Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin

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Food

Microbiology

Occurrence

and

characteristics

of

extended

spectrum

beta-lactamases-producing

Enterobacteriaceae

from

foods

of

animal

origin

˙Ismail

Hakkı

Tekiner

,

Haydar

Özpınar

DepartmentofFoodEngineering,IstanbulAydınUniversity,FloryaCampus,Sefaköy,Küc¸ükc¸ekmece,Istanbul,Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received16April2015 Accepted12November2015 Availableonline2March2016 AssociateEditor:AnaLúciadaCosta Darini Keywords: Blagene Enterobacteriaceae ESBL Publichealth Turkey

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Presenceofextendedspectrumbeta-lactamases(ESBL)inbacteriaisagrowinghealth con-cernofglobalsignificance.Thelocal,regional,national,andinternationalepidemiological studiesforextended spectrumbeta-lactamases-producing Enterobacteriaceaeand their encodinggenesinfoodsarestillincomplete.Theobjectiveofthisstudywastodeterminethe occurrenceofextendedspectrumbeta-lactamases-producingEnterobacteriaceaeandthe characteristicsoftheirencodinggenesfromatotalof250samplesofvariousfoodsof animal-origin(100rawchickenmeat,100rawcowmilk,and50rawcowmilkcheese)soldinTurkey. Overall,55isolateswerepositiveasextendedspectrumbeta-lactamases-producing Entero-bacteriaceae.The mostprevalent extendedspectrum beta-lactamases-producing strain wereidentifiedasEscherichiacoli(80%),followedbyEnterobactercloacae(9.1%),Citrobacter braakii(5.5%),Klebsiellapneumoniae(3.6%),andCitrobacterwerkmanii(1.8%)byVitek®_MS.The

simultaneousproductionofextendedspectrumbeta-lactamasesandAmpCwasdetectedin fiveisolates(9.1%)inE.coli(80%)andE.cloacae(20%).ThefrequencyratesofblaTEM,blaCTX-M,

andblaSHV were96.4%,53.7%,and34.5%,respectively.Theco-existenceofbla-geneswas

observedin82%ofextendedspectrumbeta-lactamasesproducerswithadistributionof blaTEM&blaCTX-M(52.7%),blaTEM&blaSHV(20%),blaTEM&blaCTX-M&blaSHV(12.7%),andblaSHV

&blaCTX-M(1.8%).ThemostprevalentvariantofblaCTX-MclusterswasdefinedasblaCTX-M-1

(97.2%),followedbyblaCTX-M-8(2.8%).Insummary,theanalysedfoodswerefoundtobe

pos-ingahealthriskforTurkishconsumersduetocontaminationbyEnterobacteriaceaewitha diversityofextendedspectrumbeta-lactamasesencodinggenes.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:ihakkitekiner@aydin.edu.tr(˙I.H.Tekiner).

http://dx.doi.org/10.1016/j.bjm.2015.11.034

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

Introduction

Antibioticsareusedasveterinaryandhumanmedicinesfor treatment,controlandpreventionofinfectiousdiseases. How-ever,theirrepeatedoff-labeloverusecanhaveun-anticipated adverseeffectsincludingdevelopmentofantibioticresistance inthebacteriatowardsmodernbeta-lactamantibiotics.1

Oneoftheantibioticresistancemechanismsinthebacteria istheproductionofspecificenzymessuchasbeta-lactamase tobreakdownthefour-atombeta-lactamringofthe antibi-otics.Whenthisringisopenedthroughhydrolysisbythese enzymes,the antimicrobialpropertiesoftheantibioticsare completely lost.2 _{The extended spectrum beta-lactamases}

(ESBL)are the rapidly evolving plasmid-mediated group of beta-lactamases.3 _{ESBL canhydrolyse penicillins, first,}

sec-ondandthird-generationcephalosporins,andaztreonam(but notcephamycinsorcarbapenems).TheactivityofESBLcanbe inhibitedbybeta-lactamaseinhibitorssuchasclavulanicacid (CLA).4 _{Theantibioticresistanceleadstoincreased}

morbid-ity,mortalityandthecostoftreatinginfections,inparticular, thosecausedbyESBL-producingbacteria.5_{Themostprevalent}

extendedspectrumbeta-lactamasesareTEM,SHV,andCTX-M variants.6_{TheCTX-Mvariantsarealsoclusteredinfivemajor}

groups:1,2,8,9,and25.7_{After2000s,CTX-Mtypeenzymes}

haveexhibitedarapidgrowthoverawiderangegeographic areas,andhavebecomemuchmoreprevalentthanTEMand SHVtypeenzymes.8

TheESBLs are mainlyencoded byplasmids and mobile genetic elements such as integrons, insertion sequences, transposonsandplasmids.Thesegeneticelementsare eas-ilytransferabletootherbacteriasuchasthosebelongingto Enterobacteriaceae.5,9 _{Thewidespread use ofthird}

genera-tioncephalosporinsandaztreonamhasledtotheemergence anddisseminationofESBLproducingstrainsandtheir encod-inggenes,inparticular,amongEnterobacteriaceaeassociated withsevereentericillnesses.10–13

ThemajorgenesresponsibleforESBLproductioninclude TEM genes (blaTEM), SHV genes (blaSHV), and CTX-Mgenes (blaCTX-M).14 Thiscontinued evolution isaserious threatto the public health by limiting the ability to treat bacterial infections.15,16_{Mostofthemedicinesunderhuman}

consump-tion are antibiotics. Moreover, antibiotic use in veterinary medicineandforgrowthpromotionanddiseaseprevention inagriculture, aquaculture and horticulture is alsoa huge problemofantibioticresistance,sincemostoftheantibiotics manufactured(100,000–200,000tonnesperyear)isestimated to be massively consumed by these sectors, especially in the food-producing animals for last 60 years.17,18

There-fore,food-producinganimalsandfoodsofanimal-originare under suspicion forbeing transmission vectors for coloni-sation and infection of the humans with ESBL-producing Enterobacteriaceae.11,16

The potential contribution of foods to human health risks by dissemination of plasmid-born ESBLs should be epidemiologicallyassessedbecauselocal,regional,national andinternationalinformationrelatedtothisemerging phe-nomenon is largely incomplete in various geographical locations,includingTurkey.9,19_{TheTurkishauthoritiesaccept}

thattheuseofantibioticsinthefoodanimalstomakethem

growfaster and/ortopreventdisease cannotbecontrolled effectively due to the economic benefits of food-animals, as the sector largely ignores risks associatedwith human andanimalhealth.20_{Therefore,chickenmeat,rawmilkand}

cheese can be identified ashigh risk-carrying contributors for ESBL-producing Enterobacteriaceae and their encoding genes.9

Theobjectiveofthisstudywastodeterminetheoccurrence ofESBL-producingEnterobacteriaceaeandthefrequencyrates ofcorrespondingencodinggenesinvariousfoodsofanimal originsoldinTurkey.

Materials

and

methods

Referencecultures

AnESBL-negativestrain Escherichia coliATCC®_{25922and an} ESBL-positivestrainKlebsiellapneumoniaeATCC®_700603were used asnegative and positive controls,respectively, in the phenotypicandgenotypicmethods.

Sampling,samplepreparation,isolationandidentification ofESBLsuspiciousbacteria

DuringtheperiodofMay2014toNovember2014,atotalof250 foodsamplesofanimalorigin(100packedrawchickenmeat frompublicbazaars,marketsandpoultryfarms,100rawcow milkfromcontainersandholdingtanksinbazaarsanddairy farms,and50unpackedrawcowmilkcheesefrombazaarsand markets)wererandomlycollectedindifferentcitiesof Mar-mara,AegeanandBlackSearegions.Allsampleswereplaced intosamplingbags,andtakentothelaboratoryinathermobox containerat4◦C.Thesamplepreparationandfurther exami-nationswerequicklystartedafterthesampling.

Tenmillilitresofrawmilkin90mLofEnterobacteriaceae EnrichmentBroth(LABM,Lancashire,England),and25geither ofrawcow milk,cheese orrawchicken meatin 225mLof thementionedbrothwashomogenisedinasterilebag (Inter-science,SaintNom,France)for2minbyastomacher(EasyMix, AESChemunex,Rennes,France),andfollowedbyanaerobic incubationat37◦Cfor18–24h.21_{Afterthat,10␮Lofthe}

pre-enrichedsuspensionwasdirectlystreakedontoChromaticTM ESBLselectivemedia(Liofilchem,Istanbul,Turkey).The inoc-ulatedplateswereincubatedat37◦Cfor18–24hunderaerobic conditionaspertothemanufacturer’sinstructions.The pre-sumptiveESBLcolonieswerethensub-culturedontoTryptic SoyAgar(LABM,Lancashire,England),followedbyan incuba-tionat37◦Cfor18–48h.Thespeciesidentificationofsuspected ESBLisolateswasperformedbyVitek®_{MS(bioMérieux,Marcy} l’Etoile,France).

ScreeningandconfirmationofESBLs

Presumptive ESBL-producers were screened using cefpo-doxime(CPD;10␮g),cefotaxime(CTX;30␮g),andceftazidime (CAZ;30␮g)antibioticdiscs(MASTGroup,UK).A0.5 McFar-landstandardinoculumoftheisolatewasspreadontoMueller Hinton Agar(LABM, Lancashire, England) plates.Thediscs wereinsertedontheplate,andallowedforincubationat37◦C

Table1–Primerdesign,sequences,ampliconsizesandthermocyclerconditionsforbla-genesaccordingto23,24.

Primer(F/R) Sequence Ampliconsize(bp) Thermocyclercondition(TC)

blaTEM-F 5-GCTCACCCAGAAACGCTGGT-3 686 TC-1

blaTEM-R 5-CCATCTGGCCCCAGTGCTGC-3

blaSHV-F 5-CCCGCAGCCGCTTGAGCAAA-3 733 TC-2

blaSHV-R 5-CATGCTCGCCGGCGTATCCC-3

blaCTX-M-F 5-SCSATGTGCAGYACCAGTAA-3 585 TC-3

blaCTX-M-R 5-ACCAGAAYVAGCGGBGC-3

blaCTX-M-1-F 5-AAAAATCACTGCGCCAGTTC 415 TC-4

blaCTX-M-1-R 5-AGCTTATTCATCGCCACGTT

blaCTX-M-2-F 5-CGACGCTACCCCTGCTATT 552

blaCTX-M-2-R 5-CCAGCGTCAGATTTTTCAGG

blaCTX-M-8-F 5-TCGCGTTAAGCGGATGATGC 666

blaCTX-M-8-R 5-AACCCACGATGTGGGTAGC

blaCTX-M-9-F 5-CAAAGAGAGTGCAACGGATG 205

blaCTX-M-9-R 5-ATTGGAAAGCGTTCATCACC

blaCTX-M-25-F 5-GCACGATGACATTCGGG 327

blaCTX-M-25-R 5-AACCCACGATGTGGGTAGC

TC-1:1cyclefor15minat95◦C,followedby35cyclesfor1minat95◦C,1minat63◦C,and1minat72◦C,andfinally10minat72◦C. TC-2:1cyclefor15minat95◦C,followedby30cyclesfor30sat94◦C,30sat58.5◦C,and45sat72◦C,andfinally10minat72◦C.

TC-3:1cyclefor15minat95◦C,followedby30cyclesfor30sat94◦C,30sat55◦C,and45sat72◦C,andafinalelongationat72◦Cfor10min. TC-4:1cyclefor15minat94◦C;5cyclesfor25sat94◦C,40sat52◦C,and50sat72◦C,followedby22cyclesfor25sat94◦C,40sat54◦C,and 50sat72◦C,andafinalelongationat72◦Cfor6min.

for18–24h.Thebreakpointswithzonediameterswere eval-uatedaccordingtothecriteriaestablishedby.22_TheESBLSet

D67C(MASTGroup,UK)withacombinationofCPD,CTX,and CAZ±clavulanate(CLA,10␮g)wasusedfordiscdiffusion con-firmationtest.Thediscinsertedplateswereincubatedat37◦C for18–24h.Thezoneofinhibitionwasevaluatedaccordingto thecriteriadescribedby22.

Determinationofminimalinhibitoryconcentration(MIC) Micronaut-S beta-lactamase VII Plate (Merlin Diagnostika, Berlin,Germany) was usedforthe phenotypicdetection of ESBL-production, including AmpC, metallo-beta-lactamase (MBL), and carbapenemase (KPC) according to the criteria describedby22.A50␮Laliquotof0.5McFarland-standardised microbialsuspensionoftheisolatewasinitiallyvortexedin 10mLofMuellerHintonBroth(Merck,Darmstadt,Germany). Subsequently,100␮Lofthissuspensionwaspipettedintoeach welloftheplate.Afterthat,theplatewasincubatedovernight at37◦C.ThereadingwasrecordedusingaThermofisher Mul-tiskanFCSpectrometer.TheMICanalysiswasautomatically performedbytheMCN6Software(Sifin,Berlin,Germany). ExtractionofbacterialDNA

TotalDNAofESBL-producingisolateswasextractedusingthe GENESpin DNA Isolation Kit(Eurofins, Hamburg, Germany) accordingtothe manufacturer’sinstructions.Theextracted DNAsampleswerestoredat–20◦Cforfurthermolecular anal-yses.

Characterisationoftheblavariants

Theprimerpairsaspreviouslydescribedby23wereusedfor screeningofblaTEM,blaSHVandblaCTX-M.TheblaCTX-Mvariants (blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, and blaCTX-M-25)

weresub-characterisedaccordingto24.Allprimerswere pre-paredbyFullgenBiotechnologyand ˙Iontek(˙Istanbul,Turkey). TheamplificationwasperformedbyaThermalCycler PTC-0200G DNA Engine (BioRad, Istanbul, Turkey). The blaTEM,

blaSHVandblaCTX-MwereamplifiedinasingleplexPCRassay while a multiplex PCR assay was performed for blaCTX-M variants. The PCR mixtures were prepared in a final vol-ume of25␮L. All PCR conditions were optimised again as showninTable1.Gel-electrophoresisofthePCRproductswas performedona1.5%agarose-gel.Theampliconswere photo-graphed by GelDoc 2000imagingsystem (BioRad, Istanbul, Turkey).Finally,theanalysiswascarried-outusingQuantity One4.6.3GelDocXRSoftware(BioRad,Istanbul,Turkey). Statisticalanalysis

TheKruskal–WallisH-testwasusedformultiplecomparison oftheproportionsofESBLproducersintheirsamplinggroups. TheteststatisticHwasapproximatedbya2_{distribution.All} statisticalanalyseswereperformedusingSPSS19(SPSSInc., USA)statisticalpackageprogramme,andaP-value<0.05was consideredasstatisticallysignificant.

Results

Inthisstudy,wescreened,forESBL-producing Enterobacte-riaceae, 100 raw chicken meat samples, 100 rawcow milk samples, and50rawcow milkcheesesamplesfrom differ-ent cities of Marmara, Agean and Black Sea regions, and subsequentlycharacterisedtheirencodingblaTEM,blaSHVand

blaCTX-Mvariants.

Phenotypicresults

Thephenotypictestingindicatedthatatotalof55isolates(29 inrawchickenmeat,24inrawcowmilk,and2inrawcowmilk

Table2–ScreeningresultsofthesamplesforESBL-producingEnterobacteriaceae.

Origin Noofsample(n,%) NoofESBL+isolates(n,%) Species-baseddistribution(n,%)

E.coli E.cloacae K.pneumoniae C.brakii C.werkmanii

Rawchickenmeat 100(40%) 29(52.7%) 24(82.8%) 2(6.9%) 2(6.9%) – 1(3.4%)

Rawcowmilk 100(40%) 24(43.6%) 18(75%) 3(12.5%) – 3(12.5%) –

Rawcowmilkcheese 50(20%) 2(3.7%) 2(100%) – – – –

Total 250(100%) 55(100%) 44(80%) 5(9.1%) 2(3.6%) 3(5.5%) 1(1.8%)

cheese)from250foodsamplesofanimaloriginwerepositive forESBL-producing Enterobacteriaceae.Themostprevalent phenotypewasE.coli(80%), followedbyEnterobactercloacae

(9.1%),Citrobacterbraakii(5.5%),K.pneumoniae(3.6%),and Cit-robacterwerkmanii(1.8%)(Table2).

Thedisc approximation tests were performed by using CPD, CAZ, and CTX±CLA containing discs according to the criteria set by 22. The average zone differences of CAZ±CLA, CTX±CLA and CPD±CLA were found as 6.83±3.66mm, 11.97±6.08mm and 12.76±6.06mm in chicken meat, respectively, and 7.92±5.07mm, 13.33±4.78mmand12.63±7.11mmincowmilk,respectively, while10.00±0.00mm, 15.00±4.24mm and 14.50±2.12mm incowmilkcheese,respectively(Table3).

TheMICdeterminationrevealedthat62.1%oftheisolates fromrawchickenmeatwereresistanttoCTX,55.2%toCAZ, 51.7%toCEP,20.6%toCOX,and6.9%toCMC,whileamong theisolatesfromrawcowmilk,79.2%wereresistanttoCAZ, 75%toCTX,66.6%toCEP,54.2%toCOX,4.2%toERT,and4.2% toCMC.Two isolatesfrom raw-cow-milkcheesewere100% resistanttoCTX,CAZ,CEP,andCMC.

Amongthe55ESBLproducers,fiveisolates(9.1%)werealso positiveforAmpC.Basedonthespecies,theAmpC-producing isolateswereE.coli(80%)andE.cloacae(20%)fromrawchicken meat(n=1),rawcow milk(n=3),and raw-cow-milkcheese (n=1).TheywereresistanttoCAZ(100%),CTXandCEP(80%), CMC(40%),andCOX(20%).

Genotypicresults

Inthegenotypicanalysis,thePCRproductswereidentified:53 asblaTEM,36asblaCTX-M,and19asblaSHV.Inthe36blaCTX-M, 35 were clustered asblaCTX-M-1, and one asblaCTX-M-8. The

blaTEMwasfoundinmultiplecombinationswithblaCTX-M-1&

blaCTX-M-8in29,withblaSHVin11,andwithblaSHV&blaCTX-M-1

in7.TheblaSHVwasdetectedinacombinationwithblaCTX-M-1 onlyinoneinstance.Ofthe44E.coliisolates,representingthe mostprevalentESBL-producingphenotype,aco-existing dis-tributionofblageneswasobservedasblaTEM&blaCTX-M-1in25,

blaSHV&blaTEMinsix,blaSHV&blaTEM&blaCTX-M-1in6,blaSHV &blaCTX-M-1inone,blaTEM&blaCTX-M-8inone,blaTEMalonein four,andblaCTX-M-1aloneinone,respectively(Table4).

Statisticalresults

ThemultiplecomparisonoftheproportionsofESBLproducers intheirsamplinggroupswasperformedusingKruskal–Wallis H-test (H=5.935; P<0.05). Thetest statistic H was approxi-matedby2_{distribution(CI95%;}2_{=12.5291;P=0.001).Since}

Hwaslessthan2_{,nostatisticallysignificantdifferencewas} observedfortheassociationbetweenESBLproportionsand theirsamplinggroups(P<0.05)withrespecttothefoodtypes ofanimalorigin.

Discussion

Inthisstudy,wescreenedforESBL-producing Enterobacteria-ceaefromrawchickenmeat,rawcowmilkandraw-cow-milk cheese samples. Subsequently, we characterised genotypi-cally their encodinggenes. The resultshighlighted that 55 isolateswerepositiveforESBL-producingEnterobacteriaceae; ESBL-producingE.coli(80%)wasthemostfrequentspecies;a combinationofblaTEM&blaCTX-M(52.7%)wasthemost com-monpattern;andthemajorvariantofblaCTX-Mwasdefinedas

blaCTX-M-1(97.2%),respectively.

The ESBL encoding genetic elements are transferable betweenthesameanddifferentbacterialspecies.4,25Astudy reported that the antibiotic residues in calf urine resulted in amplification of the populations of antibiotic resistant

Table3–ZonediametersofCPD,CTX,andCAZ±CLA.

Antibioticagent Rawchickenmeat(n=29) Rawcowmilk(n=24) Raw-cow-milkcheese(n=2) Mean(mm) Std.deviation(±mm) Mean(mm) Std.deviation(±mm) Mean(mm) Std.deviation(±mm)

CAZ 18.24 5.125 16.25 4.057 14.5 3.536 CAZCLA 25.07 6 24.17 4.603 24.5 3.536 1 6.83 3.665 7.92 5.073 10 0 CTX 15.59 6.196 11.29 4.428 12 1.414 CTXCLA 27.55 6.3 24.63 4.817 27 5.657 2 11.97 6.08 13.33 4.779 15 4.243 CPD 7.03 5.723 2.13 4.047 6 0 CPDCLA 19.79 5.87 14.75 7.837 20.5 2.121 3 12.76 6.063 12.63 7.113 14.5 2.121

Table4–genotypiccharacteristicsof55ESBL-producers isolated. Source Species Noof isolates ESBL-type detectedbyPCR Rawchicken meat

E.coli 3 blaTEM

E.coli 12 blaTEM&

blaCTX-M-1

E.coli 1 blaTEM&

blaCTX-M-8

E.coli 6 blaTEM&blaSHV

E.coli 2 blaTEM&blaSHV&

blaCTX-M-1

E.cloacae 1 blaTEM

E.cloacae 1 blaTEM&blaSHV

C.werkmanii 1 blaTEM

K.pneumoniae 2 blaTEM&blaSHV&

blaCTX-M-1

Rawcowmilk E.coli 1 blaTEM

E.coli 13 blaTEM&

blaCTX-M-1

E.coli 1 blaSHV&

blaCTX-M-1

E.coli 3 blaTEM&blaSHV&

blaCTX-M-1

E.cloacae 3 blaTEM

C.brakii 3 blaTEM&blaSHV

Raw-cow-milk cheese

E.coli 1 blaTEM&blaSHV&

blaCTX-M-1

E.coli 1 blaCTX-M-1

bacteriainthepensoil;andpresumably,thisincreasedthe probabilityoftransmissionoftheseresistantpopulationsto calvesandthentheanimal-derivedfoodchain.26_Thismight

leadto arisk forinfection and colonisation ofthe human intestinalmicrobiomewithESBLproducingbacteriasuchasE. coli,E.cloacae,Klebsiellaspp.,Salmonellaspp.,Proteusspp.and Citrobacterspp.,throughvariousfoodsofanimalorigin27–30_;

whenthesefoodsaredirectlyconsumedwithoutundergoing anythermalprocess,orusedforrawmilkcheeseproduction.31

Duetothisthreat,theCodexAlimentariusCommission estab-lishedanintergovernmentaltaskforce.32 _{Followingthat, in}

2014,theStandardCommitteeofEuropeanDoctors(CPME), theCouncil ofEuropeanDentists(CED)andthe Federation of Veterinarians of Europe (FVE) issued a common press releaseforalltheauthoritiestomanagetheproblemof ESBL-producingEnterobacteriaceae(www.fve.org).Inourstudy,a highoccurrenceofESBLproducersandtheirencodinggenes intheexaminedfoodsofanimaloriginindicateahealthrisk fortheTurkishconsumers.

Sincethe early2000s, E. colihasbecomethe most con-cerned member for infection and colonisation of the food animals.33–36 _{AnESBL-producing E.coliassociatedmortality}

wasthree-timeshigherthannon-ESBLproducingE.coli.37_The

NationalSurveillance Networkbythe MinistryofHealth in Turkey(www.uhes.saglik.gov.tr) has reported an increasing prevalenceofESBL-producingE.coli(33.2%in2008and48.83% in2013)andESBL-producingK.pneumoniae(40%in2008and 49.69%in2013).However,theroleoffoodsinthe dissemina-tionofESBL-producingbacteriaandtheirencodinggenesto humanshasnotbeenaddressedsofarinTurkey.Inourstudy, thefrequencyofESBL-producingE.coliwassimilartothose

reportedinPortugal(85%)38_{andSpain(93.3%),}33_whileitwas

higherthanthosereportedinDenmark(1.3%),39_India(3.1%),40

China(12.4%),41_{Germany(53.9%),}42_{Belgium(45%),}43_Austria

(24%)44_{andTaiwan(10.5%).}36_{OurstudyalsopointsE.colibeing}

animportantfoodborneESBLcarrier.

Numerousphenotypic methods havebeen developedto detectESBLproductionbyEnterobacteriaceae.However,itis not still clearwhich tests are the mostsensitive.45 _{In our}

study,thephenotypicscreeningandconfirmationofESBL pro-ductionwereperformedbythediscapproximationtests.All oftheuseddisc combinationsshowed goodsensitivitiesin accordancewiththecriteriaestablishedby22.Afterthedisc approximation tests,MIC wasdetermined duetothe pres-ence ofESBL encoding genes on the same plasmid.46 _The

MIC revealedthat55 ESBLproducerswereresistant toCTX (MIC≥4␮g/mL) and CAZ (MIC≥16␮g/mL), and partly vari-able to CPD (MIC≥8␮g/mL). Basically, TEM and SHV type beta-lactamases are resistant to CAZ, and variable toCTX whileCTX-Mtypebeta-lactamaseexhibitsresistancetoCPD and variable to CAZ because CTX-M type beta-lactamase may escape from screening by CAZ. A survey in Europe reportedthatupto33%ofESBLsstrainsremainedundetected, therefore, in this case, genotypic confirmation is further needed.47–50_{Inourstudy,thephenotypicresultsindicatedthat}

TEMand CTX-Mtypebetalactamaseswere themost com-monlyencounteredESBLs.

The co-existenceofESBL and AmpC beta-lactamasesis a worldwide growing concern.5,51 _{ESBL and AmpC}

produc-ing enterobacteriahavebeenclinicallyreportedas19.5%in Europe,andas13.9%inTurkey.52,53 _{Inourstudy,AmpC}

pro-ducers were E. coliand E. cloacae,asreported earlier.52,54,55

Failure to detect these beta lactamaseshas contributed to their uncontrolled spread and sometimes the consequent therapeutic failures.56 For Enterobacteriaceae, the use of either cefepime (CEP) or cefpirome (CPO)±CLA is recom-mended sincetheyareun-affectedbyAmpC.51_Incontrast,

ourresultsrevealedthatAmpCproducerswere100% resis-tant to CEP+CLA. This indicates that the techniques to detectAmpCbeta-lactamasearestillevolvingandarenotyet optimised.57

The genotypic characterisation by PCR assays revealed thatblaTEMwasthemostfrequentgene,followedbyblaCTX-M and blaSHV.InTurkey,the clinicallyreportedmostfrequent beta-lactamasetypewasCTX-M,followedbyTEMandSHV, whileanotherstudyreportedafrequencyofSHVhigherthan TEM.15,58,59_{Ourgenotypicresultsreportingthemostfrequent}

genesasblaTEM&blaCTX-Mwerealsoepidemiologically simi-lartothemostcommonlyencounteredESBLencodinggenes inchickenmeatfromEasternEuropeandNearEast,7

South-ern America,30,60 _Tunisia,61 _{Northern Europeancountries,}33

Japan,62,63_Germany,64_China,35_Austria65_andIndia,40_followed

bytherawmilkfromSpain,21_England66_andTaiwan36_,and

bythecheeseanddairyproductsfromPortugal,38_England,66

Iran67 _{and Egypt,}68 _{respectively. However, the foodborne}

ESBL-producingbacteriaandtheirencodinggenesinTurkey havenotbeen addressedtilldate,andthe foodborne beta-lactamases have been studied only bydisc approximation testing,excludinggenotypiccharacterisation.69–71 _Therefore,

ourstudyconfirmsthatthefoodsofanimalorigininTurkey arepotentialreservoirsofdiverseESBLencodinggeneswith

aco-resistancepattern,whichcanbedisseminatedtoclinical andcommunitysettings.

WedetectedCTX-M-1clusterasthemostprevalent sub-typeofCTX-Mtypebetalactamases.Chromosomalblagene, kluC,presentinKluyveracryocrescensistheancestorof CTX-M-1cluster.72 _{TheCTX-MtypeESBLsarecurrentlyrecognised}

as the part ofthe mostthreatening mechanism of antibi-oticresistanceinclinical andcommunitysettings,virtually invadingallhumanandanimalcompartmentsaswellasthe environmentallovertheworld,andareincreasinglybeing pre-dominantforminEnterobacteriaceaeworldwide.47,72–74_South

AmericaisknownasanimportantsourceofCTX-MtypeESBL originatedfromclinicalsettings.75_{InTurkey,CTX-M-1group}

wasfoundin86.8%oftheclinicalE.coliisolates.76_Ourresults

showedthatthedisseminationofCTX-M-1clusterwasnot restrictedtotheclinicalsettings,butalsoinvolvedthefoods ofanimalorigin,causingrapid,importantandunpredictable changesintheepidemiologyofantibioticresistance.75

In 2007, European Food Safety Authority (EFSA) recom-mendedthatbacterialstrainscontainingtransferable resis-tancegenesshouldnotbeusedinanimalfeedsandfermented andprobiotic foods forhumanuse.77,78 _{However,theissue}

ofantibioticsresistancebacteriainfoods hasnotyetbeen seriouslyraisedasahealthsafety indicator.Inconclusion, thefoodsofanimaloriginsoldinTurkeyhavebeenfoundas potentialreservoirsfordiverseESBL-producing Enterobacteri-aceaeandtheirencodinggeneswithaco-resistancepattern, thusposingacriticalhealthriskfortheTurkishconsumers.

Disclaimer

Thisarticleis basedon the Doctoralthesis entitled “Char-acterization of Extended Spectrum Beta-Lactamases in Enterobacteriaceaefrom FoodsUsing MolecularMethod”by ˙IsmailHakkıTekiner,2015,DepartmentofFoodEngineering, InstituteofNaturalandAppliedSciences,IstanbulAydın Uni-versity,TR-34295Istanbul,Turkey.

Conflicts

of

interest

Theauthorsdeclarethatthereisnoconflictofinterest.

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s

1.WangH,McEntireJC,ZhangL,LiX,DoyleM.Thetransferof

antibioticresistancefromfoodtohumans:facts,

implicationsandfuturedirections.RevSciTech.

2012;31:249–260.

2.ThenmozhiS,MoorthyK,SureshkumarBT,SureshM.

AntibioticresistancemechanismofESBLproducing

Enterobacteriaceaeinclinicalfield:areview.IntJPureAppl

Biosci.2014;2:207–226.

3.PatersonDL,BonomoRA.Extended-spectrum␤-lactamases:

aclinicalupdate.ClinMicrobiolRev.2005;18:657–686.

4.RuppME,FeyPD.Extendedspectrumbeta-lactamase

(ESBL)-producingEnterobacteriaceae:considerationsfor

diagnosis,preventionanddrugtreatment.Drugs.

2003;63:353–365.

5.EFSA.Scientificopiniononthepublichealthrisksof

bacterialstrainsproducingextended-spectrum␤-lactamases

and/orAmpC␤-lactamasesinfoodandfood-producing

animals.EFSAJ.2011;9:2322–2417.

6.LiebanaE,CarattoliA,CoqueTM,etal.Publichealthrisksof

enterobacterialisolatesproducingextended-spectrum

␤-lactamasesorAmpC␤-lactamasesinfoodand

food-producinganimals:anEUperspectiveofepidemiology,

analyticalmethods,riskfactors,andcontroloptions.Clin

InfectDis.2013;56:1030–1037.

7.BonnetR.Growinggroupofextended-spectrum

␤-lactamases:theCTX-Menzymes.AntimicrobAgents

Chemother.2004;48:1–14.

8.FernandesR,AmadorP,OliveiraC,PrudêncioC.Molecular characterizationofESBL-producingEnterobacteriaceaein NorthernPortugal.SciWorldJ.2014;2014:782897,

http://dx.doi.org/10.1155/2014/782897.

9.Giedraitien ˙eA,Vitkauskien ˙eA,Naginien ˙eR,PavilonisA.

Antibioticresistancemechanismsofclinicallyimportant

bacteria.Medicina(Kaunas).2011;47:137–146.

10.MesaRJ,BlancV,BlanchAR,etal.Extended-spectrum

␤-lactamase-producingEnterobacteriaceaeindifferent

environments(humans,food,animalfarmsandsewage).J

AntimicrobChemother.2006;58:211–215.

11.ReulandEA,alNaiemiN,RaadsenSA,SavelkoulPHM,

KluytmansJAJW,Vandenbroucke-GraulsCMJE.Prevalenceof

ESBL-producingEnterobacteriaceaeinrawvegetables.EurJ

ClinMicrobiolInfectDis.2014;33:1843–2184.

12.GhafourianS,SadeghifardN,SoheiliS,SekawiZ.Extended

spectrumbeta-lactamases:definition,classificationand

epidemiology.CurrIssuesMolBiol.2014;17:11–22.

13.vonSalviatiC,LaubeH,GuerraB,RoeslerU,FrieseA.

EmissionofESBL/AmpC-producingEscherichiacolifrompig

fatteningfarmstosurroundingareas.VetMicrobiol.

2015;175:77–84.

14.HabeebMA,SarwarY,AliA,SalmanM,HaqueA.Rapid

emergenceofESBLproducersinE.colicausingurinaryand

woundinfectionsinPakistan.PakJMedSci.2013;29:

540–544.

15.SharmaM,PathakS,SrivastavaP.Prevalenceand

antibiogramofextendedspectrum␤-lactamase(ESBL)

producingGramnegativebacilliandfurthermolecular

characterizationofESBLproducingEscherichiacoliand

Klebsiellaspp.JClinDiagnRes.2013;7:2173–2177.

16.WHO.Integratedsurveillanceofantimicrobialresistance: guidancefromaWHOAdvisoryGroup;2013ISBN978924 1506311,Switzerland.

17.LaxminarayanR,DuseA,WattalC,etal.Antibiotic

resistance–theneedforglobalsolutions.LancetInfectDis.

2013;13:1057–1098.

18.CapitaR,Alonso-CallejaC.Antibiotic-resistantbacteria:a

challengeforthefoodindustry.CritRevFoodSci.

2013;53:11–48.

19.ArslanS,ÖzdemirF.Extendedspectrumbeta-lactamasesin

Escherichiacolistrainsisolatedfromhomemadewhite

cheeses:prevalenceandantibioticssusceptibility.WorldJ

MicrobiolBiotechnol.2008;24:2361–2364.

20.YıbarA,SoyutemizE.Antibioticsuseinfood-producing

animalsandpossibleresidualrisk.AtatürkÜniversitesiVetBil

Derg.2013;8:97–104.

21.GeserN,StephanR,HächlerH.Occurrenceand

characteristicsofextended-spectrum␤-lactamase(ESBL)

producingEnterobacteriaceaeinfoodproducinganimals,

mincedmeatandrawmilk.BMCVetRes.2013;8:21.

22.CLSI.Performancestandardsforantimicrobialsusceptibility

testing.Twenty-ThirdInformationalSupplement.CLSIDocument;

23.OjdanaD,SachaPB,WieczorekP,etal.Theoccurrenceof

blaCTX-M,blaSHV,andblaTEMgenesinextended-spectrum

␤-lactamase-positivestrainsofKlebsiellapneumoniae,

Escherichiacoli,andProteusmirabilisinPoland.IntJAntibiot.

2014;2014:935842.

24.WoodfordN,FaganEJ,EllingtonMJ.MultiplexPCRforrapid

detectionofgenesencodingCTX-Mextended-spectrum

b-lactamases.JAntimicrobChemother.2006;57:154–155.

25.HaqueA,YoshizumiA,SagaT,IshiiY,TatedaK.

ESBL-producingEnterobacteriaceaeinenvironmentalwater

inDhaka,Bangladesh.JInfectChemother.2014;20:735–737.

26.MitchellSM,SubbiahM,UllmanJL,FrearC,CallDR.

Evaluationof27differentbiocharsforpotential

sequestrationofantibioticresiduesinfoodanimal

productionenvironments.JEnvironChemEng.2015;3:162–169.

27.AllenHK,DonatoJ,WangHH,Cloud-HansenKA,DaviesJ,

HandelsmanJ.Callofthewild:antibioticresistancegenesin

naturalenvironments.NatRevMicrobiol.2010;8:251–259.

28.LupoA,Papp-WallaceKM,SendiP,BonomoRA,EndimianiA.

Non-phenotypicteststodetectandcharacterizeantibiotic

resistancemechanismsinEnterobacteriaceae.Diagn

MicrobiolInfectDis.2013;77:179–194.

29.StefaniS,GiovanelliI,AnacarsoI,etal.Prevalenceand

characterizationofextended-spectrum

␤-lactamase-producingEnterobacteriaceaein

food-producinganimalsinNorthernItaly.NewMicrobiol.

2014;37:551–555.

30.BonelliRR,MoreiraBM,PicãoRC.Antimicrobialresistance

amongEnterobacteriaceaeinSouthAmerica:history,

currentdisseminationstatusandassociatedsocioeconomic

factors.DrugResistUpdate.2014;17:24–36.

31.OverdevestI,WillemsenI,RijnsburgerM,etal.

Extended-spectrumbeta-lactamasegenesofEscherichiacoli

inchickenmeatandhumans,TheNetherlands.EmergInfect

Dis.2011;17:1216–1222.

32.DoyleMP,LoneraganGH,ScottHM,SingerRS.Antimicrobial

resistance:challengesandperspectives.ComprRevFoodSci

FoofSaf.2013;12:234–248.

33.Aidara-KaneA,AndremontA,CollignonP.Antimicrobial

resistanceinthefoodchainandtheAGISARinitiative.JInfect

PublicHealth.2013;6:162–165.

34.LahlaouiH,BenHajKhalifaA,BenMoussaM.Epidemiology

ofEnterobacteriaceaeproducingCTX-Mtypeextended

spectrum␤-lactamase(ESBL).MédMaladiesInfect.

2014;44:400–404.

35.RaoL,LvL,ZengZ,etal.Increasingprevalenceof

extended-spectrumcephalosporin-resistantEscherichiacoli

infoodanimalsandthediversityofCTX-Mgenotypes

during2003–2012.VetMicrobiol.2014;172:534–541.

36.SuY,YuCY,TsaiY,WangSH,LeeC,ChuC. Fluoroquinolone-resistantandextended-spectrum

␤-lactamase-producingEscherichiacolifromthemilkofcows withclinicalmastitisinSouthernTaiwan.JMicrobiolImmunol Infect.2014,http://dx.doi.org/10.1016/j.jmii.2014.10.003.

37.MelzeraM,PetersenI.Mortalityfollowingbacteraemic

infectioncausedbyextendedspectrumbeta-lactamase

(ESBL)producingE.colicomparedtonon-ESBLproducingE.

coli.JInfect.2007;55:254–259.

38.AmadorP,FernandesR,PrudêncioC,BritoL.Resistanceto

␤-lactamsinbacteriaisolatedfromdifferenttypesof

Portuguesecheese.IntJMolSci.2009;10:1538–1551.

39.Garcia-MiguraL,HendriksenRS,FraileL,AarestrupFM.

Antimicrobialresistanceofzoonoticandcommensal

bacteriainEurope:themissinglinkbetweenconsumption

andresistanceinveterinarymedicine.VetMicrobiol.

2014;170:1–9.

40.KarD,BandyopadhyayS,BhattacharyyaD,etal.Molecular

andphylogeneticcharacterizationofmultidrugresistant

extendedspectrumbeta-lactamaseproducingEscherichiacoli

isolatedfrompoultryandcattleinOdisha,India.InfectGenet

Evol.2015;29:82–90.

41.ZhengH,ZengZ,ChenS,etal.Prevalenceand

characterisationofCTX-M␤-lactamasesamongstEscherichia

coliisolatesfromhealthyfoodanimalsinChina.IntJ

AntimicrobAgents.2012;39:305–310.

42.SchwaigerK,HutherS,HölzelC,KämpfP,BauerJ.Prevalence

ofantibiotic-resistantEnterobacteriaceaeisolatedfrom

chickenandporkmeatpurchasedattheslaughterhouseand

atretailinBavaria,Germany.IntJFoodMicrobiol.

2012;154:206–211.

43.SmetA,MartelA,PersoonsD,etal.Diversityof

extended-spectrumbeta-lactamasesandclassC

beta-lactamasesamongcloacalEscherichiacoliisolatesin

Belgianbroilerfarms.AntimicrobAgentsChemother.

2008;52:1238–1243.

44.PetternelC,GallerH,ZarfelG,etal.Isolationand

characterizationofmultidrug-resistantbacteriafrom

mincedmeatinAustria.FoodMicrobiol.2014;44:41–46.

45.GarrecH,Drieux-RouzetL,GolmardJL,JarlierV,RobertJ.

Comparisonofninephenotypicmethodsfordetectionof

extended-spectrum␤-lactamaseProductionby

Enterobacteriaceae.JClinMicrobiol.2011;49:1048–1057.

46.PerezF,EndimianiA,HujerKM,BonomoRA.Thecontinuing

challengeofESBLs.CurrOpinPharmacol.2007;7:459–469.

47.NaasT,OxacelayC,NordmannP.Identificationof

CTX-M-Typeextended-spectrum-␤-lactamasegenesusing

real-timePCRandpyrosequencing.AntimicrobAgents

Chemother.2007;51:223–230.

48.BradfordPA.Extended-spectrum␤-lactamasesinthe21st

century:characterization,epidemiology,anddetectionof

thisimportantresistancethreat.ClinMicrobiolRev.

2001;14:933–951.

49.SrisangkaewS,VorachitM.Theoptimumagentforscreening

andconfirmatorytestsforextended-spectrum

beta-lactamasesinEscherichiacoliandKlebsiellapneumoniae

inRamathibodiHospital,Thailand.JInfectDisAntimicrob

Agents.2004;21:1–5.

50.TanejaN,SharmaM.ESBLdetectioninclinicalmicrobiology:

why&how?IndianJMedRes.2008;127:297–300.

51.ThomsonKS.Extended-spectrum-␤-lactamase,AmpC,and

carbapenemaseissues.JClinMicrobiol.2010;48:

1019–1025.

52.TurnerPJ.MYSTICEurope2007:activityofmeropenemand

otherbroad-spectrumagentsagainstnosocomialisolates.

DiagnMicrobiolInfectDis.2009;63:217–222.

53.KortenV,UlusoyS,ZarakoluP,MeteB,TurkishMYSTICStudy

Group.Antibioticresistancesurveillanceovera4-yearperiod

(2000–2003)inTurkey:resultsoftheMYSTICprogram.Diagn

MicrobiolInfectDis.2007;59:453–457.

54.Pérez-PérezFJ,HansonND.Detectionofplasmid-mediated

AmpCbeta-lactamasegenesinclinicalisolatesbyusing

multiplexPCR.JClinMicrobiol.2002;40:2153–2162.

55.KiratisinP,HenprasertA.Genotypicanalysisof

plasmid-mediatedbeta-lactamasesamongst

EnterobacteriaceaeotherthanEscherichiaspp.andKlebsiella

spp.thatarenon-susceptibletoabroad-spectrum

cephalosporin.IntJAntimicrobAgents.2010;36:343–347.

56.ThomsonKS.Controversiesaboutextended-spectrumand

AmpCbeta-lactamases.EmergInfectDis.2001;7:333–336.

57.JacobyGA.AmpCbeta-lactamases.ClinMicrobiolRev.

2009;22:161–182.

58.NazikH,BektoreB,OngenB,etal.Plasmid-mediated

quinoloneresistancegenesinEscherichiacoliurinaryisolates

fromtwoteachinghospitalsinTurkey:coexistenceofTEM,

SHV,CTX-MandVEB-1Type␤-lactamases.TropJPharmRes.

59.ZanianiFR,MeshkatZ,NasabMN,etal.Theprevalenceof

TEMandSHVgenesamongextended-spectrum

betalactamasesproducingEscherichiacoliandKlebsiella

pneumoniae.IranJBasicMedSci.2012;15:654–660.

60.FerreiraJC,FilhoRACP,AndradeLN,JuniorAB,DariniALC.

IncI1/ST113andIncI1/ST114conjugativeplasmidscarrying

blaCTX-M-8inEscherichiacoliisolatedfrompoultryinBrazil.

DiagnMicrobiolInfectDis.2014;80:304–306.

61.ChouchaniC,MarrakchiR,ElSalabiA.Evolutionof

␤-lactamsresistanceinGram-negativebacteriainTunisia.

CritRevMicrobiol.2011;37:167–177.

62.HiroiM,YamazakiF,HaradaT,etal.Prevalenceof

extended-spectrum␤-lactamase-producingEscherichiacoli

andKlebsiellapneumoniaeinfood-producinganimals.JVet

MedSci.2012;74:189–195.

63.KawamuraK,GotoK,NakaneK,ArakawaY.Molecular

epidemiologyofextended-spectrum␤-lactamasesand

Escherichiacoliisolatedfromretailfoodsincludingchicken

meatinJapan.FoodbornePathogDis.2014;11:104–110.

64.ReichF,AtanassovaV,KleinG.Extended-spectrum

␤-lactamase-andAmpC-producingenterobacteriain

healthybroilerchickens,Germany.EmergInfectDis.

2013;19:1253–1259.

65.ZarfelG,GallerH,LuxnerJ,etal.multiresistantbacteria

isolatedfromchickenmeatinAustria.IntJEnvironResPublic

Health.2014;11:12582–12593.

66.RandallL,HeinrichK,HortonR,etal.Detectionofantibiotic

residuesandassociationofcefquinomeresidueswiththe

occurrenceofextended-spectrum␤-Lactamase

(ESBL)-producingbacteriainwastemilksamplesfromdairy

farmsinEnglandandWalesin2011.ResVetSci.

2014;96:15–24.

67.KhoshbakhtR,ShahedA,AskiHS.Characterizationof

extendedspectrumB-lactamase-producingEscherichiaecoli

srainsisolatedfromdairyproducts.JMicrobiolBiotechnolFood

Sci.2014;3:333–336.

68.AhmedAM,ShimamotoT.Molecularanalysisofmultidrug

resistanceinShigatoxin-producingEscherichiacoliO157:H7

isolatedfrommeatanddairyproducts.IntJFoodMicrobiol.

2015;193:68–73.

69.GundoganN,YakarU.Siderophoreproduction,serum

resistance,hemolyticactivityandextendedspectrumbeta

lactamaseproducingKlebsiellaspeciesisolatedfrommilk

andmilkproducts.JFoodSaf.2007;3:251–260.

70.GundoganN,CıtakS,YalcinE.Virulencepropertiesof

extendedspectrumbeta-lactamase-producingKlebsiella

speciesinmeatsamples.JFoodProt.2011;74:

559–564.

71.GundoganN,AvciE.Prevalenceandantibioticresistanceof

extendedspectrumbeta-lactamase(ESBL)producing

EscherichiacoliandKlebsiellaspeciesisolatedfromfoodsof

animalorigininTurkey.AfrJMicrobiolRes.2013;7:4059–4064.

72.CantónR,González-AlbaJM,GalánJC.CTX-Menzymes:

originanddiffusion.FrontMicrobiol.2012;3:110.

73.PriyadharsiniRI,KavithaA,RajanR,MathaviS,RajeshKR.

PrevalenceofblaCTXMextendedspectrumbetalactamase

geneinEnterobacteriaceaefromcriticalcarepatients.JLab

Physicians.2011;3:80–83.

74.HawkeyPM.Multidrug-resistantGram-negativebacteria:a

productofglobalization.JHospInfect.2015;89:241–247.

75.RossoliniGM,D’AndreaMM,MugnaioliC.Thespreadof

CTX-M-typeextended-spectrum␤-lactamases.ClinMicrobiol

Infect.2008;14:33–41.

76.GonulluN,AktasZ,KayacanCB,etal.Disseminationof

CTX-M-15␤-lactamasegenescarriedonIncFIandFII

plasmidsamongclinicalisolatesofEscherichiacoliina

UniversityHospitalinIstanbul,Turkey.JClinMicrobiol.

2008;46:1110–1112.

77.VankerckhovenV,HuysG,VancanneytM,etal.Biosafety

assessmentofprobioticsusedforhumanconsumption:

recommendationsfromtheEU-PROSAFEProject.TrendsFood

SciTechnol.2008;19:102–114.

78.SharmaP,TomarSK,GoswamiP,SangwanV,SinghR.

Antibioticresistanceamongcommerciallyavailable