4–6 Similarities inresistanceprofiles
amongisolateshavebeendescribed,andthemolecular
char-acterizationoftheseisolateshasdemonstrated substantial
differencesdependingonthepopulationandthegeographic
regionoforigin.Ingeneral,comparisonsofresistanceprofiles
are performedonsamplesofindividuals who are not
nec-essarilycohabiting and are therefore notepidemiologically
related. Further studies are needed tocharacterize related
humanand dog isolatesin Brazil aswell as toinvestigate
thepossibilityofhumansandcaninessharingmultiresistant
strains.Theaimofthisstudywastocompareresistance
pro-filesofisolatesofE.colifromtheintestinaltractsofdogsand
theirownersandtoassessthepresenceofextended-spectrum
beta-lactamase(ESBL)genesinE.coliisolatesrecoveredfrom
dogsandhumanslivinginthesamehousehold.Inaddition,
weintendedtocharacterizethegeneticrelatednessamong
antimicrobial-resistantisolates.
Materials
and
methods
Studydesign
Atotalof134fecalsamplepairsfromdogsandtheirownersof
householdslocatedinthecityofRiodasOstras(RiodeJaneiro,
Brazil)werestudied.Everyowner(>18yearsold)chosenfor
thestudyhadonlyonedog.Acontrolgroupof44fecal
sam-plesfromindividualswhoclaimedhavenocontactwithdogs
wasalsoincludedinthestudy.Thesampleswerecollected
foroneyear(2010),andthehumananddogparticipantshad
notbeenusingantimicrobialsforatleastthreemonthsprior
tothe study.Bacterial strains were selectedusing a
proto-colforgrowthinselectivemediacontainingantibiotics.The
protocolofthisstudywassubmittedtotheResearchEthics
CommitteeofHUAP/UFF(CAAE0146.0.258.000-09)andthe
Ani-malEthicsCommitteeoftheNAL/UFF(NAL00126-09),andit
wasapprovedandcertifiedbybothcommittees.
Bacterialsamples
Fecalsamplesonswabsfromdogsandhumanswereobtained
andprocessedfortesting.TypicalE.colicolonieswereselected
andtestedforantimicrobialsusceptibility.Afterenrichment
growth inE. colibrothcontaininggentamicin(8g/mL) and
cephalothin (32g/mL),the sampleswere culturedon
Mac-Conkey agar (Himedia, Mumbai, India) supplementedwith
gentamicin(8g/mL)andcephalothin(32g/mL)topromote
thegrowthofpotentiallymultidrug-resistantE.colistrains.7
Samplesthatdidnotgrowwere discarded.Forquality
con-trolpurposes,eachsamplewasalsoinoculatedintomedium
withoutantibiotics.Threecolonies,believedtobeE.coli,were
chosenfromtheMacConkeyagarcontainingantibioticsand
identifiedbasedonstandardbiochemicaltests.8
Thecharacterizationportionofthisstudy includedonly
isolates that displayed multidrug resistance, which was
definedasresistancetotwoormoreclassesofdrugs.With
ourprotocolofselectiveisolationandsusceptibilitytesting,
onehundredandthreemultidrug-resistantE.coliisolateswere
identifiedfrom dogs(n=42),theirrespectiveowners(n=42)
andcontrolsubjects(n=19).
Antimicrobialsusceptibilitytesting
E.coliisolatesweretestedforantimicrobialsusceptibilityto
the drugsampicillin(AMP),amoxicillin+clavulanate(AMC),
cephalexin (CEF), chloramphenicol (CLO),
trimethoprim-sulfamethoxazole (SUT), streptomycin (EST)+gentamicin
(GEN), doxycycline (DOX)+tetracycline (TET), ciprofloxacin
(CIP),ceftazidime (CAZ),ceftriaxone (CRO),cefepime(CPM),
cefotaxime(CTX)andaztreonam(ATM)usingClinical
Labora-toryStandardsInstitute(CLSI)methodologiesandinterpretive
criteria.9,10
DiskapproximationtestsusingthedrugsAMC,CAZ,ATM,
CTXandCPMwereperformedonallisolatestoscreenforESBL
producersaspreviouslydescribed11andaccordingtoCLSI
rec-ommendations(2012 S22).ThebacterialstrainsE.coliATCC
25922andKlebsiellapneumoniae700603wereusedascontrol
strains.
Characterizationandidentificationofbeta-lactamase genes
Thepresenceofthebeta-lactamase(bla)genesblaTEM,blaCTX-M
and blaSHV was detected by polymerase chain reactions
(PCR)inthird-andfourth-generationcephalosporin-and
ATM-resistantstrainsclassifiedasthoseexceedingthebreakpoints
recommendedbytheCLSI(2012)andthosethattestedpositive
indiskapproximationexperiments.11Theprimersand
ampli-ficationconditionsusedinPCRassaysarelistedinTable1.The
bacterialstrainsusedaspositivecontrolsinPCRreactionsfor
thedetectionofblageneswere:E.coliH21,E. coliA41
(pre-viouslycharacterized12),andK.pneumonia(ATCC700603)for
genesblaTEM,blaCTX-MandblaSHV,respectively.E.coliDH5␣was
Table1–PrimersandamplificationconditionsusedinPCRreactionsforthedetectionofESBLgenesandphylogenetic characterization.
Geneortargetregion Primers Ampliconsize(bp) Annealingtemperature(◦C) Reference
blaTEM T1ATTCTTGAAGACGAAAGGGCCT 1100 55 Wiegandetal.14 T3TTGGTCTGACAGTTACCAATGC
blaSHV S1ATGAGTTATATTAGAATGGT 860 58 Fuetal.15
S2GTTAGCGTTGCCAGTGCTCG
blaCTX CTM-MACGCTTTGCGATGTGCAG 550 60 Bonnetetal.16
CTX-MBACCGCGATATCGTTGGT chuA GACGAACCAACGGTCAGGAT 279 55 Clermontetal.13 TGCCGCCAGTACCAAAGACA yjaA TGAAGTGTCAGGAGACGCTG 211 ATGGAGAATGCGTTCCTCAAC tspE4.C2 GAGTAATGTCGGGGCATTCA 152 CGCGCCAACAAAGTATTACG
Determinationofphylogeneticgroups
TheE. coli isolateswere subjected tophylogenetic
classifi-cationaccordingtothemethodologydescribedbyClermont
etal.13basedonstandardPCRamplificationofthegenesyjaA,
chuAandaDNAfragmentTspE4.C2.Theprimersand
amplifi-cationconditionsusedareshowninTable1.
PFGEassays
Paired isolates of E. coli from dogs and owners presenting
similarprofilesofresistanceandESBLresistancegeneswere
selected forpulsed field gel electrophoresis (PFGE) ofXbaI
digested total DNA according to a standardized
method-ology of the Centers for Disease Control and Prevention
(CDC) (http://www.cdc.gov/pulsenet/pathogens/index.html).
ThegeneratedPFGEprofilesofwithin-householdisolateswere
compared according criteria proposed by Tenover17:
indis-tinguishable(I) – nodistinct bands; closely related (CR)-2-3
distinct bands; possibly related (PR) – 4–6 distinct bands;
different(D)–morethansevendistinctbands.Restriction
frag-mentprofileswerealsocomparedbyusingtheBioNumerics®
software version6.6 (Applied Maths, Austin, TX), using the
DicesimilarityindexandtheUPGMA(UnweightedPairGroup
MethodwithArithmeticMean)clusteringmethodtoconstruct
the dendrograms. E. coliisolates with≥94% PFGE similarly
wereconsideredtobeclonal.5
Statisticalanalysis
Theprevalenceofresistancetoantimicrobials,thepresenceof
ESBLgenesandthedistributionofphylogeneticgroupsamong
canine,humanand control groupsamples were compared
usingtheChi-squaredtestwithp≤0.05usedasthecriterion
forstatisticalsignificance.
Results
and
discussion
Multiresistant E. coli strains were selectively isolated from
42 (31.3%)fecal sample pairs from dogs and their owners,
totaling84 isolates,andfrom 19(43.1%)controlgroup
sub-jects.Thefrequencyofmultiresistant E. coliobtainedfrom
fecalsamplescanreach89%,accordingtosomestudies,with
frequenciesthatvaryaccordingtotheselectioncriteria.18–21
To recover strains that exhibited potential resistance to
multiple antimicrobials, we used cephalothin. Cephalothin
resistance is clinically significant because it is frequently
found insituations without strong selectivity (water
reser-voirs and non-hospital locations). Cephalothin resistance
is alsoassociatedwithother resistancemarkers, including
markersforchloramphenicol,tetracycline,and
trimethoprim-sulfamethoxazole,whichareconsideredcommonmarkersin
ourcategorization.Gentamicinwasalsousedinour
identi-ficationofmultidrugresistance,duetothehighcorrelation
betweengentamicinresistanceandmultiresistance profiles
in nosocomial strains.7 Because of this selective isolation
protocol, some samples did not grow in the presence of
the antimicrobialsmentioned,and thus we may have lost
some multiresistant strains sensitive to the drugs used in
theselection.Alloftheisolatesincludedinourstudy,
how-ever,wereresistanttoatleasttwodrugclasses,presumably
becausetheywereallsubjectedtoaselectiveisolation
pro-cedure. When the same resistanceprofilewas observedin
a dogand ownerpair, adifferent methodofselectionwas
used toenhance the detection of lower prevalenceclones.
Thismethod,describedbyDamborgandcolleagues,22entails
selecting coloniesthatgrowinthe inhibitionzonesformed
bythedrugsamikacin,gentamicin,ampicillin,ciprofloxacin,
cefotaxime, and sulfamethoxazole-trimethoprim on
Mac-Conkey agar, and submitting these for identification. This
wasdonetoenhancetheidentificationofmultidrug-resistant
strainscommontobothdogsandownersofsubjectpairs.
Among the resistant E. coli fecal isolates, 74 patterns
were observed, each with resistance to at least two drug
classes (data not shown). Across all groups, the most
fre-quentresistancepatternswereAMP/EST(13.6%)followedby
AMP/EST/SUT (3.9%). There were no significant differences
intheresistancefrequenciesamongthetesteddrugsexcept
for sulfamethoxazole-trimethoprim for which the human
isolatesfromownersandcontrolgroupsubjectshad
statis-ticallyhigherresistancefrequenciesthanthecanineisolates
(p≤0.05).Otherstudiesshowedfewpatternscomparableto
our data.21–23 Inasimilar study,Skurniket al.24 compared
fecalE.colifromdifferentanimalspeciesandnotedthatthe
Table2–NumbersofresistantE.coliisolatesoffecalorigin(humansanddogs)forthedrugstested.
Antimicrobials Numberofresistantisolates(%) Dogs n=42 Humans(owners) n=42 Humans(control) n=19 AMP 36(85.7) 31(73.8) 17(89.4) AMC 15(35.7) 10(23.8) 4(21.1) CFE 14(33.3) 13(30.9) 8(42.1) CAZ 7(16.6) 3(7.1) 4(21.1) CPM 4(9.5) 3(7.1) 3(15.8) CTX 8(19.4) 6(14.3) 2(10.5) CRO 9(21.4) 6(14.3) 5(26.3) ATM 7(16.6) 6(14.3) 5(26.3) GEN 13(30.9) 17(40.5) 11(57.8) EST 28(66.6) 27(64.3) 11(57.8) CLO 10(23.8) 14(33.3) 5(26.3) CIP 5(11.9) 9(21.4) 2(10.5) SUT 13(30.9) 23(54.7) 12(63.2) TET 21(50.0) 22(52.3) 11(57.8) DOX 14(33.3) 11(26.2) 5(26.3)
AMP,ampicillin;AMC,amoxicillin+clavulanicacid;CFE,cephalexin;CAZ,ceftazidime;CPM,cefepime;CTX,cefotaxime;CRO,ceftriaxone; ATM,aztreonam;GEN,gentamicin;EST,streptomycin;CLO,chloramphenicol;CIP,ciprofloxacin;SUT,sulfamethoxazole+trimethoprim;TET, tetracycline;DOX,doxycycline.
thehighestlevelsofmultiresistantstrainslikethosefoundin humans.Thesedataaresimilartothoseobtainedinourstudy. In our study, the highest frequency of resistance was recorded for ampicillin, tetracycline, streptomycin, and trimethoprim-sulfamethoxazole (Table 2). Resistance
to streptomycin, ampicillin, tetracycline, chloramphenicol
and trimethoprim-sulfamethoxazole has been observed in
otherstudiesofdogfecalisolates18–21,23,25andhumanfecal
isolates.26,27 Aminopenicillins and aminoglycosides appear
most often among the three classes of drugs with high
frequencies of resistance in studies of samples of animal
origin.6,21,23,28–34Strainsresistanttothecarbapenemstested
inthisstudywerenotdetected.
Importantly, the strains analyzed in this study were
selected using media containing antibiotics, and although
resistancerates may havebeen higher than reported
else-where, the prevalence data, especially for ampicillin and
streptomycin,weresimilartothoseobservedinotherstudies.
Stenskeetal.5alsocomparedtheresistanceprofilesoffecal
strainsfromdogsandtheirowners,obtaininghigh
frequen-ciesofresistancetocephalothin,ampicillin,andstreptomycin
inbothgroups,aresultcomparabletoourcurrentfindings,
althoughourfrequencieswerehigherandweusedcephalexin
ratherthancephalothin.Haradaetal.35observedsimilarities
amongisolatesfromdogsandhumans;however,theyshowed
significantdifferencesbetweenthegroupsanalyzedinterms
ofwhichdrugscorrespondedtothehighestratesofresistance.
Comparisonsof strains of clinical origin has also been
afocus ofstudy oftheepidemiologyofantimicrobial
resis-tanceandhasshownthatthedrugsampicillin,streptomycin
andsulfamethoxazole-trimethoprimexhibithighfrequencies
ofresistancein strains ofbothhuman origin36–38 and dog
origin.6,28–33 With regard to isolates from dogs, resistance
tothefirst-generationcephalosporins,includingcephalothin
andcephalexin,wasalsoidentifiedinthestudiescited.
Dif-ferences in the proportions of resistancehave been noted
in relationtothe site ofinfection, forexample,in urinary
infection,pyometraandosteomyelitis,inwhichdifferent
lev-elsofresistancetothesamedrugswereencountered.18,39–41
Some studies characterizing resistance in strains
originat-ing from humanpatients haveanalyzed the prevalenceof
resistant infections in samples from hospital patients and
outpatients.42Thesestudiesshowmanydifferencesin
resis-tanceprevalence,relatednotonlytotheoriginofthesamples
but also to the site of infection, the continent and
coun-trywherethestudytookplaceaswellastheprofilesofthe
patientsfromwhichthesamplesoriginated.Differencesare
alsoattributedtotherepertoireofantibioticsusedinthestudy.
In the current study, 28.5% of the E. coli isolates from
dogs were resistant to atleast one antibiotic belongingto
thethird-orfourth-generationcephalosporinsandwere
posi-tiveforESBLbydisk-approximationtest.Thisprofileoccurred
in 16.6% and 36.8% of isolates from dog owners and
con-trol subjects,respectively.These differences,however,were
not significant. Resistance to third-and fourth-generation
cephalosporinsissuggestiveofbeta-lactamaseproductionby
theisolates.Inourtests,wedetectedbeta-lactamresistance
genesoftheblaTEM,blaCTX-MandblaSHVfamiliesinisolatesof
bothhumananddogorigin(Table3).
WedetectedatleastoneESBLencodinggenein26.2%of
theE.coliisolatesandallofthemwere alsopositiveinthe
phenotypictests.However,fiveisolates(8H,9H,57H,22F,38F),
despitepresentingphenotypessuggestiveofESBLproduction,
didnottestpositiveforanyofthethreeblagenessurveyedin
ourstudy.OtherESBLgenesmaybepresentinthesestrains
becausetheOXAandCMYgenefamiliesarealsocommonin
E.coli.43
The blaCTX-M gene was significantly more prevalent in
strainsfromthecontrolgroupthanintheothertwogroups;
furthermore,allESBLpositiveisolatesinthisgroupwere
pos-itiveforCTX-M.Indogisolates,theblaTEMgenewaspresent
Table3–PhylogeneticgroupsandESBLgenesamongfecalE.coliisolatesfromdogs,theirowners,andcontrolhuman subjects.
Class Type NumberofIsolates(%) Total(%)
Dogs(n=42) Owners(n=42) Controls(n=19)
Phylogeneticgroup A 21(50) 24(57.1) 10(52.6) 55(53.4) B1 09(21.4) 08(19.1) 02(10.5) 19(18.4)
B2 03(7.2) 04(9.5) 00(0) 07(6.9)
D 09(21.4) 06(14.3) 7(36.9) 22(21.3)
ESBLgenea blaTEM 3 4 0
blaTEM/blaCTX-M 4 1 3
blaCTX-M 4 2 4
blaTEM/blaCTX-M/blaSHV 1 1 0
Total 12(28.6) 8(19.0) 7(36.8) 27(26.2)
a AllisolatescarryinganESBLgenewerealsopositiveinphenotypictests.
theblaCTX-Mgenewasdetectedin75%oftheseisolates.Only
8.4%ofthedogisolateswerepositivefortheblaSHVgene.In
humanstrains,theprevalentESBLgenefamilywastheblaTEM
family.TheblaCTX-Mgenewaspresentinsimilarproportions
instrainsofhumananddogoriginfromthesamehousehold.
TheblaSHVgenewasdetectedin8.4%ofthehumanisolates.
ThedetectionofESBLgenesinisolatesofpetdogswasfirst
publishedin1988.43Otherstudieshavealsoreportedthe
pres-enceoftheblagenesinisolatesfromdogs,especiallyisolates
of clinical origin. Different subtypes of the bla gene
CTX-M are oftendetectedin extraintestinal clinical isolates44,45
andinfecalisolates.22,29,46Thebla
TEMgene,detectedin
clin-icalstrainsfromdogssince2002,6,28,44,45,47wasalsofoundin
intestinalisolates.19,21,29,46Forthegenebla
SHV,dataare
simi-lar,withdetectioninbothclinical29,44andfecalisolates.6,29,48
TheprevalenceofthesegenesindogE.colistrains,however,
isnotwellestablished.
The prevalenceof ESBL genes in human clinical
speci-mens varies considerably, depending on the origin of the
strains,whethertheinfection originatedinahospitalorin
acommunitysetting,andonthesiteofinfection.Generally,
isolatesfrom infections acquired inhospitalsexhibit more
complexprofileswithbroaderrepertoiresofresistance
com-paredtoisolatesofcommunity-acquired infections.Recent
data indicate a prevalence of ESBL blaCTX-M genes in the
bloodstream49,50andinotherinfectionsinhospitalsettings.51
Less than a decade ago,TEM and SHV ESBL isolates were
thoughttobelimitedtothehospitalenvironment,and
CTX-MESBLisolates weredetectedin urinarytract infections in
community settings.52 Currently, the most prevalent ESBL
geneidentifiedinisolatesacrossmanycountriesisthe
CTX-Mgene,particularlytheCTX-M-15gene,whichisassociated
withIncFvariantplasmids.53 Theepidemiologicalprofileof
highly mutable enterobacteria can be considered an
addi-tionalfactorinthisvariability,whichgreatlyinfluencedthe
spread of plasmids and other transposable elements
act-ing asmobile carriersof multipleresistancegenes against
drugs,includingaminoglycosides,chloramphenicol,
tetracy-cline,andtrimethoprim-sulfamethoxazole.54
Inourstudy,thedistributionofphylogeneticgroupsinthe
hostspeciesexaminedwassimilarinallgroups,withthe
phy-logeneticgroupAbeingthemostprevalent(Table3),occurring
in50%ofthecaninestrains,57.2%ofthehumanstrainsand
52.6% ofthecontrolgroup.For fecalstrains, this findingis
consistentwithliteraturethatdescribesthemajorityof
iso-latesfromcommensalstrains,regardlessoftheirrepertoireof
resistance,belongingtogroupsAorB1.55
Thedistributionofphylogroupsamongdogisolates,
how-ever, has been shown to differ in terms of prevalence
in extraintestinal, commensal and clinical samples. In the
caseofextraintestinalisolates,wherethepredominant
phy-logroupswereexpectedtobegroupsB2andD,Gibsonetal.32
foundno isolatesbelongingtogroupB2,whereasMaynard
et al.6 reported a 63% frequency of the B2 phylogroup in
extraintestinal samples.Moreover,commensalstrains from
animalsingeneralpredominantlyincludedthegroupsAand
B1.FecalstrainsfromdogsinastudybyHaradaetal.35showed
a highprevalenceofgroup B2.Daviset al.25 alsoobserved
a predominanceofB2(and D)intheirisolatesfrom
differ-entanatomicalregionsofhealthydogs,includingtherectal
area.
Studies comparing humanand canine isolatesin terms
ofantimicrobialresistanceprofiles usedsamplesofclinical
origin, whichhad ahigher prevalenceofgroups B2and D,
as expected. However, Harada et al.35 analyzed fecal
sam-plesfromdogsandtheirownerswithoutusingantimicrobial
agents for selective isolation, and they observed a higher
prevalenceofgroup B2,whereas Damborget al.22 reported
that intheir experiments,humanfecalisolatesmoreoften
belongedtogroupAandthedogisolatestogroupB1.Astudy
byClermont etal.4 showedthatextraintestinal infectionis
mostlycausedbyE.colistrainsofgroupB2.Anassessment
ofextraintestinalclinicalisolatesfromdogsrevealedthatthe
twomaingroupsofE.coliresistanttofluoroquinolonesareA
andD.56DatareportedinastudybySkurniketal.,57however,
suggestedthatclinicalisolatesfromgroupB2arelessresistant
toantimicrobialsthannon-B2isolates.
Inourtests,12ESBLisolatesbelongedtogroupAand10
togroupD,andnoESBLisolatescarryingthegenesblaTEM,
blaSHVandblaCTX-MwereincludedingroupB2.Ourdata
sug-gestthatresistantcommensalE.colistrainsdonotnecessarily
belongtothephylogroupsexpectedfromresistantpathogenic
strains.Theexplanationforthisobservationrequiresfurther
Table4–Resistance,ESBLgenesandgeneticrelatednessofpairsofE.coliisolatesoffecalorigin(dogsandhumans withinahousehold)withsimilarprofiles.
Isolatea Hostspecies Resistancephenotypeb ESBLgenes Phylogroup PFGEc
blaTEM blaCTX-M blaSHV
15F Dog AMPAMCCFECAZCTXATM − + − D −
15H Human AMPAMCCFECTXCRO − + − A −
23F Dog AMPESTCLOSUTTET − − − D I
23H Human AMPESTCLOSUTTET − − − A I
27F Dog AMPCFEEST − − − B1 D
27H Human AMPCFEEST − − − A D
48F Dog AMPCFECAZCTXCROGENESTCLOSUT + + − A I
48H Human AMPCFECTXCROGENESTCLOSUTTET + + − B1 I
49F Dog AMPAMCCFECPMCTXCROGENEST
CLOCIPSUTTET
− + − B1 D
49H Human AMPCFECPMCTXCROGENESTCLOSUT TET
+ − − B1 D
59F Dog AMPEST − − − B1 D
59H Human AMPEST − − − B1 D
61F Dog AMPAMCESTCLOSUTTETDOX − − − B2 I
61H Human AMPAMCESTCLOSUTTETDOX − − − B2 I
58F Dog AMPAMCESTCLO + − − B1 −
58H Human AMPESTCLO − − − B1 −
92F Dog AMPCAZATMGENESTCIPSUTTETDOX + + + B1 I
92H Human AMPATMGENESTCIPSUTTETDOX + − + B1 I
a TheletterFdesignatescaninefecalisolates;H,humanfecalisolates(dogowners).
b AMP,ampicilin;AMC,amoxicillin+clavulanicacid;CFE,cephalexin;CAZ,ceftazidime;CPM,cefepime;CTX,cefotaxime;CRO,ceftriaxone;
ATM,aztreonam;GEN,gentamicin;EST,streptomycin;CLO,chloramphenicol;CIP,ciprofloxacin;SUT,sulfametoxazol+trimethoprim;TET, tetracycline;DOX,doxycycline.
c Tenovercriteriaappliedforeachisolatepairfromthesamehousehold:I,indistinguishable;D,different;−notpossibletocompareduethe
DNAdegradationofoneisolateduringthePFGErestriction.
ThepresenceofESBLgeneswassimilarinE.coliisolates
fromdogsandfromtheirowners,especiallyinsomehuman
anddog isolatesfromthe samehousehold. Thus,18E. coli
isolatescomprisingnineisolatepairsfromdogsandowners
thatpresentedsimilarantimicrobialresistance,ESBLgenes
andphylogeneticgroupprofileswereselectedforPFGEassays
(Table4).ByapplyingtheTenovercriteriatoeachisolatepair
fromthesamehousehold,wedetectedfourisolatepairswith
identicalpatterns.ThedendrogramconstructedwiththePFGE
profilesoftheseisolatesreinforcestherelatednessbetween
thesefourstrainpairs,anditshowsastrongsimilaritytoan
isolatepairwithanotherdogisolate(Fig.1).
Some strains recovered from dogs and owners showed
phenotypicandgenotypicsimilaritiessuggestingaclonal
rela-tionship.Indeed,wedemonstratedinthisstudythesharing
ofmultiresistantE.colistrainsin9.5%(4/42)ofthepairsof
isolatesfromthedogsandtheirownersthatwestudied.
IncomparisonsbetweenE.coliisolatesofhumananddog
origin, many similarities have been identified in the
vari-oustraitsstudied,especiallywithregardtotherepertoireof
Fig.1–DendrogramshowinggenomicPFGEfingerprintpatternsoffecalE.coliisolatesfromdogs(F)andowners(H)with
similarresistanceprofiles.ThedendrogramwasconstructedusingtheDicesimilaritycoefficientandUPGMAclustering
virulenceofthestrains.5,6,35,58,59 Ithasbeensuggestedthat
the hostspecificity of E. colistrains is determined by
spe-cificadhesions.4Studieshaveshownasimilaroccurrenceof
virulencefactorsassociatedwithextraintestinalpathogenic
E.coliinstrainsisolatedfromdogsandhumans.6,58,60Some
studiessuggestthaturopathogenicE.colistrainsareshared
betweendogsandhumans,58whereasothersshowa
signif-icantdiversityinthevirulencerepertoiresofstrainsisolated
fromdistinctspeciesorevenwithinthesamespecies.6,35
Regardingthegeneticsofresistance,the dataare
some-whatheterogeneousanddifferdependingonthepopulation
andthegeographicregionoforiginaswellasonwhetherthe
strainsarecommensal orpathogenic.5,35,48 Despitethefact
thatapositivecorrelationbetweenthepresenceofplasmids
andintegronswithamultiresistantphenotypeisdescribedin
severalstudies,24,61 additionalfactorsthatshapethegenetic
structureofthesestrainsareimportant.55,62Comparisonsof
resistanceprofilesarenotalwaysperformedonstrainsfrom
cohabitingindividuals,andsamplesarethereforenotalways
epidemiologicallyrelated.Thecharacterization,therefore,of
relatedhumanandcanineisolates,aswellasthepossibility
ofsharingmultiresistantstrainsbetweenhumansanddogs,
requiresfurtherstudyinBrazil.
Conclusion
We detected the simultaneous presence of
multidrug-resistantE.coliindogsandtheirowners,albeitwithdifferent
phenotypic profiles of antibiotic resistance, especially for
ESBLs.Furthermore,somestrainsfromdogsandhumansof
the same householdhad similar resistance patterns,ESBL
genes,identicalphylogeneticgroupsandidenticalorclosely
relatedPFGEprofiles.These datademonstratehigh degrees
ofhomologyandthereforethepossibilitythatresistantE.coli
clonesarecirculatingbetweenindividualsandanimalsinthe
sameenvironment.Indeedsomestrainsshowedclonal
rela-tionshipsindicatingwithin-householdsharing.
In addition, our data suggest that the prevalence of
antimicrobial-resistantcommensal E.coli isvery similar in
dogsandhumansandthatthereisnodifferenceamongthe
resistantE.coliisolatedfromdog-ownersandhumanswithout
petdogs.
Thisareaneedsmoreaccurateinvestigations andmany
uncertainties remain regarding how resistance arises and
spreadsintheenvironmentandamongcohabitinghumans
anddomesticanimals.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Thisstudy was supportedbya Grantfrom State ofRiode
JaneiroResearchFoundation(FAPERJProcE-26/111.520/2010).
WealsothankDr.FelipeP.Gonc¸alvesNevesfortheirsupport
inthedendogramsconstructions.
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