h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Short
communication
Characterization
of
avian
pathogenic
Escherichia
coli
isolated
from
free-range
helmeted
guineafowl
Mariana
Monezi
Borzi
a,∗,
Marita
Vedovelli
Cardozo
a,
Elisabete
Schirato
de
Oliveira
a,
Andressa
de
Souza
Pollo
b,
Elisabete
Aparecida
Lopes
Guastalli
c,
Luis
Fernando
dos
Santos
d,
Fernando
Antonio
de
Ávila
aaUNESP–UniversidadeEstadualPaulista,FaculdadedeCiênciasAgráriaseVeterinárias,DepartamentodePatologiaVeterinária,
ProgramaemMicrobiologiaAgrícola,Jaboticabal,SP,Brazil
bUNESP–UniversidadeEstadualPaulista,FaculdadedeCiênciasAgráriaseVeterinárias,DepartamentodeMedicinaVeterinária
PreventivaeReproduc¸ãoAnimal,SãoPaulo,SP,Brazil
cInstitutoBiológico,CentroAvanc¸adodePesquisaTecnológicadoAgronegócioAvícola,SãoPaulo,SP,Brazil dInstitutoAdolfoLutz,CentrodeBacteriologia,SãoPaulo,SP,Brazil
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r
t
i
c
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e
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o
Articlehistory:
Received7February2018
Accepted19April2018
Availableonline11August2018
AssociateEditor:NiltonLincopan
Keywords: APEC Virulencefactors Colibacillosis Zoonoticpotential Numidameleagris
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AvianpathogenicEscherichiacoli(APEC)isolatesfromapparentlyhealthyfreerangehelmeted
guineafowlwerecharacterized.Mostofthemhada highfrequencyofvirulence
associ-atedgenes,multidrugresistanceandhighpathogenicity.Wedemonstratedthathelmeted
guineafowlhavepotentialtotransmitantibioticresistantAPECtootherspeciesincluding
humans.
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
The Avian Pathogenic Escherichia coli (APEC) strains are
responsibleforalargenumberofextra-intestinaldiseasesin
birds,eitherlocallyorassystemicinfections;thosearetheso
calledaviancolibacillosisandareresponsible foreconomic
lossesfortheworld’spoultryindustries.1Inaddition, APEC
strains are a subpathotype of pathogenic extra-intestinal
bacteria(ExPEC),categoryresponsiblefordiseasesinhumans
∗ Correspondingauthor.
E-mail:[email protected](M.M.Borzi).
and animalssuchasurinary tractinfection (UTI),neonatal
meningitis and septicemia,2 suggesting that APEC strains
maybeapotentialzoonoticpathogens.3
Healthybirdscaneliminatestrainsthatharborsvirulence
genestotheenvironment,thusbecomingasourcesof
infec-tion tootheranimals andtohumans.4,5 Inthis regard,the
increaseofdrugresistantstrains,duetotheindiscriminate
https://doi.org/10.1016/j.bjm.2018.04.011
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
There is no consensus in the literature to define the
APECpathotypeand thedevelopmentofmethodologiesfor
itsdiagnosis arefault;mainlyduetoit beavery
heteroge-neousgroupofmicroorganisms, inwhich differentisolates
canharboradifferentassociationsofvirulencefactors(VFs),
eachcapableofinducingaviancolibacillosis.9Also,free-range
chickens and human often share the same environment,
in this sense, these birds and their products (meat and
eggs) can act as ExPEC sources of infection to humans.10
Like any source of animal protein, helmeted guineafowl
may act as a possible carrier of pathogenic
microorgan-isms to humans; however, until now, no studies focused
on this species as been possible carriers. Thus, we
per-formedthisstudy todetect andcharacterizedrug-resistant
pathogenic E. colicarrying virulencegenes relatedto APEC
pathotype infree-range helmeted guineafowl and clarified
howthesebirdscanberelevantinhumansandotheranimal’s
infections.
ThisstudywasapprovedbytheCommitteeonEthicsfor
theUseofAnimalsof(CEUA),protocolnumber19.008/16.
Sam-pleswerecollectedfrom56freerangehelmetedguineafowl
withouthistoryoftheuseofantibiotics,ofunknowngenetic
origin and of different age groups from 4 small farms in
Jaboticabalcity, Sao Paulo State, Brazil.In the first farm 6
sampleswascollected,12samplesinthesecondfarm,48
sam-plesinthethirdfarmand 46inthefourthfarm,totalizing
56 samples from the cloaca and 56 from the
oropharyn-gesthatwereobtainedwiththeaidofsterileswabs,which
were placed insterile tubes, containing 5mLof BHI broth
(BrainHeartInfusion)andkeptinunder refrigerationuntil
processed.
From the swabs inoculated into BHI and incubated at
37◦C for 16h, a Polymerase Chain Reaction (PCR)
screen-ing was performed for the detection of the cvaC,iroN, iss,
iutA, ompT and hlyF genes. Protocols for the DNA
tem-plate preparation, PCR and primer oligonucleotides for
thesegenes weretaken from the EcLprotocol,availableat
http://www.apzec.ca/en/APZEC/Protocols/APZECPCRen.aspx.
Then,samplesthatwerepositiveforatleastfiveoftheabove
citedgeneswereusedtodetectE.coliisolates.11,12Inaddition,
obtainedisolatesweresubjectedtoanewPCRstodetectthe
followingadditional 11 virulence genes: sitA, tsh, traT, vat,
astA,iucC,iucD, papC, irp2, fimHand fyuA.Thefrequencies
ofVAGswerecomparedwithFisher’sexacttestusingPrism
forWindowsversion6.01(GraphPadSoftware).Associations
were considered statistically significant if the calculated
P-valuewas<0.05.
E.coliphylogroupsweretypingbyPCRphylotypingmethod
of Clermont et al. (2013)13 targeting chuA and yjaA genes
andTspE4.C2DNAfragment.SerotypingoftheAPECstrains
were performedusing somatic(O1-O181)and flagellar
(H1-H56)antigensthatwereproducedattheBacteriologyCenter
oftheAdolfoLutzInstitute–SaoPaulo,Brazil.Additionally,
the isolates antimicrobial susceptibility was performed by
disc diffusionmethod.14 Antimicrobials testedwere:
ampi-cillin (amp) (10g), cephalothin (cep) (30g), streptomycin
(str)(10g),gentamicin(gen)(10g),ciprofloxacin(cip)(5g),
(25g),ceftiofur(ctf)(30g),ceftriaxone(cro)(30g),amikacin
(ami)(30g),cefoxitin(cfo)(30g),kanamycin(kan)(30g),
amoxicillin+clavulanic acid (amc) (30g), norfloxacin(nor)
(10g) andfosfomycin(fos)(50g).E.coliisolateswerealso
screenedforextended-spectrumbeta-lactamase(ESBL)genes
for blaCTX-M genotype groups 1,15 216 and 9,17 blaTEM and
blaSHV.18,19
PathogenicitytestwasdeterminedaccordingtoGuastalli
et al.(2013).20 Bacterial culture(0.1ml) wasinoculated into
theleftthoracicairsacofday-oldchicks.Forinoculum
prepa-ration,acolonyofeachbacterialstrainwasseededin10ml
of BHIbroth, incubated for 18h at37◦C and subsequently
dilutedtoa1:10ratio.Inoculumconcentrationwas
standard-izedto107CFU/mL.TheE.coli(serogroupO1)belongingthe
LaboratoryofOrnithopathologyofUSP,wasusedasa
posi-tivecontrol.NegativecontrolbirdswereinoculatedwithBHI
brothonly.For each strain, aswellasforthe negativeand
positivecontrolgroups,tenmalechicksfromacommercial
lineagewereused.Thestrainswereclassifiedduetoits
mor-talityasfollow:high(≥80%),intermediate(>50%and<80%),
lowpathogenicity(≤50%)andnon-pathogenic(zero
mortal-ity).
Genomic DNAdigestionwithXbaI(Invitrogen,USA)was
performedasdescribedbyRibotetal.(2006)21with
modifica-tionsandaSalmonellaBraenderupH9812strainwasusedas
amolecular weightreference.Theelectrophoresisoccurred
at14◦C,ona1%PulsifieldCertifiedagarosegel,withinitial
timeof2.2s,finaltimeof54.2s,onagradientof6Vcmx−1,
anangleof120◦,for23h.Thefragmentssimilaritywas
com-pared using the Dicecoefficient at1% tolerance and 0.5%
optimizationandthedendrogramwascalculated usingthe
neighbor joining method with BioNumerics software
ver-sion7.1(AppliedMaths,Sint-Martens-Latem,Belgium).The
housekeepinggenesadk,fumC,gyrB,icd,mdh,purAandrecA
were amplified byPCRforthe MSLTanalysis. These genes
werechosenbasedontheMLSTprotocolforE.colifromthe
University ofWarwick, USA.22 The specific oligonucleotide
as well as the PCR amplification conditions are
avail-ableathttp://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/
primersColihtml. ThePCRproductswere sequenced inan
ABI 3100 sequencer (Applied Biosystems, Waltham, USA)
with Big Dye Terminator v3.1 kit (Applied Biosystems,
Waltham,USA).Theresultingsequenceswereanalyzedbythe
Phred/Phrap/Consedprogrampackage.23–25
AlltheresultsareshowninFig.1.Fromthe112samples,
21isolateswereobtained,20werefromcloacaandonefrom
theoropharynx.Theseisolateswerepositiveforatleastfive
oftheAPECrelatedgenes(cvaC,hlyF,iss,iroN,ompTandiutA)
andwere,further,subjectedtoanotherPCRforthedetection
ofadditionalvirulencegenes.IsolatedE.coligeneprofileis
describedatTable1.
Phylogenetic analysisrevealed that mostofthe isolates
(33.3%)belongtogroupB1,followed bygroupsA(19.0%),D
(14.3%),B2(9.5%),F(9.5%)andC(4.8%)andthattwoisolates
(4and7)couldnotbetyped.IsolatesbelongingtogroupsB2
andFwereassociatedwithahighernumberofvirulence
fac-tors,withameanof14.5and13.5perisolateofeachgroup,
Fig.1–AssociationbetweendendrogramanalysisofgeneticdiversitybyPFGEandvirulenceindicatorsofthe21APEC isolates.No:isolatenumber;Pa:pathogenicity;Ph:philogeny;H:high;I:intermediate;NP:non-pathogenic;L:low;unk: unknown;ST:stypes;CC:clonalcomplex,AR:antimicrobialresistance;amp:ampicillin;cep:cephalothin;str:streptomycin; gen:gentamicin;cip:ciprofloxacin;chl:chloramphenicol;tet:tetracycline;nit:nitrofurantoin;sut:
sulfamethoxazole+trimethoprim;ctf:ceftiofur;cro:ceftriaxone;ami:amikacin;cfo:cefoxitin;kan:kanamycin;amc: amoxicillin+clavulanicacid;nor:norfloxacin;fos:fosfomycin;ESBL:extended-spectrumbeta-lactamasegenes.
Table1–Frequencycorrelationofeach
virulence-associatedgene(VAGs)inthe21potentially APECisolates.
Function VAGs Frequency(%)
fimH 100.0%
Adhesion papC 4.8%
tsh 71.4%
fyuA 42.8%
iroN 95.2%
Ironacquisition iucC 61.9%
iucD 80.9%
irp2 42.8%
iutA 85.7%
sitA 100.0%
Hemolysis hlyF 100.0%
Serumresistance iss 95.2%
traT 100.0%
Toxins astA 9.5%
vat 14.3%
Other cvaC 90.4%
ompT 100.0%
Asingleisolateshowedresistancetoonlyone antimicro-bial.The remaining20 isolateswere resistanceto atleast threeantimicrobialsimultaneously and19oftheseisolates wereresistanttothreeormoreantimicrobialclasses,which represent90.4%ofmultidrugresistant.Thehighestdrug resis-tance was foundagainst cephalotin (100.0%),streptomycin (90.5%),ampicillin(71.4%)andtetracycline(61.9%).Asforthe antimicrobialclasses,42.8%oftheisolateswereresistantto penicillins,32.2%tocephalosporinsand40.5%to aminoglyco-sides.ESBLgenesforblaTEMwerefoundinresistantisolates3,
4,9,19and21andblaSHVinisolates5and20.
Thirteenofthe21samplesanalyzedwereOantigentypable (61.9%) and were within the following six serogroups: O2 (14.3%), O51 (19.0%), O11 (9.5%), O7 (3.2%), O8 (3.2%) and O9(3.2%).Oftheremainingstrains,seven(33.3%)werenon typable(NT)andtwo(9.5%)could notbecharacterizeddue toitsrough-lookingcolonies(RL).TheHantigencouldnotbe determinedinsixsamples(28.5%)duetotheybeenimmobile (IS).Theother16(76.2%)strainsweretypableandfive anti-genswereidentified:H4(28.5%),H14(23.8%),H25(9.5%),H9 (3.2%)andH10(3.2%).ThemostprevalentO:Hserotypeswere: O51:H14(19.0%)andNT:IS(19.0%)
The pathogenicity test revealed that 14 (66.6%) strains werehighlypathogenic(HP),three(14.8%)strainswere inter-mediate pathogenic (MP), three (14.8%) strains were low pathogenic (LP) and one (4.7%) was a nonpathogenic (NP) strain. Itwas observed thatall ofthe birds inthe positive control group died, while all birds of the negative control grouplived.Theclinicalsignsandmacroscopiclesionswere observedwithhigherfrequencywithhighandintermediate pathogenicstrains.NoneoftheVAGswerestatistically signif-icanttodifferentiatedHP/MPfromLP/NPstrainsbasedFisher’s test.
Thedendogramshowedtwolargeclusterswiththree sub-divisions and the 21 possible APEC isolates generated 18 differentpulsotypes,ofwhichonlythreewereshared,those been:1and2,10and11,13and15.Sixteensequencetypes (ST)wereidentifiedasfollow:ST624,ST58,ST1406,ST46,ST 88,ST6496,ST155,ST1727,ST93,ST3851,ST3580,ST117,ST 83,ST641andanewone(ST)thatcorrespondedtotheisolate 21.
Therearenodataabouthelmetedguineafowlpotentialas sourcesofAPECinfectionforotheranimals.MoreoverAPEC virulencemechanismsarenotyetfullyunderstoodanditis knownthatAPECstrainshavedifferentvirulenceprofilesthat correlate tothe animal species and the regionin which it wasisolated.12,26,27Inseveralstudiesauthorshaveshownthat
APEC-relatedgenesiutA,hlyF,iss,iron,ompTandcvaCoccur
morefrequentlyand areassociatedwithhighlypathogenic
strains.12,28,29 All 21 isolates showed at least five of these
andothersimportantgenesassociateswithExPECthathave
beendetectedinotheranimalslikepigeons30andhumans.31
Thesefindingssuggestthatwildanddomesticbirdmayact
assourcesofhumanpathogenicE. coli,thusreinforcing its
zoonoticnature.3
Mostoftheisolateswerefoundhighlypathogenicbythe
pathogenicity test, with low and intermediate pathogenic
strainscomposingonly29.6%andonlyoneisolatewas
non-pathogenic.Thehighnumberofhighpathogenicisolatesin
apparently healthy birds can beattributed tothe fact that
these geneswere notexpressedbecauseaccording toWon
etal.(2009),32thepathogenicityofAPECisbasednotonlyon
thegenepresence,butalsoonitsexpression.Inaddition,APEC
infections are multifactorial and highly dependent on the
worthmentioningthatasignificantgeneticmaterialexchange
betweenbacteriamayoccur,makingpossiblethathorizontal
genetransferfrompathogenictonon-pathogenicstrainlead
totheemergenceofnewvirulentstrainsbetweenthenormal
humanmicrobiota.7
OftodayconcernisthefactthatalargenumberofE.coli
strainscausinghumaninfections,includingthoseresistantto
antimicrobials,areofanimalorigin.5,34 AccordingtoMellata
etal.(2013),35E.colisamplesisolatedfrombirdsarecommonly
resistanttomorethanoneantimicrobial.Thisprofilewasalso
overservedherewith95.2%oftheisolatesexhibiting
resis-tancetoatleastthreeantimicrobials,factsimilartoothers
studies.36,37Thehighestdrugresistancewasobservedagainst
cephalothin,streptomycin,ampicillinandtetracyclinewhich
arethemostcommonlyusedantibioticsinhumaninfections.
Inaddition,findingESBLgenesintheseanimalsisaconcern
becausetransmissionofESBLisolatesbetweenhumansand
otherspecieshasbeenreported.38
Phylogeneticanalysis revealed thatmost ofthe isolates
fromgroupsB1,and B2andFstrainshaveahighest
num-berofVAGs.Althoughrecentstudiesthatutilizetheupdated
methodbyClermont et al. (2013)13 are scarce,Logue et al.
(2017)39 classified APECisolates accordingto thenew
phy-logenetic typing and concluded that strains in A and B1
group were of lower pathogenic potential. In contrast, in
this study, three samples from group A were classified as
high pathogenic and one as intermediate. The only
non-pathogenic strain belonged to the B1 group, as well as a
low pathogenicitystrain and,interestingly, two
intermedi-atepathogenicitystrainsandthreehighpathogenicstrains
wereincludedinthisgroup.Thus,itwasnoticedthatstrains
of low and high pathogenic potential can be of the same
phylogeneticgroupandmorestudiesareneededinorderto
clarify, effectively, the phylogeny relationshipof APEC
iso-lates.
Strainsinvolvedincolibacillosisareoftenassociatedwith
threemajorserogroups:O1,O2andO78,withtheotherones
beensporadic,butalsooccurring.3,40 Inthisstudy,14.3%of
the isolated belongedto the O2 group, which is in
agree-ment with Schouler et al. (2012),9 whose study found out
thatthisserogroupharboredmainlypathogenicsamples.In
our findings,all O2samples were pathogenic. In addition,
theserogroupH4wasthemostcommonlyfound,withthe
O51:H14serotypehasbeenthemostprevalent.
PFGE analysis revealed a great intra-specific variability.
Amongtheisolatesthatsharedthesamepulsotype,samples
1and2,fromthesameanimal,havegeneticsimilaritiesand
sharedtheotherevaluatedcharacteristicsbutdifferamong
themselvesinthepresenceofthecvaC. Isolates13 and15,
whichwerefromthesameanimal,hadasimilargenotypic
profile,pathogenicityandphylogeny,buttheyhaddifferences
inothersparameters.Samples10and11,isolatedfrom
differ-entbirds,weresimilarinallcharacteristicsand,possibly,there
arebacterialclones.Theseresultsthatcanbeexplainedbythe
factthatthesebirdssharethesameenvironmentandwere
in close proximity, facilitating genetic material exchange,7
fact that suggesting the possibility of free-range helmeted
anewST,correspondingtoisolate21,whichdifferedinonly
onelocusfromtheST155(datanotshown).Therewas
sim-ilarity amongisolateswhichshare the same STaswell as
betweenisolateswithdifferentSTs.Ingeneral,phylogenetic
analysisbyMLSThasshownthat,infact,APECconstitutesa
heterogeneousgroup,factthatwasalsoobservedinanother
study.41
Maluta et al. (2014)29 verified that APEC and ExPEC in
humans cansharedST 155,ST88 and ST117,which were
found infree-rangehelmetedguineafowl, withtheST 117,
beingthemostcommonamongisolatesrelatedtointensive
careunits.TheST46andST83,alsoassociatedwithhumans
and otheranimalslikedogs42and cats43 wasfoundinthis
studytoo.HelmetdguineafowlAPECisolatessharingthesame
phylogeneticbackgroundwithExPecstrainsprovesthatthese
animals canbeasourceofinfection inhumans and other
animals,includepetcompanions.
The diversity of factors combinations that may lead to
virulenceand toantimicrobialsresistancerevealsthegreat
importanceofAPECzoonoticpotential.Ourdatashowedthat
healthy free-range helmeted guineafowl are an
underesti-matedhownaturalreservoirsofAPECwithhighpathogenic
potential and multi drug resistance. This is aggravated
becauseofthefactthatthesebacteriacanbeeliminatedby
the respiratorytract andbyfeces, thusmaking thesebirds
asourcesofinfection ofExPECtoother animals, including
humans.
Acknowledgments
The authors would like to thank Fundac¸ão de Amparo à
Pesquisa do Estado de São Paulo (FAPESP – [grant number
2014/06313-3]andCoordenac¸ãodeAperfeic¸oamentode
Pes-soaldeNívelSuperior(CAPES)forallresearchsupportgranted.
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