Diarrheagenic Escherichia coli

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

Diarrheagenic

Escherichia

coli

Tânia

A.T.

Gomes

_,

_Waldir

_P.

_Elias

_,

_Isabel

_C.A.

_Scaletsky

_,

_Beatriz

_E.C.

_Guth

_,

Juliana

F.

Rodrigues

_,

_Roxane

_M.F.

_Piazza

_,

_Luís

_C.S.

_Ferreira

_,

_Marina

_B.

_Martinez

a_{UniversidadeFederaldeSãoPaulo,EscolaPaulistadeMedicina,DepartamentodeMicrobiologia,ImunologiaeParasitologia,SãoPaulo,}

SP,Brazil

b_{InstitutoButantan,LaboratóriodeBacterologia,SãoPaulo,SP,Brazil}

c_{UniversidadedeSãoPaulo,InstitutodeCiênciasBiomédicas,DepartamentodeMicrobiologia,SãoPaulo,SP,Brazil}

d_{UniversidadedeSãoPaulo,FaculdadedeCiênciasFarmacêuticas,DepartamentodeAnálisesClínicaseToxicológicas,SãoPaulo,SP,}

Brazil

a

r

t

i

c

l

e

i

n

f

o

Received8October2016 Accepted27October2016 Availableonline5November2016

Keywords: Escherichiacoli Diarrhea Pathogenicmechanisms Virulencefactor Epidemiology

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MostEscherichiacolistrainsliveharmlesslyintheintestinesandrarelycause diseasein healthyindividuals.Nonetheless,anumberofpathogenicstrainscancausediarrheaor extraintestinaldiseasesbothinhealthyandimmunocompromisedindividuals.Diarrheal illnessesareaseverepublichealthproblemandamajorcauseofmorbidityand mortal-ity ininfantsandyoungchildren,especially indevelopingcountries.E.colistrainsthat causediarrheahaveevolvedbyacquiring,throughhorizontalgenetransfer,aparticularset ofcharacteristicsthathavesuccessfullypersistedinthehost.Accordingtothegroupof virulencedeterminantsacquired,specificcombinationswereformeddeterminingthe cur-rentlyknownE.colipathotypes,whicharecollectivelyknownasdiarrheagenicE.coli.Inthis review,wehavegatheredinformationoncurrentdefinitions,serotypes,lineages,virulence mechanisms,epidemiology,anddiagnosisofthemajordiarrheagenicE.colipathotypes.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

The genus Escherichia, which was named after the Ger-manpediatricianTheodor Escherich,consistsoffacultative anaerobic Gram-negative bacilli that belong to the family Enterobacteriaceae.1_{ThegenustypespeciesEscherichiacoliis}

widelydistributed,whereitisthemajorfacultativeanaerobe inhabitingthelargeintestineofhumansandwarm-blooded animals.2 _{Although most E. coli strains live harmlessly in}

thecolonand seldomcausedisease inhealthy individuals,

∗ _{Correspondingauthor.}

E-mail:tatg.amaral@unifesp.br(T.A.Gomes).

a number of pathogenic strains can cause intestinal and extraintestinaldiseasesbothinhealthyand immunocompro-misedindividuals.3

Diarrhealillnessesareaseverepublichealthproblemand amajorcauseofmorbidityandmortalityininfantsandyoung children.4_{Low-andmiddle-incomecountriesinAfrica,Asia}

andLatinAmericaarethemostaffectedregionswithdiarrheal diseasesoccurringmoreoftenwithlethaloutcomesmainly

http://dx.doi.org/10.1016/j.bjm.2016.10.015

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

due to poor living conditions (inadequate water supplies, poorenvironmentalhygieneandsanitation,andinsufficient education).5

E.colistrainsinvolvedindiarrhealdiseasesareoneofthe mostimportantofthevariousetiologicalagentsofdiarrhea, wherestrainshaveevolvedbytheacquisition,through hori-zontalgenetransfer,ofaparticularsetofcharacteristicsthat havesuccessfullypersistedinthehost.3,5,6_{Accordingtothe}

groupofvirulencedeterminantsacquired,specific combina-tions were formeddeterminingthe currentlyknown E. coli

pathotypes, which are collectively known asdiarrheagenic

E.coli(DEC).6_{TheDECpathotypesdifferregardingtheir}

prefer-entialhostcolonizationsites,virulencemechanisms,andthe ensuingclinicalsymptomsandconsequences,andare clas-sifiedasenteropathogenic E.coli(EPEC),enterohemorrhagic (Shigatoxin-producing)E.coli(EHEC/STEC),enteroaggregative

E.coli(EAEC),enterotoxigenicE.coli(ETEC),andenteroinvasive

E.coli(EIEC).

Each of these pathotypes represents a group of clones thatsharespecificvirulencefactors.Nevertheless,itshould bepointedout thattheplasticityoftheE. coligenome has hindered the identification of certain E. coli isolates as a pathotype,becausesomeisolatescombinethemainvirulence characteristicsofdifferentpathotypesandare thus consid-eredpotentiallymorevirulenthybridpathogenicstrains.5

Anotherlesswell-definedpathotypehasbeendescribed, thatis,thediffusely-adherentE.coli(DAEC)pathotype,which comprisesstrainsthatadheretoepithelialcellsinadiffused distribution.6 _{Despitetheirclassificationasagroupdistinct}

fromtheotherpathotypes,thedesignationofDAECasa differ-entDECpathotyperequiresfurtherepidemiologicalstudies, whichhavebeenhamperedbythedifficultiesinits identifi-cationandclassification.5_{Furthermore,certainE.colistrains}

thathavebeenclassifiedastheadherentinvasiveE.coli(AIEC) pathotype,compriseoneofthepotentialagentsforCrohn’s disease(CD).CDisaninflammatoryboweldisease(IBD),which isthoughttobecausedbyacombinationoffactors(genetics, theintestinalmicrobiota,environmentalfactors,andenteric pathogens).7,8

DiarrhealepisodesduetoDECinfectionsareanimportant publichealth issueamong childrenand adultsin develop-ingcountries,becauseoftheirassociationwithmorbidityand mortalityofchildrenlessthanfiveyearsofage.Itwasouraim withthisreviewtogatherinformationoncurrentdefinitions, serotypes,lineages,virulencemechanisms,epidemiology,and diagnosisofthemajorDECpathotypeswithemphasisonthe studiesconductedinBrazil.

Typical

and

atypical

enteropathogenic

E.

coli

Theterm enteropathogenic E. coli (EPEC) was first used in 1995byNeteretal.,9_{todescribeanumberofE.colistrains}

epidemiologicallyrelatedtoaseriesofoutbreaksofinfantile diarrheainthe1940sand1950s.10,11 _{Originallyidentifiedby}

serotype,EPECarenowdefinedasthoseE.colistrainshaving theabilitytocausediarrhea,toproduceahistopathologyon theintestinalepitheliumknownastheattachingand effac-ing(AE)lesion,andtheinabilitytoproduceShigatoxinsand heat-labile(LT)orheat-stable(ST)enterotoxins.6

Improvementsintechniquesallowingabetter understand-ing ofthe genomeand virulencemechanisms amongEPEC strainsovertheyearshaveledtothesub-classificationofEPEC intotypicalEPEC(tEPEC)andatypicalEPEC(aEPEC).3,12

Typi-calEPECstrainscausinghumaninfectiousdiarrheapossess alargevirulenceplasmidknownastheEPECadherence fac-tor(EAF)plasmid(pEAF),whichencodesthetypeIVfimbriae called the bundle-formingpilus (BFP), while aEPEC donot possessthisplasmid.6,12

ThemajorityoftEPECstrainsfallintowell-recognizedO serotypes.ClassicalEPECOserogroupsincludeO55,O86,O111, O114,O119,O127, andO142.ThemostcommonHantigens associatedwithEPECaretheH6andH2antigens.12–15_Aless

common EPEC typeisH34, and anumber oftEPEC strains areclassifiedasnon-motile(H-)inconventionaltests.Typical EPECstrainsbelongingtonon-classicalserotypeshavealso beenreported.12,16

Based on multilocus enzyme electrophoresis analysis (MLEE) of allelic differences between housekeeping genes, tEPEC strains havebeen subtypedinto twomajorlineages, previouslydesignatedEPEC1andEPEC2.13,14 _{EPEC1includes}

widespreadserotypessuchasO55:H6andO119:H6,whereas EPEC2 consists of serotypes with more limited occurrence such as O111:H2 and O114:H2. Based on a whole-genome phylogeny and analysis oftypeIII secretion system (T3SS) effectors, tEPEC strains havebeen demonstrated to cluster inthreemainlineages,designatedEPEC1,EPEC2,andEPEC4, whichprobablyacquiredthelocusofenterocyteeffacement (LEE)regionandpEAFindependently.17

In turn, aEPEC belong to a large diversity of classical and non-classical serotypes.12,16,18 _{Over 20% of strains of}

non-classicalEPECserotypesareOnon-typeableandthe O-typeablestrainsbelongtomorethan4200differentserotypes, withmanynon-motileandHnon-typeablestrains.12,18

Inter-estingly,ithasbeenfoundthat35%oftheaEPECstrainsalso belong tothe tEPEC lineages.17 _{Thus, it hasbeen}

hypothe-sizedthatatleastsomeaEPECmayhaveoriginatedfromtEPEC strainsthatlostpEAFinthehostorintheenvironment.17,19,20

Virulencefactors,mechanismsandpathogenesis

TypicalEPECstrainsadheretoHeLa,HEp-2,andothercelllines andtoorganculturesinvitroinadistinctivepatternof three-dimensional microcolonies,aso-calledlocalized adherence (LA)pattern.6,21 _{Asimilaradherencepattern hasbeen seen}

intissuebiopsiesofEPEC-infectedhumans.22

The LA phenotype is mediated by the BFP,23 _which

alsocontributestoantigenicity,autoaggregation,andbiofilm formation.23–27_{Anoperonof14genescontainedonthepEAF}

isnecessaryforBFPexpression,withbfpAencodingthemajor structural subunit (bundlin)28 _{and being highly conserved}

amongEPEC1andEPEC2strains.

The self-transmissible pEAF pMAR2 is found among strainsoftheEPEC1lineageandcontainsanintacttransfer region,unlikepB171,whichismorecommonamongEPEC2 strains.29,30_{Besidesthebfpgenecluster,encodingBFP,}23_the

pEAFcarriestheperlocus,encodingthetranscriptional activa-torcalledplasmid-encodedregulator(Per).29_BetweenpMAR2

andpB171,thebfpandperlocishare99%sequencesimilarity,30

virulence in human volunteers.24 _{Recent comparative}

genomics ofthe EAF plasmids from diverse EPEC phyloge-nomic lineages demonstrated significant plasmid diversity evenamongisolateswithinthesamephylogenomiclineage.31

TypicalEPEChavetheabilitytoformtight,spherical, bac-terialautoaggregateswhengrowninliquidculture.32_LikeLA,

autoaggregationrequiresBFP.TypicalEPECalsoformbiofilms onabioticsurfacesunderstaticconditions,orinaflowthrough continuous culture system, and a model of EPEC biofilm formation has been proposed.26 _{Mutagenesis analysis has}

identified adhesivestructures suchas the common type 1 pilus(T1P),antigen43,BFPandtheEspAfilament(seebelow) asparticipantsinbacterialaggregationduringbiofilm forma-tiononabioticsurfaces.26

AhallmarkphenotypeofbothtEPECandaEPECisthe abil-itytoproduceAElesions.33_{Thisphenotypeischaracterized}

byeffacementofintestinalepithelial-cellmicrovilliand inti-mateadherencebetweenthebacteriumandtheepithelialcell membrane.Directlybeneaththeadherentbacterium,marked cytoskeletalchangesareseenintheepithelialcellmembrane, particularlytheformationofanactin-richcup-likepedestalat thesiteofbacterialcontact.AElesionsareobservedinmodel EPECinfectionswithculturedcellsandmucosalexplants,as wellasinintestinal biopsiesfrom EPEC-infectedinfantsor animals.6

AE lesions are encoded by LEE, which is a ∼35-kb pathogenicityisland(PAI)34 _{thatisorganizedintofive}

oper-ons (LEE1 to LEE5).35–37 _{The LEE1, LEE2, and LEE3 operons}

encodecomponentsofaT3SS,andthe globalregulatorLer (LEE-encoded regulator).38 _{LEE4 encodes the T3SS-secreted}

proteinsEspA,EspB,andEspD(EPEC-secretedprotein),which arecomponentsofthetranslocationapparatusbywhichother effectorproteinsaretranslocatedintothecell.LEE5encodes theadhesinintiminanditstranslocatedreceptor,Tir.39

Intiminisa94-kDaproteinencodedbytheeaegeneand requiredforintimateadherenceofEPECtohostcellsatthe sites of AE lesions.6 _{The N-terminus of intimin is highly}

conserved,whereastheC-terminusishighlyvariable.40

Dif-ferencesinthe C-terminusofintiminhavebeenusedas a basisforclassificationintoseveral distinctsubtypes, repre-sentedbytheGreekletters␣(alpha)through␨(zeta)41,42_;the␣

subtypeisexpressedbyEPEC1strainswhilesubtype␤is asso-ciatedwithhumanEPEC2strains.TheN-terminusofintimin anchorsthe proteininthe EPECouter membrane,whereas the C-terminus extends from the EPEC surface and binds tothe Tir.Intimin–Tir interaction leadsto intimate adher-enceandpedestalformationbeneathadherentbacteria,39_and

inhibitsNF-␬Bactivity throughtumornecrosisfactoralpha (TNF-␣) receptor-associatedfactors.43_{InadditiontoTir,the}

EPECgenomecontainssixotherLEE-encodedeffectorproteins thataretranslocatedintothecell(Map,EspF,EspG,EspZ,EspH, andEspB),whichinterferewithdifferentaspectsofthecell’s physiology.13,36,37,44

In addition to the LEE effectors, various non-LEE (Nle)-encoded effector genes (cif, espI/nleA, nleB, nleC, nleD,nleE, nleH)36,44_{havebeendescribed,whicharelocatedoutsidethe}

EPEC LEE region, in at least six chromosomal PAIs, or in prophageelements.45,46 _{TheNleproteinshavebeenshown}

todisrupt thecytoskeleton and tightjunctions ofthe host cell, and to modulate or prevent the host inflammatory

response.45–47 _{Althoughtheyare notrequiredforAElesion}

formation,itisunderstoodthattheycontributetoincreased bacterialvirulence.44

IntracellulartEPEChavebeenobservedbothintissue cul-tureand insmall intestinalbiopsiesfrom anEPEC-infected infant.6_{TwostudieshavereportedthatO111:NMstrains}

con-tainplasmidsequencesthatconferinvasivenessuponE.coli

K12strainscontainingtheclonedfragments.48,49_Sequences

homologoustotheseclonedgenesarepresentinonlya minor-ityoftEPECstrains(Scaletskyetal.,unpublisheddata).

Typical EPEC strains encode a large surface protein, lymphocyteinhibitoryfactor(LifA),whichinhibitsthe expres-sion of multiple lymphokines and inhibits lymphocyte proliferation.50 _{Two related genes efa1 and toxB have been}

implicated inadhesionto epithelial cells.51,52 _{There is}

evi-denceindicatingthatEfa1/LifAcontributestoepithelialcell adherenceinvitro53_{andisrequiredforintestinalcolonization}

ofmicebytherelatedAEpathogenCitrobacterrodentium.54

SometEPECstrainspossessotherfimbriaeorpiliin addi-tion toBFP.Type1fimbriaeofEPEChavebeenfoundtobe antigenicinvolunteerstudies;however,theydonothavearole inadherencetoepithelialcellsinvitro.6_{Inaddition,someEPEC}

strainshaveconservedfimbrialgenesencodinghomologsof longpolarfimbriae(LPF),55 _{butanumberofpolymorphisms}

within the lpfA genes have been identified.56 _Initial

stud-ies haveindicatedthat LPFisapparently notnecessaryfor adherenceandAElesioninhumanbiopsies.55_TheE.coli

com-monpilus(ECP)hasalsobeenshowntoactasanaccessory adherence factor inEPEC, playinga role duringcell adher-enceand/orinbacterium-bacteriuminteractions.57_However,

the significanceofECPto EPECpathogenesishasnot been determined.Interestingly,ithasbeenshownthatsometEPEC strainsmayproduceahybridadherencephenotypeinHeLa cells,i.e.,LAandaggregative(AA)-likepattern concurrently (LA+/AA-like+).58_{Recently,itwasshownthatatleastsomeof}

theseLA/AA-like+strainsbearlargeplasmids,distinctfrom the pEAF, that encode a so far unknown adhesin.59 _Ithas

been proposedthatthe abilityofsuchstrainsinproducing AElesionsandanAA-associatedbiofilmconcomitantlycould worsenthepatient’sclinicalcondition,leadingtopersistent diarrhea.59

FlagellamayalsobeinvolvedintEPECadherenceto epithe-lialcells,60_{sincecertainEPECmutantsaremarkedlyimpaired}

intheirabilitytoadhereandtoformmicrocolonies. Further-more,inonestudy,purifiedEPECflagellaandanti-flagellum antibodieswere botheffectiveinblockingtheadherenceof severalEPECserotypes.60_{However,anotherstudycouldnot}

confirmtheroleofflagellainEPECadherence.61

SometEPECstrainsharbortheastAgene,whichencodes the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1).62,63_{Arecentstudyreportedthat11of70(16%)tEPEC}

strains testedharbored anintact astAgene.64 _TypicalEPEC

strains of serotype O86:H34 produce cytolethal-distending toxin (CDT).65_{ThesignificanceofEAST1and CDTtoxinsin}

EPECpathogenesisremainsunknown.

Autotransporter (AT) proteins, which have been associ-atedwithbacterialadherence,aggregation,biofilmformation, invasion,andtoxicity66_{inGram-negativebacteria,havealso}

beendescribedamongEPECstrains.67_{Onesuchprotein,EspC,}

injectedbytheT3SSinepithelialcells,hasanIgA protease-like activity and, once in the host cytoplasm, has various cytopathiceffects,includingcytoskeletaldamage,68_enhanced

lysozymeresistance,70_{hemoglobindegradation,}69_hydrolysis

ofpepsin,factorV,andspectrin,70_{andfodrinandfocal}

adhe-sion protein degradation.71 _{In addition, oligomerization of}

EspCgivesrisetorope-likestructuresthatserveasa substra-tumforadherenceandbiofilmformationaswellasprotecting bacteriafromantimicrobialcompounds.72

A three-stage model of tEPEC adhesion and pathogen-esis, consisting of LA, signal transduction, and intimate attachmentwithpedestalformation,wasproposed.73 Simul-taneously with intimate attachment, a series of bacterial effectorproteinsareinjectedintohostcells,wherethey sub-vertactinpolymerizationandotherhostcellprocesses.37,44

Inthe earlieststageand undercorrectenvironmental con-ditions,tEPECexpressBFP,intimin,andtheT3SS/translocon apparatus.Next, EPECadheretothe surface ofthe intesti-nal epithelium via BFP and EspA filaments, and the T3SS injectsthe bacterialtranslocated intiminreceptor(Tir)and effectorproteins(EspB,EspD,EspF,EspG,and Map)directly into the host cell.37 _{The effectors activate cell-signaling}

pathways, causing alterations in the host cell cytoskele-ton and resulting in the depolymerization of actin and the lossofmicrovilli. Finally, bacteriaintimately adhereto hostcell byintimin–Tir interactions,causinga cytoskeletal rearrangementthatresultsinpedestal-likestructures.Tir pro-motes cytoskeletalreorganization through interaction with neuralWASP(Wiskott-Aldrich syndromeprotein) (N-WASP) and subsequent activation of the Arp2/3 complex,45

lead-ing tothe effacement ofthe microvilliand the production ofpedestals.44,74 _{Thetranslocatedeffectorsdisrupthostcell}

processes, resulting in loss of tight-junction integrity and mitochondrialfunction,leadingtobothelectrolytelossand eventualcelldeath.45

For actin dynamics subversion, tEPEC usually recruits Nck to the adhesion site in a Tir phosphorylated Y474-dependentmechanism.Inturn,TirEHEC (enterohemorrhagic

E.coli[EHEC]O157:H7)isdevoidofanY474equivalentand employs EspFU/TccP (Tir-cytoskeleton coupling protein), a

T3SS-translocatedeffectorproteinthatbindsN-WASP,leading toNck-independentactinpolymerization.45

aEPECare devoidofpEAF and donotproduceBFP. Itis importanttopointoutthatEPECstrainsofserotypesO128:H2 andO119:H2containapEAFwithdefectivebfpoperons,which containpart ofthe bfpA gene but havethe rest ofthe bfp

geneclusterdeleted.Thus,theyareclassifiedasaEPEC.12,75

MostaEPECproduceadherencepatterns categorizedas LA-like,withloosenedmicrocoloniescomparedtothoseofthe tEPEC LA pattern.12,76,77 _{In addition, some isolates express}

the aggregative(AA) ordiffuse(DA) patterns ofadherence, whicharecharacteristicsoftheEAECandDAECpathotypes, respectively,20,78_{oradhereinundefinedpatternsorare}

non-adherent.20,78–80 _{Remarkably, the epithelial cell adherence}

phenotype displayedby aEPECis determined in prolonged assays(6h)ofbacteria-cell interaction.12,76,77 _{Inaddition, it}

hasbeensuggestedthatlackofthepEAF-encoded Per pro-teinsintheregulatorycascadeoftheaEPECvirulencegenes maypromotedelayedAElesionformation,probablymakingit difficultforsuchstrainstocausedisease.81

TheprevalenceofintiminsubtypesamongaEPECstrains hasbeenreviewed.18,67_{Intiminsclassifiedasbeta1,epsilon1}

andthetaappearasthemostfrequentamongaEPEC.78,82–85_In

addition, someaEPECstrainsbearadhesive-encodinggenes thathavebeenoriginallydescribedinotherDECpathotypes and/or inextraintestinal pathogenic E. coli.79,80,82,86–88 _This

observation suggests that aEPEC could employ additional adherence mechanisms besidesthe Tir-intimininteraction. The only adhesin first characterized in an aEPEC strain (serotypeO26:H11)isthelocusofdiffuseadherence(LDA),which is an afimbrialadhesin that confers the diffusepattern of adherenceonHEp-2cells,whenclonedinE.coliK-12strains.89 TheT3SS-transloconhasbeenalsoshowntocontributetothe adherenceefficacyofanaEPECstraininvitro.90_{Theprevalence}

ofthesedifferentadhesinsamongaEPEChasbeenrecently reviewed.18,67,91

Moreover, it has been recentlyshown that the flagellar capproteinFliDofanaEPECstrain(serotypeO51:H40)binds tounknownreceptorsonintestinalCaco-2cellmicrovilli.92

Interestingly, ananti-FliD serum and purified FliD reduced adherenceoftheaEPECaswellasthatoftEPEC,EHECandETEC prototypestrainstothesamecellline.92_{Furthermore,ithas}

beensuggestedthatadherenceofaEPECofserotypeO26:H11 maybemediatedbybindingoftheflagellinproteinFliC(the subunitoftheflagellashaft)tocellularfibronectin.93_However,

theroleoftheflagellainaEPECinvivocolonizationhasyetto beinvestigated.

AtypicalEPECstrainshavealsobeenshowntoadhereto abioticsurfaces(polystyreneandglass).94,95_{Thenon-fimbrial}

adhesincurliandtheT1Phavebeenshowntomediatebinding tothesesurfacesinsomeaEPECatdifferenttemperatures.90,96

The LEEregion ofsomeaEPEC strainsdisplay agenetic organization similar to that found in the tEPEC prototype E2348/69strain.97_{AlthoughtheT3SS-encodinggenesare}

con-siderablyconserved,97,98_{theeffectorprotein-encodinggenes}

displayimportantdifferences,andremarkabledifferencescan bedetectedatthe5and3flankingregionsofaEPEC, suggest-ingtheoccurrenceofdifferentevolutionaryevents.99_Atypical

EPECstrainsmaycarrytwotccPvariants,tccPand/ortccP2, sug-gesting thatsomeaEPECstrainsmay usebothTir-Nck and Tir-TccPpathwaystopromoteactinpolymerization.100

Inter-estingly,Rochaandcolleagues101_{showedthattransformation}

ofanon-adherent aEPECstrain(serotypeO88:HNM) witha TccPexpressing-plasmid,conferredthisstraintheabilityto adheretoandtoinduceactin-accumulationinHeLacells.

TheoccurrenceandprevalenceofNleinaEPECstrainshave been recentlyreviewed.67 _{Ithasbeensuggestedthat}

differ-entisolatescanemploydistinctstrategiestopromotedamage tothe hostandcause disease.45 _{In addition,the Nle}

effec-tors Ibe(invasionofendothelialcells) andEspT have been originallydescribedandcharacterizedinaEPECstrains.102,103

IbeappearstoregulateTirphosphorylationandtoenhance actinpolymerizationandpedestalformation,103_whileEspT104

modulatesactindynamics,leadingtomembranerufflingand cell invasion, and induces macrophages to produce inter-leukinsIL-8andIL-1␤andPGE2.102

Invasionofepithelialcellsinvitroinanintimin-dependent pathwayhasbeendescribedinanaEPECstrain,105_butfurther

studiespointedoutthattheinvasivephenotypeisnota com-mon characteristic among aEPEC.106 _{Despite their invasive}

potentialinvitro,107_{mostaEPECareconsideredextracellular}

pathogens.5

Ithasbeenshownthatapicalinfectionofculturedhuman mucin-secreting intestinal HT29-MTX cells by some aEPEC strainsmay induceincreasedproductionofsecretedMUC2 andMUC5ACmucinsandmembrane-boundMUC3andMUC4 mucins.108_{Thisobservationsuggeststhattheapically}

adher-ingbacteriacouldexploitlargeamountsofmucinstogrow moreefficientlyinthehostintestines,characterizinga puta-tivenewvirulencemechanisminaEPEC.108

ATproteinshavealsobeenshowntobeproducedbysome aEPECstrains.67_{Abreuandcollegues}109_{haveshownthatthe}

ATproteinencodedbythe ehaCgene,which isinvolvedin biofilmformationinEHECstrains,wasthemostfrequent,with asignificantlyhigherprevalencethanintEPEC.Althoughthe prevalenceoftheATproteinPic(proteininvolvedinintestinal colonization),formerlyidentifiedinEAEC,isnotacommon findinginaEPECstrains,italsoappearstomediate coloniza-tionofmouseintestines,hemagglutination,mucincleavage, andcomplementcomponentsdegradation.110_{Morerecently,}

some aEPEC strains were shown to cause cell damage by secretingtheATproteinPet(plasmidencodedtoxin)tothe extracellularenvironment.111

Epidemiology

TheprevalenceofEPECinfectionsvariesbetween epidemio-logicalstudiesonthebasisofdifferencesinstudypopulations, agedistributions, andmethods(serotyping, adherence pat-terns,andpresenceoftheeaeorconservedLEEgenes)used fordetectionanddiagnosis.112_{Inaddition,differencesin}

geo-graphicregions,periodsoftimeandsocioeconomicclassmay alsocontributetodifferencesinthe epidemiologyof EPEC-induceddiarrhealdisease.113_{Lackofdiscriminationbetween}

tEPECandaEPECinsomestudiesalsomakessuchanalysis difficult.

DiarrheaduetotEPECdecreaseswithage,andinfectionsin adultsarerarelyreported.Thisapparentresistanceinadults andolderchildrenhasbeenattributedtothelossofspecific receptorswithageordevelopmentofimmunity.6

For many decades, studies conducted worldwide have shownthattEPECserotypesarestronglyassociatedwith diar-rheainchildren<1 yearofage,mainly inpoorchildrenin urbancenters.6,12,15_{Theassociationwithdiarrheawas}

partic-ularlystrongininfantslessthan6monthsofage.Studiesin Brazil,Chile,Mexico,andSouthAfrica,showedthat30–40%of infantilediarrheacaseswereduetotEPECserotypes.15,112,114

However, the epidemiology of EPEC infections has shifted. Innumerousdevelopingcountries,wherethe prevalenceof EPECinfectionhadbeenhighuntilthe1990s,recentstudies havenotidentifieda significantassociation betweentEPEC and infantile diarrhea. In Brazil, 92% of EPEC isolates col-lectedfromchildrenbetween2001and2002wereatypical,115

comparedto38%ina1998–1999study.79_{However,other}

stud-iesstillreporttEPECbeingmoreprevalent thanaEPECasa causeofdiarrhea.116_{Inaddition,insomelessdevelopedareas}

(Africa and Asia),tEPEC are still someof the most impor-tantenteropathogens.117–122_{Basedontherecentlycompleted}

Global Enteric Multicenter Study(GEMS) involving children

lessthan5yearsofagefromsevensitesinAfricaandAsia, tEPEC wassignificantlyassociatedwithmoderate tosevere diarrheainchildrenunder2yearsofageinKenya,whereas aEPECwasnotassociatedwiththistypeofdiarrhea.118

Transmission of tEPEC follows a fecal-oral process throughcontaminatedsurfaces,weaningfluids,andhuman carriers.123 _{Although rare, outbreaks among adults seem}

to occur through the ingestion of contaminated food and water;however,nospecificenvironmentalreservoirhasbeen identified.6_{Theinfectivedoseinadultvolunteersishigh,at}

108_to1010_organisms,124_{whiletheinfectivedosethatcauses}

disease inchildrenisunknown. EPECoutbreakshave been reportedtoshowaseasonaldistributionwithpeaksduring thewarmmonths.6,125_{Humansaretheonlyknownreservoir}

fortEPEC,withsymptomaticandasymptomaticchildrenand asymptomaticadultsbeingthemostlikelysource.6

IncontrasttotEPEC,aEPEChavebeenfoundindiarrheic patientsofallagesandinadultswithHIV-AIDS.82,126–128

Fur-thermore,theproportionofaEPECstrainshasincreased,and aEPECstrainshaveoutnumberedtEPECstrainsandhavealso beenassociatedwithchildhooddiarrheainsomedeveloping anddevelopedcountries.12,18,67,91,112,129_{However,theincrease}

inprevalenceofaEPECmayalsoreflecttherefined discrimi-nationbetweentEPECandaEPEC.12,18,91

TheroleofaEPECindiarrheaisnotclearbecauseofits detectionatsimilarratesinbothdiarrheicandnon-diarrheic patientsinvariousgeographicalareas.18,91,128_Instudies

con-ducted in the last five years, aEPEC have been found at rates varying from ∼0.05 to ∼12% in diarrheic versus 0 to ∼14%innon-diarrheicpatients.67_{Somerecentstudieshave}

alsoimplicatedaEPECasthecauseofpersistentandbloody diarrhea.18,91 _{Moreover,aEPECstrainshavebeenassociated}

with diarrheal outbreaks in Finland, United States, Japan, China18,91,112_andBrazil.85

IncontrasttotEPEC,whichareseldomfoundinanimals,12

many aEPECstrainshavebeenfoundinbothdiarrheicand healthyanimals.18,67_{Interestingly,animalaEPECserogroups}

associated withhumandiarrhea have been identified (e.g., O26, O103, O119, O128, O142and O157).18,130,131 _Serotyping

andmolecularmethodssuchasmultilocussequencetyping (MSLT)andpulsedfieldgelelectrophoresis(PFGE)have con-tributedtodemonstratingthatdomesticandwildanimalsand the environmentare potentialsourcesofaEPECforhuman infectionsinseveralregions.18,67,91,131_{Therefore,althoughno}

directtransmissionfromanimalstohumanshasbeenshown sofar,itisreasonabletosuggestthatsomeaEPECstrainsare potentially zoonotic pathogens,withalargevarietyof ani-malspeciesservingasimportantreservoirs.67,91_Inaddition,

foods includingrawmeat, pasteurizedmilkand vegetables andwaterhavealsobeenimplicatedasvehiclesofaEPECin humaninfections.67

aEPECstrainscompriseaveryassortedgroupwithvarious additionalvirulencemechanismsthataltogethercan modu-latethediseaseoutcomeortheiroccurrenceinasymptomatic persons.Therehavebeencontinuousadvancesinour knowl-edgeofthegeneticbackgroundandpathogenicityofaEPEC aswellasintheinformationgatheredfromepidemiological studies,and may contributetothediscrimination between strains that cause diarrhea and those that cause asymp-tomaticinfections.

Detectionanddiagnosis

EPEC canbe detected byDNA probes or PCRassays using primerstargetingtheeaeandstxgenes.132,133_{Alleae-positive}

andstx-negativeE.colistrainsarefurthertestedbyPCRfor thepresenceofthebfpAgeneencodingbundlin6_and/orthe

EAF plasmid todifferentiatetEPEC from aEPEC.134,135

How-ever,thismay failtoidentifyall bfpA-positiveEPECstrains, sincemultipleallelesofbfpAhavebeenidentified,136

suggest-ingthatsomecurrent PCRmethodsmay failtoidentifyall

bfpA-positiveEPECstrains.

However,in routine microbiology laboratories,all E. coli

coloniesobtainedfromprimaryisolationplatesare tradition-allyscreenedbyslideagglutinationassaysusingseraagainst theclassicalEPECserogroupsO26,O55,O86,O111,O114,O119, O125,O126, O127,O128,O142, andO158.137 _Thismethodis

practicalandeasytoperform,themainadvantageofwhichis thecommercialavailabilityofthesera.However,the disadvan-tageofthismethodistheheterogeneityofEPECserogroups thatcancomprisecategoriesotherthanEPEC,theinabilityto distinguishtEPECfromaEPECwithintheseserogroups,and theoccurrenceofEPECstrainsbelongingtoserogroupsother thantheclassicalEPECserogroups.12,18,138,139

SinceEPEC strainsare definedbased on their virulence properties,asetofproteins,includingintimin,BFPandT3SS secretedproteinscanbeconsideredtargetsfordiagnosis.BFP expressionhas been considered the phenotypic marker of tEPEC.18,78,140_{Immunofluorescenceandimmunoblottingtests}

usingmonoclonalorpolyclonalantibodiesagainstBFPhave beenemployed.141,142_{Thesecitedauthorsdetectedthe}

pro-ductionof BFPon different media, inwhich theyreported that 91% ofthe tEPEC strains tested produced BFP in Dul-becco’sModifiedEagleMedium(DMEM),89%inMacConkey, and83% inEMB agars.Theseresultsare particularly inter-esting,sinceMacConkeyand EMBagars are routinely used for the identification of lactose-fermenting E. coli isolated fromdiarrhealstools.AcolonyimmunoblotassayfortEPEC detection based on BFP expression was also standardized usingarabbittEPECanti-BFPpolyclonalserum. Standardiza-tionwasdoneaftergrowingthebacterialisolatesonDMEM agarcontainingfetal bovineserumortrypticsoyagar con-taining5%washed sheepblood (TSAB).Thistestshowed a positivityof92and83%andspecificityof96and97%, respec-tively,whentheculturewasdoneinDMEMandTSAB.This method combines the simplicity of an immunoserological assaywiththehighefficiencyoftestingalargenumberofEPEC colonies.140

Concerning intimin detection, a rabbit polyclonal sera raisedagainsttheconservedregionofintimin(Int388-667)143 wasemployedinordertodetecttEPECisolatesexpressing␣, ␤,␥,␦and␧intiminreportedanapplicationof immunoblot-tingwith100%specificityand97%sensitivityinthedetection of eae positive E. coli strains.144–146 _{These authors clearly}

demonstrated thatpolyclonalrabbitantiseraissuitable for immunoblottingasadiagnostictool,andshowedthatprotein denaturation and linearization is a critical step for anti-intimin antibody accessibility.Indeed, even employing the recombinantantibodysuchassinglechainfragmentvariable (scFv-intimin),147,148_{merelybyimmunofluorescencethe}

scFv-intiminwasabletodetecttEPEC,aEPEC,andEHECisolates,

showingthatintimincanbeatargetforEPECandEHEC diag-nosisafterbacterialpermeabilization.148

Regardingsecretedproteins,Luetal.149_{developedanew}

practical method to identify EPEC by detecting the E. coli

secreted protein B (EspB) in the culture supernatant by reversedpassivelatexagglutination(RPLA),afterthestrains havebeencultivatedinDMEM.Inaddition,Nakasoneetal.,150

establishedarapidimmunochromatographic(IC)testto iden-tify the presence of EspB in EPEC and EHEC isolates. The detectionlimitofthetesthasbeenreportedtobe4ng/mL,and theresultsshowed96.9%sensitivityand100%specificity.The ICtestforthedetectionofEspBmaybeapracticalmethod todefineEPECorEHECbothinclinicallaboratoriesandthe field.150

In addition, arapidagglutination testusing latexbeads coatedwithanti-EspBmAbwasstandardized,showing97% sensitivity, 98% specificity and 97% efficiency, which is requiredforthediagnosisofenteropathogenicdiseasesand canbeemployedindevelopingcountrieswithpoorlyequipped laboratories.151

Enterohemorrhagic

(Shiga

toxin-producing)

E.

coli

(EHEC/STEC)

EHEC/STEC represent a well-known group of foodborne pathogensdistributedworldwide.Theabilitytoproduceone or more ofthe Shiga toxin (Stx) family cytotoxins152

con-stitutes the mainvirulence attribute ofthis pathogroupof

E. coli. A wide array of infections from mild and almost unapparent diarrhea to more serious manifestations such as hemorrhagic colitis(HC) and the developmentofa life-threateningsyndromeknownashemolyticuremicsyndrome (HUS)arecausedbyEHEC/STEC.Infantsandchildrenarethe mainaffectedpatients,andalthoughtheincidenceof infec-tion variesindifferentregions,the impactandimportance ofEHEC/STECinfectionsinpublichealthisimmense,being the main cause ofacute renal failure in childrenin many countries.TheperspectiveofEHEC/STECinfectionshasbeen previouslydescribed,153,154_{butaconsiderableamountof}

infor-mationhasbeenobtainedinmorerecentyearsrelatedtothe epidemiology,ecologyandvirulencepropertiesofthese bacte-ria.

E. coliO157:H7serotype wasthefirst tobelinkedtoHC and HUScasesintheearly1980s,andhasbeensincethen responsible fornumerous outbreaks and sporadic casesof severediseasesallovertheworld,thereforeconsideredtobe theprototypeofthispathogenicgroupofbacteria.155_Itiswell

knownthathundredsofotherE.coliserotypescanharborthe

stxgenes,butepidemiologicalstudiescarriedoutworldwide haveproventhatonlysomeofthemhavebeenresponsible forcausinghumandiseases.SomeserogroupsincludingO26, O45,O103,O111, O121andO145canbehighlightedamong thosemostcommonlyrelatedtohumaninfections.156

More-over,inrecentyearstheemergenceofsomeparticularclones suchasthehybridO104:H4enteroaggregativeE.colicarrying Stx2 genes, responsibleforasevereoutbreak ofHUS start-inginGermanyin2011,157_{thespreadofanewO26:H11clone}

themobilityofgenesandcertainlythehostbackgroundare importantfeaturesimplicatedintheirpathogenicpotential.

Virulencefactors,mechanismsandpathogenesis

ThecommonfeatureamongEHEC/STECisolatesisthe abil-ity to produce Stx. This family oftoxins has a conserved AB5 subunit structure, composed of one active A subunit

linkedtoapentameric Bsubunit responsibleforthe bind-ingofthetoxintospecificglycolipidreceptorsonthesurface oftarget cells. Thestx operon is usuallyfound withinthe sequence foraninducible, lysogenic,lambda-like bacterio-phage.Stxsinhibitproteinsynthesisbyremovinganadenine residuefromthe28SrRNAofthe60Sribosome.152_However,

besidesthisactivity,studieshavedescribedthatStxalsoacts oncellsignaltransductionandimmunemodulationcausing proinflammatoryandpro-apoptoticresponses.159_Twomajor

families, Stx1 and Stx2, havebeen recognized, and on the basisofsequencediversity,eachiscomposedofseveral vari-ants.TheStx1familyismorehomogenousandincludesStx1a, Stx1candStx1d;whiletheheterogenousStx2groupis com-posedofStx2a,Stx2b,Stx2c,Stx2d,Stx2e,Stx2f,andStx2g.160 Itshould bementioned that the association of some vari-ants such as Stx2a, Stx2c or Stx2d with HC and HUS has beenhighlighted comparedtosomeothers thatseemed to bemorerelatedtouncomplicatedcasesofdiarrheasuchas Stx1variantsorevenStx2e,Stx2fandStx2g,whichare uncom-monlyfoundcausinghumaninfectionssofar.161,162 _Indeed,

thehigherassociationofStx2withseverediseaseshasbeen extensivelystudiedbyusingVeroand endothelialcelllines aswellassomeanimalmodels.159 _{Moreover,knowledgeof}

stxphagecharacteristicsandbehaviorhashelpedour under-standing of how differences in expression ofStx between EHEC/STEC isolates may contribute to pathogenesis and disease.163

The ability to adhere to intestinal epithelial cells is another key event in EHEC/STEC pathogenesis. The pres-ence ofthe chromosomal pathogenicityisland LEE,164 also present in isolates belonging to the EPEC pathotype, is common. Although LEE has been described in the major EHEC/STEC serotypes responsible for a high proportion of HCandHUScasesinseveralcountries,itspresenceisnota requiredconditionfortheoccurrenceofmoreserious infec-tionsasinitiallythought,becausesomeLEE-negativestrains arealsocapableofcausingoutbreaksandsporadiccasesof HUS.165,166

Therefore,itisclearthatEHEC/STECpathogenesisisa mul-tistepprocess,andbesidestheproductionofStxtoxinsand theAElesion,otherfactorsincludingdifferenttypesoftoxins andadhesinshavebeendescribedandfoundtobeinvolvedin virulence.159

One should also consider that as a pathogen of the human gastrointestinal tract the ability of EHEC/STEC to monitor nutrients in the gut milieu, and translate this information to sense the host physiological state in order to program the expression of its virulence mark-ers has a pivotal role on the development of infection.167

In addition, it has been shown that EHEC/STEC can also cross-communicate with the host by exploiting the autoinducer-3 (AI-3)/epinephrine/norepinephrine signaling

systemtoexpresstwoimportantvirulencetraits,motilityand A/Elesion,requiredatdifferenttimepointsduringintestinal colonization.168

Theabilitytoadhere,colonizeandformbiofilmonfood andseveraltypesofsurfacesmaybeawaytobean impor-tantsourceand/orvehicleoftransmissionofEHEC/STEC.In addition,biofilmmayalsoactasbacterialprotectionagainst adverseenvironmentalconditions.Astudyconductedby Bis-cola et al.,169 _{evaluated the capacity of biofilm formation}

inEHEC/STECstrainsisolatedfrom differentreservoirsand serotypes. Theauthors observed that the abilitytoadhere to abiotic surfaces formingbiofilms, under definedculture conditions,occurredinanarrayofwild-typeO157and non-O157 strains. Biofilm production was identified in several non-O157 STECserotypes ofhuman, animal, andfood ori-gin.Ontheotherhand,amongtheO157strains,onlythose isolatedfromtheanimalreservoirandfromawatersample produced biofilm. A close correlation between biofilm for-mation and expressionofcurli fimbriaeand cellulose was observedamongO157strains.However,inadditiontocurli, thepresenceofotherfactorssuchastype1fimbriaeandAT proteinsmaybeassociatedwiththeabilitytoformbiofilmin non-O157strains.Matheus-Guimarãesetal.,170_studiedO157

andnon-O157EHEC/STECstrainsisolatedfrombovinehides andcarcassesandshowedthatdifferentsetsofgeneswere involvedinthe interactions ofthebacteria withbioticand abiotic surfaces. Moreover, the detectionof anO157 strain thatwasabletoformbiofilmonbothglassandpolystyrene and thatadheredtoandinvadedhumancells,suggestsan importantabilityofthisisolatetopersistintheenvironment and interact with the host. In fact, cell invasion and sur-vivalofsomeEHEC/STECstrainsinculturedhumanintestinal epithelialcellshasbeenpreviouslydescribed.171_Itshouldbe

mentionedthatthisinvasivecharacteristichasbeenidentified insomeEHEC/STECserotypes,manyofwhichare responsi-ble forhumaninfections.170–173 _{Therefore, it isconceivable}

that this virulencestrategymay helpbacteria toovercome hostdefensemechanismsandcertainlycontributestotheir persistenceinthezoonoticreservoir,ensuringefficient envi-ronmentalandfoodtransmission.

Anothertopicofinteresthasbeentheanalysisand com-parisonofthevirulenceprofileofEHEC/STECstrainsisolated fromtheanimalreservoirandenvironmentwithstrains recov-eredfromhumaninfections.Ingeneral,thesestudieshave shownthatdespiteserotypediversity,thestxsubtypes and thevirulenceprofileidentifiedamongisolatesfromtheanimal reservoirandenvironmentaresimilartotheisolates recov-eredfrompatients.173,178–180_{Therehasbeenparticularinterest}

insomeSTECserotypesthathavebeenresponsibleforcausing severehumaninfections,suchasO113:H21,butunlikeothers, theydonotproduceadhesinsencodedbyLEE.ByusingaPCR microarray,41virulenceorgeneticmarkersweretestedina panelof65O113:H21strainsisolatedfromclinicalinfections, environmentandfoodfromvariouscountries.174_Theresults

obtainedshowednocleardifferencesinthesegenetic mark-ersbetweenthepathogensrecoveredfromHUScasesandthe environmentalstrains.Moreover,onlystxsubtypesassociated withhumaninfectionswereidentifiedinallisolates,therefore suggestingthattheenvironmentalisolateshavethepotential tocausehumandiseases.

Epidemiology

TheincidenceofHUScasesinBrazilislow,175_andalthough

somehypothesishasbeenproposedtoexplainthisfact,there arelimiteddataontheimmuneresponseagainstStx.Inan attempttoovercomethis gap,prevalenceofanti-Stx2 anti-bodiesinseraofchildrendiagnosedwithHUSandofhealthy childrenwasrecentlydetermined.176_{Thepercentageof}

indi-vidualsshowingantibodiesagainstStx2 washigheramong HUSpatients thancontrols, andthe resultsalsoconfirmed thatSTECstrainsarecirculatinginoursettingsdespitethe lownumberofidentifiedHUScases.

Among the several serotypes associated with human infections,O157:H7isresponsibleformoreseverecases. Epi-demiologicalinvestigationsofdiarrhealoutbreaksconducted infourBrazilianstatesshowedthatO157:H7strainswere iso-latedfromtwohospitalizedpatients,onewithHUSandthe otherwithbloodydiarrhea.177_{Besides,O157:H7,EHEC/STEC}

strains belongingto the top sixmost important non-O157 serogroupssuchasO26,O103,O111 andO145 were identi-fied,allofwhichwererecoveredfromambulatorypatients. Inaddition,someuncommonserogroupsincludingO1,O24 and O77 among others were also detected, but they were all associatedwithacute diarrhea. Itisinteresting tonote thatthemajorityofpatientsfromwhomSTECwasisolated were female (57%), and that patients’ ages ranged from 8 monthsto80years,withmostbeinglessthanfiveyearsold (54%).177

ThedistributionofEHEC/STECinthegastrointestinaltract ofawide varietyofanimals indicatesthezoonotic charac-terofitsinfections.Theroleofdifferentanimalspeciesas asymptomatic carriers of EHEC/STEC has been extensively studied in the last years in Brazil. Besides cattle, which are their most common natural reservoir,173,178 _the

pres-ence of these pathogens has been identified in the feces of dairy buffaloes,179 _sheep,180,181 _pigs,182,183 _birds,184 _and

fishes.185_{Itisnoteworthythatsomerelevantserotypeslinked}

to human infections such as O103:H2 and O157:H7 have been recovered from the feces of sheep186 _{and cattle}173

respectively. Additionally, the high prevalence of O157:H7 EHEC/STEC strains identified in hides of cattle sent to slaughter in a Brazilian processing plant178 _certainly

rep-resents a relevant issue that should be considered when thinkingaboutinterventionstargetingEHEC/STECrelatedto animal handling, from farm to slaughter, as well as the implementation of food safety throughoutproduction and processing.

ThepresenceofEHEC/STECintheenvironmentisanother issueofconcern,sincetheycansurviveinthesoil,manure, pasturesandwater,whichthusrepresentimportantvehicles oftransmission. The isolation ofSTEC strains from drink-ing water supplies, collected in different municipalities in northernParanáState,hasbeenrecentlydescribed, highlight-ing the importanceofdrinking water, especiallythat from untreatedwatersupplies,asasourceofSTECstrains poten-tially pathogenic for humans.187 _{Taking into account that}

chickenlitterisveryusefulasanorganicsoilfertilizerforthe productionoffruitsandvegetablesinoursettings,the detec-tionofSTECinorganicchickenfertilizerusedonfarms188_also

representsasignificantpublichealthsafetyhazard.

Although data on the detection of EHEC/STEC in foods in Brazil are stillscarce, the isolationand identification of O157:H7serotypefromagroundbeefsamplewasdescribedfor thefirsttime,189_{whileO125:H19andO149:H8STECserotypes}

were found in refrigerated raw kibbe collected from retail establishments.190 _{On the other hand, EHEC/STEChas not}

beendetectedinpasteurizedcow’smilksamplescollectedin dairiesinnorthwesternParanáState191_orinrawmilk,

pas-teurizedmilk,MinasFrescalcheeseandgroundbeefsamples collectedinMinasGerais.192_{Oneshouldbeawarethatdespite}

difficultiesinthedetectionandisolationofEHEC/STECfrom foods,theimplementationofthemostsensitivemethodsin mostlaboratoriesshouldbethemaingoalinthenearfuture tohelpintheanalysisoftheriskposedbyfoodsasvehiclesof STECtransmissiontohumans.

Detectionanddiagnosis

AnimportantconcernishowtodetectShigatoxin-producing strainseitherinstoolsofinfectedpatientsorcontaminated food, sinceselectiveenrichmentisnecessary.193,194 For rou-tinediagnosis,someprotocolshavealreadybeendescribed.139

However,thegoldstandardforStxdetectionisstillthe eval-uation ofthe cytotoxicityofbacterial culturesupernatants to eukaryoticcells.195,196 _{Thus, multiplexPCRincluding stx}

geneandothervirulencegenescouldbeusefulinscreening for STEC using bacterial confluent growth zones or sor-bitol fermenting and non-fermenting colonies taken from SMAC.197

Numerous assays for the diagnosis of STEC have been developed on the basis of the detection of Stx1 and/or Stx2, which representsthe major virulence factors of this

E.colicategory.198_{Sensitivitiesandspecificitiesvaryaccording}

to the test format and the manufacturer.199–205

Neverthe-less,thestandardbywhicheachmanufacturerevaluatesits tests also varies; therefore, a direct comparison of perfor-mancecharacteristicsofvariousimmunoassayshasnotbeen performed.198,206,207 _{Moreover,thesecommerciallyavailable}

testsarenotaffordablefordevelopingcountries.Thus,to out-linethis,previousworkshaveestablisheddifferentformatsof immunoassays,employingeitheramixtureofrabbitanti-Stx1 andanti-Stx2serabyindirectELISAorpolyclonaland mono-clonalantibodiesinacaptureELISAassayforthedetectionof STEC.207–209_{Thestandardizedmethodsarereproducible,fast,}

easytoperform,showinghighsensitivityindetectingStxby captureELISA,eveninlow-producingisolates.Theseassays havenotyetbeenevaluatedintermsofindustrialquality con-trolandcommercialavailability,buttheestimatedcostofthe assayisaroundUS$70per96detections,whichisrealistically inexpensivefordevelopingcountries.

Thesemonoclonalantibodieswererebuiltresultingin sin-glechainfragmentvariable(scFv)fragments.Stx2-scFvwas obtainedfromabacteria-inducedcultureand showed diag-nostic ability;the scFvfragmentwas able torecognize the majority of Stx2-producing strains, with 79.3% sensitivity (confidence interval of 60.3 to92%), and no reactivity was observedwiththenon-producingstrains,indicatingashigh as100%specificity(confidenceintervalof86.8–100%).210_Itis

immunoenzymatic tests forStx1/2 toxin detection employ recombinantantibodiesproducedinbacteria,whichindeed willreducethecostsofthediagnosticassays.198

Enteroaggregative

E.

coli

EAECisthediarrheagenicE.colipathotypedefinedbyshowing thecharacteristicAApatternonepithelialcellsinculture.211

TheAApatternwasdefinedin1987whenNataroetal.,212 dis-tinguishedthepreviouslydescribed“diffuseadherence”asthe trulydiffuseadherence(DA)andtheAApattern.Thestandard AAwascharacterizedbyadherentbacteriainastacked-brick arrangement onthe surface ofepithelial cells and alsoon the coverslip betweencells.Strains displaying the AA pat-tern were then categorized as “enteroadherent-aggregative

E.coli”butafterwardsthecategorywascalled enteroaggrega-tiveE.coliorEAEC,thecurrentnomenclature.Thedetection ofAAin vitroisstillthegoldstandard testtodefineEAEC; however,asdescribed before,theAApatternmaybefound instrainsofotherDECpathotypes,suchasaEPEC.Therefore, anup-to-date definitionofEAECisthe diarrheagenicE.coli

thatproduceAAinculturedepithelialcellsbutlackthemain geneticmarkersthatdefineotherDECpathotypes(EPEC,ETEC, EHEC,EIEC).AnexceptionforthatisthehybridEAEC/STEC strainresponsibleforamassiveoutbreakofdiarrheaandHUS in2011inEurope.213 _{ThisstrainconsistsofanEAECstrain}

thatacquiredtheStx2-encodingphage.Therefore,thisspecific O104:H4strainisaStx-producingEAEC.

DiarrheacausedbyEAECiswatery, oftenwiththe pres-enceofmucus,withorwithout bloodandabdominalpain, vomitingand low fever.Acute self-limiting diarrhea isthe usualpathology,but somepatientsmaydevelopprotracted diarrhea,i.e.,lasting morethan 14days.214 _Prolonged

diar-rheaoccurs depending onthe host’s immunity,nutritional statusand geneticsusceptibility.215 _{Genetic susceptibilities}

associatedwithEAECdiarrheawereidentifiedinNorth Amer-ican travelersto Mexico. Single nucleotide polymorphisms (SNP)in the IL-8 genepromoter and the promotor regions ofthegenesencodinglactoferrin,CD14andosteoprotegerin aswellwererecognizedasindicatorsforsymptomaticEAEC infection.216–219

A well-described characteristic of EAEC strains is their heterogeneous nature when serotypes, genetic markers of virulenceandphylogenetic groupsareanalyzed.220–225 _This

indicatesthat onlyEAECstrains carryingspecific virulence factors are able tocause diarrhea.While these factors are unknown,somestudieshavedemonstratedtheassociation ofspecificvirulencegeneswithdiarrhea,suchaspetoraafA

inBrazil226_{andsepAinMali.}223

Virulencefactors,mechanismsandpathogenesis

MostofourknowledgeaboutEAECpathogenesisisbasedon dataaccumulatedfromstudies withEAECstrain042, since itsassociationwithhumandiarrheainavolunteerstudy.227

Theseputativevirulencefactorsincludeadhesins,toxinsand secretedproteins.However,noneofthesefactorsarefoundin allEAECstrains.

Themajorityofthesevirulencefactorsareplasmidborne, including those mediating AA. Consequently, these high-molecular-weightplasmidsarecalledpAA.220_Baudryetal.,228

developedageneticprobe(CVD432)forEAECdiagnosisonthe basisofafragmentfrompAA1presentinEAECstrain17-2. InEAEC042,manyputativevirulencefactorsarepresentin pAA2.220

Recently,adivisionofEAECstrainsintotypicalor atypi-calsubgroupswasproposed.Thisclassificationisbasedon thepresenceorabsenceofaggR,agenethatencodesaglobal regulatorofEAECvirulencegenes.229_{Therefore,ithasbeen}

proposedthattypicalEAEChavemorepathogenicpotential bythepresenceoftheAggRregulonand,consequently,pAA virulencefactors.230_{However,atleasttwooutbreaksof}

diar-rheawerecausedbyatypicalEAEC,231,232_{andatypicalEAEC}

arecommonlyisolatedfromchildrenwithdiarrhea,insome casesmorefrequentlythantypicalstrains.233,234

Numerousadhesins,cytotoxins,enterotoxinsandsecreted proteinshavebeen characterizedinEAECstrainssincethis pathotypedefinition.211,214

Themoststudiedadhesinsaretheaggregativeadherence fimbria(AAF/I-AAF/V)family,whichincludesfivetypes.235–239

TheymediatetheAApatternandbiofilmformation.Afimbrial adhesinshavealsobeencharacterizedinEAECstrains, includ-ing outermembraneproteinsbetween30and58kDa.240–242

However, it has been shown that these structures are presentinlowfrequenciesinEAECcollectionsfromdifferent settings.221,226,243–245

Locatedin pAA2ofEAEC042 isthe aapgene, encoding an antiaggregationproteincalleddispersin.246 _Thisprotein

is secreted and linked to lipopolysaccharide, neutralizing the negativechargeofthebacterialsurfaceleading toAAF projection and consequent dispersion along the intestinal mucosa.247 _{Although immunogenic, dispersin is found in}

otherE.colipathotypesandincommensalE.coli.248

VarioustoxinshavebeendescribedinEAECinassociation with the cytotoxic or enterotoxic effects of culture super-natantsinvitro.Theheat-stabletoxinenteroaggregativeE.coli

heat-stableenterotoxin1(EAST-1)wasthefirsttoxin charac-terizedintheEAECpathotype.249_{EAST-1activatesadenylate}

cyclaseinducingincreasedcyclicGMPlevels,effectsobserved inaUssingchamberwithrabbitileum.250_{ShET1isanA:Btype}

toxinthatcausesaccumulationoffluidinrabbitilealloopsand hassecretoryresponseinUssingchamberassays.251,252

ThetwoATproteinscharacterizedinEAEC042, Petand Pic,253–254 _{are members of the serine protease}

autotrans-portersofEnterobacteriaceae,orSPATE.255_{Petisacytotoxin}

that modifies the cytoskeleton of enterocytes, leading to roundingandcelldetachment.Thecytotoxicmechanismof Petarisesfromthedegradationof␣-fodrin,amembrane pro-tein of the enterocytes.256 _{Pic is a multitask protein that}

mediateshemagglutination,mucuscleavageand hypersecre-tion, intestinal colonization in mice, cleavage of surface glycoproteinsinvolvedinleukocytetraffickingandcleavageof keycomplementmolecules.257,258Thephenotypesidentified forPicsuggestitsroleinpromotingcolonizationofthe intes-tineandimmunesystemevasion.SPATEsareimmunogenic proteins, as evidenced by the presence ofserum antibod-iesagainstPetandPicinchildrenrecoveringfromdiarrhea causedbyEAEC.259

In the years that followed the definition of EAEC as a pathotype, research in the field was dedicated to prove the pathogenic capacity of EAEC using different animal models260–262_{andhumanvolunteersreceivingoralinoculum}

ofdifferentEAECstrains.227,235,263 _{Notall volunteers}

devel-opeddiarrheaafteringestionofdifferent EAECstrains,the firstevidencethatstrainsofthispathotypeareheterogeneous. Amongthestrainstested,EAEC042(serotypeO44:H18)caused diarrheainthreeoutoffivevolunteers.227_{Sincethen,strain}

042hasbeenconsideredtheprototypeEAECstrainandis cer-tainlythemoststudiedstrainofthepathotype.264_EAEC042

wasisolatedfromacaseofacuteinfantilediarrheainPeru.265 Theclinicaldataobtainedfromthevolunteerswhodeveloped diarrheasuggestedthatEAEC042causedsecretorydiarrhea, withabundantpresenceofmucusandabsenceofbloodinthe stool.

StudiesemployingdifferentEAECstrainsinteractingwith intestinalcellsfromanimalsorhumanshavebeenperformed toelucidatethe pathogenesisofthis pathotype.Data from theseinvitro,invivoand exvivoexperimentsstrongly indi-catethatEAECcanbindtojejunal,ilealandcolonicepithelium in the characteristic aggregative pattern, forming a strong biofilminamucuslayer,followedbycytotoxicand proinflam-matoryeffects.260,266–270_{Fragmentsfromterminalileumand}

colonexcisedfrom pediatricand adultpatientswere incu-batedwithEAECstrainsthatwerecapabletocolonizetheileal andcolonicmucosainthetypicalstacked-brickpatternover anaugmentedmucuslayer.270

Alltheselinesofevidenceincombinationwiththe iden-tification of several putative virulence factors inprototype EAECstrainsallowedtheproposalofathree-stagemodelof EAECpathogenesis:(a)abundantadherencetotheintestinal mucosa,(b)productionofcytotoxinsandenterotoxins,and (c)inductionofmucosal inflammation.211_{Inthefirst stage,}

the contributionoffimbrial andafimbrial adhesinsaswell asotheradhesivestructuresisessential.Severalcolonization factorshavebeenidentifiedinEAECstrains.271_{Inthisstage,a}

characteristicincreasedsecretionofmucusontheintestinal mucosaleadstotheformationofastrongbiofilmwhereEAEC areembedded.234,266,272_{Inthefollowingstep,EAECproduce}

cytotoxiceffectsontheintestinalmucosaduetothesecretion oftoxins, inducingmicrovillusvesiculation, enlarged crypt openings,andincreasedepithelialcellextrusion.266,273

EAEC-induced inflammationresultsfrom the strong colonization ofthe intestinalmucosa;however,all bacterialfactorsthat contributetothisconditionhavenotbeenidentified. Inflam-matory markers such asIL-8, IL-1␤, interferon (INF)-␥ and lactoferrinhavebeendetectedinstoolsofchildrenandadults colonized byEAEC.274–276 _{Althoughthis modelsummarizes}

the data so far obtainedusing in vivo,in vitro and ex vivo

approachesitmaynotbevalidforallstrains.

Alargefoodborne outbreakofbloodydiarrheaand HUS occurredin2011inEurope,affectingmorethan4000patients, most of them from Germany. This outbreak was caused byaStx2-producingE.colistrain belongingtotheserotype O104:H4.Thegenomeofthatstrainwasrapidlysequenced, revealinga unique hybrid combination ofEAEC and STEC,

i.e. the EAEC strain Ec55989 harboring the Shiga toxin 2-encodingprophage.166,213_{Severalvirulencefactorsoftypical}

EAECarepresentinthatstrain,includingAggR,dispersin,Pic

andShET-1. AlsoexpressedaretwoShigellaautotransporter proteinscalledSigAand SepA,implicatedinmucosal dam-age and colonization.277,278 _{Interestingly,the EAEC Ec55989}

is the prototype strain for AAF/III.237 _{Conversely, the}

out-breakhybridstrainproducesAAF/I,showingthattheoutbreak EAEC/STECacquiredanAAF/I-encodingplasmid.166,279_Ithas

been proposedthat thepresenceofthesevirulencefactors combinedisresponsibleforthehighlyvirulentattributesof thatstrain.166,213

Epidemiology

EAECisanemergingpathogenaffectingchildrenandadults worldwide,responsibleforcasesofacuteandpersistent diar-rhea. Nevertheless,the mostimportantimpactintermsof morbidityisamongchildrenyoungerthan5yearslivingin developing countries.214 _{A meta-analysis study of the}

lit-erature on the epidemiology ofdiarrhea that included the searchofEAECshowedastatisticalassociationofEAECwith acute and persistentdiarrheaindevelopedand developing countries,withdiarrheainHIV-infectedpatientsindeveloping countries,andadulttraveler’sdiarrhea.280 Inanother meta-analysis studyEAECwas associatedwithacutediarrheain childrenlivinginSouthAsiancountries.281

It is important to mention that data on the epidemi-ology ofEAECinfection are somewhat inconsistent dueto largevariationintermsofmethodofdetection,geographical locationandpatientageandsocioeconomicstatus. Nonethe-less,EAEChasbeensystematicallyidentifiedasanemerging enteropathogen,stronglyassociatedwithacute and persis-tentdiarrheainchildrenofdevelopingcountries.Moreover,in developedcountries,EAEChavebeenfrequentlyisolatedfrom casesofdiarrheainchildrenandadultsinthelastyears.282,283

Inaddition,severalfoodborneoutbreaksofdiarrheacaused by EAEChave been reported inEurope, Japan, Mexico and India.231,232,284–286_{Oneofthemaffected2697schoolchildren}

inJapan,afterconsumptionofschoollunches.232

SeveralstudieshaveimplicatedEAECasthepredominant agentofpersistentdiarrheainchildren.287–289_{EAEC-mediated}

persistent diarrhea has been linked to malnutrition and decreaseinphysicalandintellectualdevelopmentinseveral studiesfromBrazil.274,288,290_{Notably,asymptomaticpatients}

infectedwithEAECalsoexhibitgrowthretardation.274_Since

its definition as a pathotype, high rates of asymptomatic young children carrying EAEC have been reported in sev-eralstudies,involvingsubjectswithlowsocioeconomicstatus in developing countries.214 _{The persistence of EAEC may}

inducechronicintestinalinflammation,evenintheabsence of diarrhea, reducing its absorptive function and leading to malnutrition.274,291 _{Growth impairment has also been}

observedinamousemodelofEAECoralinfection.292

Consid-ering the high number of asymptomatic EAEC-colonized children in low-income countries, this pathotype has an importantimpactonpublichealthasonecauseofimpaired physicalandcognitivedevelopment.

EAECistransmittedbythefecal-oralroutebyfoodor con-taminatedwater.232,285,286_{EAECweredetectedinmilksamples}

from infantfeeding bottles that were handled by mothers with low socioeconomic status.293 _{Also, viable EAEC were}