85 Original Articles Original articles -...

Rev Colomb Cienc Pecu 2017; 30:85-100

Revista Colombiana de Ciencias Pecuarias

Original articles

85

Original Articles

¤ Tocitethisarticle:VásquezL,RamírezNF,AkinedenÖ,FernándezJA.Presenceofextended-spectrumbeta-lactamase(ESBL)-producingEnterobacteriaceaeinbulk-tankmilkofbovinedairyfarmsinAntioquia,Colombia.RevColombCiencPecu2017;30:85-100.

* Correspondingauthor:JorgeArturoFernándezSilva.EscueladeMedicinaVeterinaria,FacultaddeCienciasAgrarias,UniversidaddeAntioquiaUdeA,Calle70No.52-21,Medellín,Colombia.Tel:(+574)2199131,Fax(+574)2199104.E-mail:[email protected]

Presence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in bulk-tank milk of bovine dairy

farms in Antioquia, Colombia¤

Presencia de Enterobacteriaceae productoras de betalactamasas de espectro extendido (BLEE) en tanques de leche de fincas lecheras de Antioquia, Colombia

Presença de Enterobacteriaceae produtoras de beta-lactamase de espectro estendido (BLEE) em amostras de leite de tanque de rebanhos de bovinos leiteiros em Antioquia, Colômbia

LauraVásquez-Jaramillo1,MV,MSc;NicolásFRamírez1,MV,MSc,DSc;ÖmerAkineden2, MV,Drmedvet;JorgeAFernández-Silva1*,MV,MSP,Drmedvet.

1Línea de Investigación en Epidemiología y Salud Pública Veterinaria, Grupo de Investigación CENTAURO, Escuela de Medicina Veterinaria, Facultad de Ciencias Agrarias, Universidad de Antioquia, Medellín, Colombia.

2Dairy Sciences, Institute of Veterinary Food Science, Justus-Liebig-University, Ludwigstr, Giessen, Germany.

(Received: June 10, 2016; accepted: October 07, 2016)

doi: 10.17533/udea.rccp.v30n2a01

Abstract

Background:antibioticresistanceisspreadingworldwide.Itisimportanttoevaluatewhetherfoodsofanimaloriginconstituteareservoirofresistancegenes.Objective:theaimofthepresentstudywastoassessthe occurrence and characterize extended-spectrumbeta-lactamase (ESBL)-producingEnterobacteriaceaefrombulk-tankmilksamplesofdairyfarmslocatedinEntrerríos,Antioquia(Colombia).Methods:atotalof120randomlyselectedrawmilksamples(onebulk-tankmilksampleperdairyfarm)werecollectedbetweenSeptemberandOctober,2013.AcommercialchromogenicagarwasusedforscreeningpresumptiveESBL-E.Identificationofgenusandspeciesofisolateswasperformedwithacommercialbiochemicalidentificationkit.TheESBLproductionwasconfirmedusingthedoublediscsynergytest.Anantimicrobialsusceptibilitytestwasperformedbytheagardiscdiffusionmethodandtheautomatizedbrothmicrodilutionmethod.TheESBL-positiveisolateswereanalyzedforthepresenceofblagenesbypolymerasechainreaction(PCR)andsequencing.Fortheexplorationofriskfactors,informationondairyfarmmanagementpracticeswasrecordedusingaquestionnaireand theassociationsofpredictorsand resultswere testedwitha logistic regression

86


Vásquez L et al. Beta-lactamase by Enterobacteriaceae in bulk-tank milk

analysis.Results:theESBL-Ewereisolatedfrom3.3%(4/120;CI95%:3-3.5%)ofthesamples.FarmsizewastheonlyfactorassociatedwiththepresenceofESBL-E(OR=11.5;CI95%:1.14-115.54;p=0.038).AllisolateswereresistanttoseveralantibioticsandharboredblaCTX-M-96(alternatenameCTX-M-12a)enzymes.Conclusion:althoughapparentfrequencyofESBL-Ewaslow,thepresenceoftheseresistantbacteriainmilkmayconstituteapublichealthriskandshouldbefurtherinvestigated.

Keywords: antibiotic resistance, CTX-M-12a, livestock, public health, raw milk.

Resumen

Antecedentes: la resistencia a antibióticos se está diseminando en elmundo.Es importante evaluarsi losalimentosdeorigenanimalconstituyenunreservoriodegenesderesistencia.Objetivo:evaluar lapresentaciónycaracterizarlasEnterobacteriaceaeproductorasdebetalactamasasdeespectroextendido(BLEE)apartirdemuestrasdelechedetanquedehatoslecheroslocalizadosenEntrerríos,Antioquia(Colombia).Métodos:cientoveintemuestrasde lechecruda(unamuestrapor tanque)dehatosseleccionadosalazarfueroncolectadasentreseptiembreyoctubrede2013.ParaeltamizajedepresuntivaBLEEseutilizóunagarcromogénicocomercial.Laidentificacióndelosaislamientosserealizóutilizandounkitdeidentificaciónbioquímicacomercial.LaconfirmacióndelaproduccióndeBLEEfueconfirmadaconlapruebadesinergiadedobledisco.Lasusceptibilidadantimicrobianafueevaluadaporelmétododedifusióndediscoenagaryporelmétododemicrodiluciónencaldoautomatizado.AislamientospositivosparaBLEEfueronanalizadosparapresenciadegenesbla porPCRysecuenciación.Paralaexploracióndelosfactoresderiesgoseregistrólainformaciónsobrelasprácticasdemanejodelhatomediantecuestionarioyseprobólaasociaciónentrepredictoresyresultadosmedianteanálisisderegresiónlogística.Resultados:lasBLEEfueronaisladasde3,3%(4/120;IC95%:3-3,5%)delasmuestrasdelechedetanqueyeltamañodelhatofueelúnicofactorqueseencontróasociadoconunincrementoensupresencia(OR=11,5;IC95%:1,14-115,54;p=0.038).TodoslosaislamientosfueronresistentesavariosantibióticosyalbergabanenzimasblaCTX-M-96(nombrealternativoCTX-M-12a)Conclusión:aunqueseobservóunabajafrecuenciaaparentedeBLEE,lapresenciadeestasbacteriasresistentesenlosalimentosdeorigenanimalpuedeconstituirunriesgoparalasaludpúblicaydebeseguirsiendoinvestigada.

Palabras clave: CTX-M-12a, ganadería, leche cruda, resistencia a los antibióticos, salud pública.

Resumo

Antecedentes:aresistênciaaosantibióticosestáseespalhandorapidamentenomundointeiro.Éimportanteavaliarseosalimentosdeorigemanimalconstituemumreservatóriodegenesderesistência.Objetivo:avaliaraocorrênciaecaracterizarEnterobacteriaceaeprodutorasdebetalactamasesdeespectroestendido(BLEE)deamostrasdeleitedetanquesderebanhosleiteiros.Métodos:umtotalde120amostrasleitecrua(umaamostraporleitedotanque)deexploraçõesleiteirasselecionadasaleatoriamentenomunicípiodeEntrerríos,Antioquia(Colombia),foramcoletadosemtresetembroeoutubrode2013.ParaorastreiodaspresumíveisBLEE foi utilizado ummeio de ágar cromogénico comercial.A identificação das espécies isoladas foirealizadautilizandoumkitcomercialdaidentificaçãobioquímica.AproduçãodeBLEEfoiconfirmadapelotestedesinergiadeduplodisco.Umtestedesusceptibilidadeantimicrobianafoirealizadopelométododedifusãoemdiscodeágaremétododemicrodiluiçãoemcaldoautomatizado.IsoladosBLEEpositivosforamanalisadosparaapresençadosgenesblaporPCResequenciação.Paraaexploraçãodosfatoresderisco,asinformaçõessobreaspráticasdegestãodorebanhoforamgravadasutilizandoumquestionário.Asassociaçõesdepreditoreseosresultadosforamtestadosporumaanálisederegressãologística.Resultados:oBLEEforamisoladasde3,3%(4/120;IC95%:3-3,5%)dasamostrasdeleitedotanque.Otamanhodorebanhofoioúnicofatorencontradoassociadocomumaumentonasuapresença(OR=11,5;IC95%:1,14-115,54;p=0,038).TodososisoladosforamresistentesaváriosantibióticosealbergavaenzimasblaCTX-M-96(nomealternativoCTX-H-12a).Conclusão: emboratenhasidoobservadaumabaixafrequênciaaparentedeBLEE,apresençadessasbactériasresistentesnosalimentosdeorigemanimalpodeconstituirumriscoparaasaúdepúblicaedevepermanecersobpesquisa.

Palavras chave:CTX-M-12A, gado, leite crua, resistência aos antibióticos, saúde pública.

87



Introduction

Antibioticresistanceisexpandingrapidlythroughouttheworldandhasbecomeaprobleminbothhumanandveterinarymedicine (FDA, 2012;Reistet al.,2013).Theemergenceofthisresistanceinbacteriafound in animals and their productshasmotivatedconsiderable interest because of the potentialfor resistance transfer to the human population(McDermottet al.,2002).

Oneof thegreatestconcerns is therapid increaseof resistantbacteria thatproduceextended-spectrumbeta-lactamases(ESBLs),whichreducetheefficacyofawiderangeofbeta-lactamantibiotics,suchasthird-generationcephalosporinsandmonobactams(Philipponet al.,1989;Bush,2010).Thisresistanceisbasedongenesofchromosomicorplasmidicorigin,whichencodeenzymesthatinactivatethesecompoundsbyhydrolysisoftheirbeta-lactamring(PatersonandBonomo,2005;Reistet al.,2013).Thesegenescanbeeasilytransferredbetweenbacteria throughmobile genetic elements,whichoftencarryadditionalgenes for resistance toothergroupsofantibiotics,compromisingtheefficacyoftreatmentsforinfectionscausedbyresistantbacteria(Smetet al.,2010).

Untilthenineties,themostprevalentESBLworldwidewereTemoniera(TEM)andsulfhydrylvariable(SHV)typeassociatedmainlytohospitaloutbreakscausedbyKlebsiella pneumoniae.However, since 2000,the enzyme cefotaxime-munich (CTX-M)becameone of themost frequent and ESBL-producingEscherichacoliemergedasan importantpathogeninthecommunity(Blancoet al.,2016).

TheCTX-Misolateshavebeenfoundinvariousspecies of Enterobacteriaceae from differentgeographic areas: mainly during nosocomialoutbreaks that occurred in Japan, Europe, SouthAmerica,Africa,China,KoreaandtheUnitedStates(Villegaset al.,2004). In2003,a studyconductedin threeColombian hospitals located inBogotá,Medellín,andCalidetectedisolatesfromK. oxytoca,K. pneumoniae, andE. coli that harboredCTX-Menzymes.ThatstudyconfirmedthatCTX-MenzymesalsooccurinColombia(Villegaset al.,2004).Furtherstudies inColombia indicated that this antibioticresistance patterns from several hospital isolates

have spread, especially inGram-negative bacteria(Sánchezet al.,2008;Villegaset al.,2011;Lealet al.,2013;Amadoet al.,2014;GonzálezandCortés,2014;Blancoet al.,2016).

TheentryofESBL-producing Enterobacteriaceaein the food chain and environment could beconsidered a possible interface for the exchangeof resistance genes between humans and animals(WHO, 2001;Walsh and Fanning, 2008; EFSA,2011).Rawmilk,forexample,canbecontaminatedintramammary with Enterobacteriaceae duringmastitis processes, directly through animal fecesor indirectly duringmilking throughmilkers ormilkingequipment(Dahmenet al.,2013),andalsobycowcontactwithfecesinthefarmenvironmentsuchaspens(FerensandHovde,2011).However,all pathogenicmicroorganisms are destroyed afterpasteurization, includingmulti-resistant bacteria,makingmilkandotherdairyproductsgenerallysafefor human consumption (Leedom, 2009;LejeuneandRajala-Schultz, 2009).Rawmilk is still usedbyalargenumberofruralfamiliesandagrowingsegmentofthepopulationwhobelieverawmilkisgenerallysafeandhasbeneficialhealtheffectsthatare destroyed by pasteurization (Zeinhomaet al.,2014).According to data from theMinisterio deAgriculturayDesarrolloRuraldeColombia,59%ofthemilkproducedinthecountryisintendedforcommercializationthroughintermediaries,processed,andusedforself-consumption,amongothers,andtheremaining41% is processedby thedairy industry(Decreto1880,2011,Ministeriode laProtecciónSocial).Thus,thetransmissionofESBL-producing Enterobacteriaceae to humans can occur throughdirectcontactand/oringestionoffoodscontaminatedwithESBL-producing strains, among other routes(Wooldridge,2008;Verraeset al.,2013).

Resistance towide-spectrumantibioticssuchascephalosporinsisincreasingamongfood-producinganimals,andfoodofanimalorigincanbeconsidereda possible reservoir ofESBLs (Mesaet al., 2006;Xian-Zhiet al.,2007;Carattoli,2008;Leverstein-vanHallet al.,2011).Theexcessiveorinappropriateuseofantibioticsinfarmanimalscangenerateadaptationof bacteria to antimicrobials (FAO, 2011); thus, ithasbecomeamatterofgreatimportanceforhealthauthorities(WHO,2001;EFSA,2011).

88



ThepresenceofESBL-producingEnterobacteriaceaeindairyanimalsinColombiaisunknown.Althoughtherearepoliciesrestrictingthesaleofantimicrobials,com pliance is minimal, which facilitates theappearance of resistance (Machado-Alba andGonzález-Santos, 2009;Vaccaet al., 2011).Thus,itisnecessarytoinvestigatethefrequencyofmulti-resistant bacteria in foods of animal origin and toestablish the possible role of dairy farms in theirdissemination(Smetet al.,2010;Timofteet al.,2014).

Thisstudyaimstocharacterizethepresenceandfrequency ofESBL-producingEnterobacteriaceaefrom bulk-tankmilk samples of dairy farms inEntrerríos (Antioquia, Colombia), and explorepotentialriskfactorsassociatedwiththesebacteriainbulk-tankmilk.

Materials and methods

Location

This study was conducted in Entrerríos,municipality inNorthernAntioquia (Colombia),which is themain dairy region of this province.Average temperature in Entrerríos is 16 °C.Thealtitudeis2,300m.o.s.l.andislocatedapproximately60KmNorthofMedellíncity,thecapitalofAntioquiaprovince. Dairy farming is themost importanteconomicactivityinthemunicipality,whichislocatedclosetothemetropolitanareaoftheValledeAburrá(Gobernación deAntioquia, 2013).The averagedailymilk production is 238,854L (GobernacióndeAntioquia,2007).Mostmilkissoldtoacoupleof dairy processors and the rest is destined fordomesticconsumptionandfeedingoffarmanimals(GobernacióndeAntioquia,2013).

Selection of farms

Fromatotalof952dairyfarmsinEntrerríos(basedon year 2013 foot-and-mouth disease vaccinationrecordsfromtheSecretaríadeAsistenciaTecnicayDesarrollodeEntrerrios),120farmswererandomlyselected.This numberwas based on an expectedfrequency of 0.10 at the farm level (Odenthal et al.,2013),a90%confidencelevelandamaximumacceptableerror rateof0.10.Thenumberofdairy

farmstobesampledfromeachdistrictwasdeterminedbyproportionalallocation.Onlyfarmshavingtheirown bulk-tankwere selected.That is, farms thatstoremilkincollectiveorcommunitytankswerenotconsidered.Anadditionallistofpossiblefarmswasrandomlyselectedtoreplacefarmswheremilkhadalreadybeencollectedbythetruckonthesamplingday.

Sample collection

Bulk-tankmilksampleswerecollectedinSeptemberand October, 2013. Each sample (30mL) wasobtaineddirectlyfromthetankinasterilecontainer,immediatelycooledandtransportedtothelaboratory(Toro,2012),wheretheywerekeptrefrigeratedandprocessedwithin24hofsampling.

Isolation and identification of ESBL-producing Enterobacteriaceae

For selective isolation (screening) of ESBL-producingbacteria,100μLofmilkwasspreadontochromogenic agar chromID™ESBL (bioMérieux,Marcy-l’Étoile, France),whichwas incubated at37ºCfor20to24h.Samplesthatdisplayedcolonygrowthwith pink-burgundy, green, blue or browncolorationswere considered presumptive ESBL-producing Enterobacteriaceae. Ineachsample,onecolonyofeachcolorationwastakenandspreadontoMcConkeyagar(bioMérieux,RiodeJaneiro,Brazil)forsubsequentidentification.Eachisolatewasweeklyreplicated inMcConkeyagar forpreservation.Theformofthecolonieswascomparedbetweensampleswithgrowthofvariouscoloniesofthesamecoloration,andtwocoloniesofdifferentmorphologyweretaken.The commercial BDBBL™Crystal™ Enteric/NonfermenterIDKit(E/NF,Sparks,MD,USA)wasused according to themanufacturer instructions toidentify genus and species ofEnterobacteriaceae.Only glucose fermenting colonieswere selected,whereasthenon-fermentingcolonieswerediscarded.

Confirmation of ESBL production

The ESBL productionwas confirmed basedon thedoubledisk synergy test.For this,Mueller-Hinton agar (bioMérieux,Rio de Janeiro,Brazil)was inoculatedwith a bacterial suspension of 0.5

89



turbiditypatternintheMcFarlandstandard,anddiscsofcefotaxime(CTX,30μg),ceftazidime(CAZ,30μg), ceftriaxone (CRO,30μg), cefepime (FEP, 30μg),andaztreonam(ATM,30μg)wereplaced20mmcenter tocenterfromacentraldiskofamoxicillin/clavulanicacid(AMC).Theagarinoculatedwithabacterialsuspension(McFarlandturbiditypatternof0.5)anddiscswereincubatedat37°Cfor18-24h.AnincreaseinthezonetowardstheAMCwasconsideredpositiveforESBLproductionaccordingtocriteriabytheClinicalandLaboratoryStandardsInstitute(CLSI2014,DocumentM24).ReferencestrainsofE. coliAmericanTypeCultureCollection(ATCC)25922andK. pneumonieae ATCC700603wereusedasnegativeandpositivecontrols,respectively,inthedoubledisksynergytest.

Antimicrobial susceptibility test

Theantimicrobialsusceptibilitytestwasperformedby the disc diffusionmethod in agar, according torecommendationsbyCLSI(2015).Forthispurpose,bacterialcoloniesweresuspendedinbrainheartbroth(bioMérieux,Marcy-l’Étoile,France)andadjustedtoaMcFarlandturbiditypatternof0.5tobeinoculatedinaplateofMueller-Hintonagar.Discsofthefollowingantibiotics were evaluated: ampicillin (10 μg),norfloxacin(10μg),imipenem(IPM,10μg),amoxicillin(10μg),cefoxitin(FOX,30μg),cefuroxime(30μg),ciprofloxacin(CIP,30μg),chloramphenicol(30μg),doxycycline (30 μg), gentamicin (GEN, 10 μg),trimethoprim/sulfamethoxazole (1.25/23.75 μg),erythromycin(15μg),andcephalexin(30μg;Oxoid,Basingstoke,Hampshire,England).After incubationfor18-24h,inhibitionhalos(marginofthezoneswherenovisiblegrowthwasobserved)weremeasured inmillimetersandinterpretedassensitive,intermediateorresistantaccordingtoCLSI(2015)standards.

Determination of minimum inhibitory concentration (MIC)

AntimicrobialMICforisolatesofESBL-producingEnterobacteriaceaewas determined by the brothmicrodilutionmethodusingtheVitek207.01system(bioMérieux,Marcy-l'Étoile,Francia).A totalof14antibioticswereevaluatedandinterpretedaccordingtoCLSI (2008)standards:ampicillin/sulbactam(SAM),FOX,CAZ,CRO,FEP,doripenem(DOR),ertapenem

(ETP), IPM,meropenem(MEM),amikacin (AMK),GEN,CIP,tigecycline(TGC),andcolistin(COL).

Characterization of ESBL by polymerase chain reaction (PCR)

PositiveESBLisolateswereadditionallyanalyzedby PCR for the presence of bla genes of ESBLsubtypesTEM,SHV,andCTX-M(groups1,2,8,9, or 25) using primers and conditions previouslydescribed(Pitoutet al.,1998;Batcheloret al.,2005;Woodfordet al.,2006).BacterialDNAwasisolatedwiththeinnuPREPbacteriaDNAkit(Analitykjena,Berlin,Germany) according to themanufacturer’sinstructions.Two strains,K. pneumoniaeATCC700603(whichharborsablaSHVgene)andanisolateofK. pneumoniae (whichharborsbothblaCTX-MandblaTEMgenes) from the strain collection oftheDepartmentofMilkSciencesof theUniversityofGiessenwere used as positiveESBL standardstrains.AnESBLnon-producingstrain E. coliATCC25922wasusedasnegativecontrol.PCRproductsweredeterminedbyelectrophoresisina2%agarosegel (Biozym,HessischOldendorf-,Germany).TheGeneRuler 100 bpDNAmolecularmarker (MBIFermentas,St.Leon-Roth,Germany)wasused.

Sequencing of bla genes

Genes coding for ESBL blaTEM, blaSHV, andblaCTX-MofESBL-positive isolateswere amplifiedwith primers and PCR conditions as describedpreviously (Pitout et al., 1998; Batchelor et al.,2005).TheresultingampliconswerepurifiedusingtheQIAquickPCRPurificationkit(Qiagen,Hilden,Germany). Sequencingwas performed in Seqlab(Goettingen,Germany).TheresultswereevaluatedusingtheBLASTalgorithmavailableathttp://blast.ncbi.nlm.nih.gov/Blast.cgi.

Collection of farm information and management practices

InformationonthecharacteristicsandmanagementpracticesthatcouldbeassociatedwiththepresenceofESBL-producing Enterobacteriaceaewasobtainedusingaquestionnaire(supplementalmaterial)atthetimeofbulk-tanksampling.Informationonvariablesrelatedtogeneralconditionsofthefarm,production,

90



sanitation, and hygiene, and the use of antibioticswas collected (Tables 1 and2). Information aboutCFUvalueswasobtainedfromthepurchaseinvoicegeneratedbythemilkprocessor.Farmswereclassifiedassmall(≤50cowsinmilking)andlarge(>50cowsinmilking).

Case definition

Bulk-tankmilkwasconsideredtheunitofanalysis.Atankwasconsideredpositivefor thepresenceofESBL-producing Enterobacteriaceaewhen at leastone(1)isolateofEnterobacteriaceaewasconfirmedasanESBLproducer.

Statistical analysis

Data were stored in Excel 2010 (Microsoft,Redmond,USA)spreadsheetsandlaterexportedtoStata 12.0 (StataCorp,Texas,USA) for statisticalanalysis.Datawereinitiallyanalyzedbydescriptivestatisticsthroughmeasuresofcentraltendencyandfrequencydistribution.Bivariatelogisticregressionanalysis was performed to evaluate associationbetweenvariable,presenceofESBLinthetank,andsomepredictorsofinterest.Associationswithp<0.25wereconsideredsignificantandwereselectedforthemultivariatemodel. In thefinalmodel, only thosevariableswithp<0.05remained.

Results

General characteristics of the farms

ThemaincharacteristicsaresummarizedinTables1and2.Intotal,60%(72/120)ofthesampledfarmsoperatedasclosedfarms;77.5%(93/120)hadatleast50cowsinmilking.Inaddition,91%(108/120)hadanimals of non-bovine species,with the presenceof domestic birds and pigs in 47% (56/119) and24.4%(29/119)ofthefarms,respectively.Moreover,51.3% (61/119) of the farmshadnomanure tank,and66.4%(79/119)reportedperformingthemilkingroutine following technical recommendations thatwere focusedongood livestockpractices.Feedingcalveswastemilkwaspracticedin40%(48/120)ofthefarms.A55%(66/120)hadonemilkerand45%(54/120)ofthefarmshadmorethanonemilker.

A totalof70%(84/120)farmsreportedantibioticusage formulatedbyaveterinarianonly50%of thetimesorless.Averagemilkproductionwas659,000±449L/day.Additionally,in75%(90/120)ofthefarms,countsofcolonyformingunits(CFUs)andsomaticcellswerebelow28.000CFU/mLand406,000cells/mL,respectively(Table2).

Characteristics of dairy farms with confirmed ESBL-producing isolates

In total, 75% (3/4) of the farmswith isolatespositive for ESBL production operated as closedfarms,4farmshad>50(average93)cowsinmilking,100%ofthefarmsalsohadhorsesandpets(dogsandcats),50%(2/4)ofthefarmsreportedthepresenceofbirds,whilepigswerereportedinonlyonefarm.Additionally,75%(3/4)ofthefarmshadamanuretank,andthenumberofmilkerswasdifferentamongpositivefarms(1,3,or5milkers).Calffeedingwithwastemilkwas reported in 100%of the positivefarms, andmilkingwasperformedunder technicalrecommendationsin75%ofthosefarms.Theuseofantimicrobialstotreatanimalsfollowingveterinaryrecommendationswasreportedasusuallyinonefarm,sometimesintwofarms,andrarelyinmorethantwofarms.Themost frequently used antimicrobials inthepastyearinthesefourfarmsincludedpenicillins(75%,3/4),macrolides(50%,2/4),andtetracyclinesandquinolones(25%,1/4).Thosefourfarmsproduced1,865L/day.TheCFUandsomaticcellcountswere39,000CFU/mLand401,000cell/mLonaverage.

Presence of ESBL-producing Enterobacteriaceae

Bacteria suspicious of producing ESBLwereisolatedin6.6%(8/120)frommilksamplesofequalnumber of bulk-tanks.One isolatewas obtainedin eachof the 8 tanks.Of these 8 isolates, 2 non-fermentingglucoseisolateswerediscarded.Ofthe6remainingisolates,ESBLproductionwasconfirmedin4;thus,theapparentfrequencyofESBL-producing Enterobacteriaceae in bulk-tankmilkwas 3.3%(4/120;CI95%:3-3.5%).Alloftheisolatesshowedsynergywith at least two of the cephalosporinsused and clavulanic acid.The isolates of ESBL-producing Enterobacteriaceaewere identified asEnterobacter cloacae, Serratia fonticola, E. coli, and Enterobacter cancerogenususingtheCrystal™

91



Table 1. Selected predictors for exploring risk factors associated with the presence of ESBL-producing Enterobacteriaceae in bulk-tank milk (n = 120) at Entrerríos, Antioquia (Colombia).

Variable Category Frequency % Description

Farm operation Open 48 40 Purchase (open) or not (closed) of animals from other farms

Closed 72 60

Farm size ≤ 50 cows in milking 93 77.5 Number of cows in milking

>50 cows in milking 27 22.5

Other production Birds 56 47 Presence of birds, pigs or other domestic animals in the farm

Pigs 29 24.4

Other 23 19.3

No 11 9.24

Milk production <370 28 23.33 Average daily milk production (L)

>369 - <850 61 50.83

>849 31 25.83

Manure tank Yes 58 48.7 Availability of tanks to collect fecal matter of cattle

No 61 51.3

Standardized milking practices

Yes 79 66.4 Milking routine under hygiene standards established by good milking practices (GMP) for

which producers have been trainedNo 40 33.6

Destination of waste milk Feeding of calves 48 40 Milk from cows with antibiotic treatment

Refuse 62 51.67

Feeding of other animal species

10 8.33

Number of milkers 1 66 55 Workers in contact with milking cows

>1 54 45

Use of antimicrobials Cephalosporins 22 19.5 Use of antibiotics in the last 12 months

Tetracyclines 32 28.3

Sulphonamides 7 6.2

Other 59 46

Table 2. Quantitative variables used to explore risk factors associated with the presence of ESBL-producing Enterobacteriaceae in bulk-tank milk (n = 120) at Entrerríos, Antioquia (Colombia).

Variable Mean Standard deviation

Maximum value

Minimum value

Percentile

25% 75%

Milk production/ dairy farm/day (L) 659 449.3 3.480 90 370 850

CFU 29.6 65.7 560 2 6 28

Somatic cell count* 316.8 164.2 800 100 201 406

CFU: colony forming units. * per mL × 1000; arithmetic mean.

92



Enteric/NonfermenterIDkit(E/NF;BectonDickinson(BD)FranklinLakes,NewJersey,USA).

Antimicrobial susceptibility

All fourESBL-producing isolateswere resistantto ampicillin, amoxicillin, cephalexin, cefuroxime,erythromycin, and trimethoprim/sulfamethoxazole.Conversely,all four isolatesweresensitive toFOX,IPM,CIP, andnorfloxacin (Table3).The isolateofE. coliwas resistant toeightantibiotics (ampicillin,amoxicillin, cephalexin, cefuroxime, erythromycin,chloramphenicol, doxycycline, and trimethoprim/sulfamethoxazole) and had the greatest resistancecomparedtotherest.TheisolatesofS. fonticolahadtheleastresistancetotheantibiotics(6/13)comparedtotherestoftheisolatesandwastheonlyisolate,inadditiontoE. cancerogenus,thatdisplayedsensitivitytogentamicin.TheS. fonticolaandE. cloacaewerethe only isolates sensitive to chloramphenicol anddoxycycline.

Minimum inhibitory concentration (MIC)

TheMICof13antibioticsobtainedthroughthebrothmicrodilutionmethod in the 07.01Vitek 2(bioMérieux)forthefourisolatesisshowninTable4.TheonlyisolatethatdisplayedsensitivitytoSAMwasE. coli,whichhad8mg/mLMIC.Fortheotherthree isolates, resistance or intermediate resistancelevelwasobserved.All isolatesshowedsensitivitytoDOR,ETP, IPM,MEM,AMK,CIP, andTGC(Table 4). For CRO, all isolates yielded aMICvaluethatwasinterpretedasresistance.Bycontrast,forFEPandCAZ,onlyE. coliwas resistant,with≤1mg/mLMIC(byautomaticmodificationofthevalues obtained by the automated expert systemVitek® 2 (bioMérieux, Durham,NC,USA) and≥64mg/mLMIC,respectively.

TheS. fonticola and E. cloacae isolatesshowedresistance toFOX,with 16 and≤ 4mg/mLMIC,

Table 3. Antimicrobial susceptibility patterns obtained by the disk diffusion method for four isolates of ESBL-producing Enterobacteriaceae in bulk-tank milk (n = 120) at Entrerríos, Antioquia (Colombia).

Antibiotics µg/disc Cut-off mm

Enterobacter cancerogenus

Enterobacter cloacae

Escherichia coli Serratia fonticola

S R S R S R S R

Ampicillin 10 ≥ 17 - 0 - 0 - 0 - 0

Amoxicillin 10 ≥ 17 - 0 - 0 - 0 - 0

Cephalexin 30 ≥ 18 - 0 - 0 - 0 - 0

Cefuroxime 30 ≥ 18 - 0 - 0 - 8 0

Cefoxitin 30 ≥ 18 20 - 23 - 21 - 23 -

Imipenem 10 ≥ 23 30 - 27 - 27 - 29 -

Erythromycin 15 ≥ 15 - 0 - 0 - 0 - 0

Chloramphenicol 30 ≥ 18 - 0 32 - - 0 30 -

Gentamicin 10 ≥ 15 16 - - 0 14 - 22 -

Doxycycline 30 ≥ 14 - 0 20 - - 12 20 -

Ciprofloxacin 5 ≥ 21 44 - 36 - 28 - 40 -

Norfloxacin 10 ≥ 17 30 - 44 - 26 - 28 -

Trimethoprim/ sulfamethoxazole

1.25/23.75 ≥ 16 - 0 - 0 - 0 - 0

S: sensitive; R: resistant.

93



respectively.ThelatterMICwasreportedasresistantby automaticmodificationof values performedbytheVitek® system. The only isolate that showedresistancetoGENwasE. cloacae with≥16mg/mLMIC.Fortherestoftheantibioticgroups,theMICvariedaccordingtotheisolatedbacteria.

Characterization of ESBL by PCR and sequencing of bla genes

ThefourisolateshostedCTX-Mgenes,basedonPCRanalysis.All of the isolateswerepositive forCTX-M-1group,andwhentheseweresequenced,allsequencesshowedstronghomologywithCTX-M96variant,whichisalternativelycalledCTX-M-12a.

Risk factors

Aunivariatelogisticregressionanalysisyieldedan association between the presence of ESBL-producingEnterobacteriaceae and the size of thefarm,andbetweenthepresenceofESBL-producing Enterobacteriaceaeandmilkproduction(Table5).Attheend,onlyfarm sizevariable remainedsignificantinthemultivariablemodelconstructedbybackwardelimination, p<0.05.The odds of having ESBL-producingEnterobacteriaceaeinbulk-tankmilkwere11.5-foldhigherinfarmswithmorethan50cowsinmilkingcomparedtofarmsequalorlessthan50cowsinmilking(p<0.038).

Discussion

Themain limitations of this studywere theabsence of previous studies on ESBL-producingEnterobacteriaceaeinbulk-tankmilkfromcomparabledairy regions of Colombia, which would haveestimatedfrequencyaccordingtonationalproductiveconditions,andalargerbudget,whichwouldallowlargersamplesizeanddifferenttimeperiods.Takingintoaccountthelownumberofpositiveisolates,theseresultsshouldbeanalyzedindetailandcarefully.

Inthisstudy,120dairyfarmswithequalnumberofmilkcoolingtankslocatedinNorthernAntioquia(Colombia)weresampledtodeterminethepresenceofESBL-producingEnterobacteriaceaeinbulk-tank

Isol

ates

Cry

stal

En

teric

/ N

onfe

rmen

ter

ID K

it (%

)

bla

gene

sA

ntib

iotic

s

SAM

FOX

CA

ZC

RO

FEP

DO

RET

PIP

MM

EMA

MK

GEN

CIP

TGC

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

CIM

Int

Ent

erob

acte

r ca

ncer

ogen

us0.

93C

TX-M

9616

I≤

4S

4S

16R

2S

≤ 0.

12S

≤ 0.

5S

0.5

S≤

0.25

S≤

2S

≤ 1

S≤

0.25

S≤

0.5

S

Ent

erob

acte

r cl

oaca

e0.

94C

TX-M

964*

R*

≤ 4

R*

≤ 1

S16

R≤

1S

≤ 0.

12S

≤ 0.

5S

0.5

S≤

0.25

S≤

2S

≥ 16

R≤

0.25

S≤

0.5

S

Esc

heric

hia

coli

0.98

CTX

-M96

8S

≤ 4

S≥

64R

8R

≤ 1

R≤

0.12

S≤

05S

≤ 0.

25S

≤ 0.

25S

≤ 2

S≤

1S

≤ 0.

25S

≤ 0.

5S

Ser

ratia

fo

ntic

ola

0.76

CTX

-M96

≥ 32

R16

R4

S≥

64R

4S

≤ 0.

12S

≤ 0.

5S

≤ 0.

25S

≤ 0.

25S

≤ 2

S≤

1S

≤ 0.

25S

1S

Am

pici

llin/

sulb

acta

m (S

AM

), ce

foxi

tin (F

OX

), ce

ftazi

dim

e (C

AZ)

, cef

triax

one

(CR

O),

cefe

pim

e (F

EP

), do

ripen

em (D

OR

), er

tape

nem

(ETP

), im

ipen

em (I

PM

), m

erop

enem

(ME

M),

amik

acin

(AM

K),

gent

amic

in

(GE

N),

cipr

oflox

acin

(CIP

), tig

ecyc

line

(TG

C),

colis

tin (C

OL)

. M

IC: m

inim

um in

hibi

tory

con

cent

ratio

n, In

t: in

terp

reta

tion,

S: s

ensi

tive,

I: in

term

edia

te, R

: res

ista

nt.

*val

ues

mod

ified

by

the

adva

nced

exp

ert s

yste

m.

Tabl

e 4.

Min

imum

inhi

bito

ry c

once

ntra

tion

(MIC

) val

ues

obta

ined

by

the

mic

rodi

lutio

n m

etho

d in

07.

01 V

itek

2 br

oth

(bio

Mér

ieux

) for

4 is

olat

es o

f ESB

L-pr

oduc

ing

Ente

roba

cter

iace

ae in

bu

lk-ta

nk m

ilk (n

= 1

20) a

t Ent

rerrí

os, A

ntio

quia

(Col

ombi

a).

94



Table 5. Unconditional logistic regression analysis of risk factors associated with the presence of ESBL-producing Enterobacteriaceae in bulk-tank milk (n = 120) at Entrerríos, Antioquia (Colombia).

Variable OR SE p CI95%

Operation of the farm 0.49 0.57 0.54 0.04 - 4.84

Milk production 7.39 8.10 0.068 0.86 - 63.27

Manure tank 3.27 3.82 0.311 0.33 - 32.40

Feeding of calves with waste milk 2.26 1.81 0.306 0.47 - 10.88

Presence of pigs 1.04 1.23 0.968 0.10 - 10.47

Presence of birds 1.14 1.16 0.892 0.15 - 8.42

Milking routine under technical recommendations 0.49 0.50 0.488 0.06 - 3.63

Number of milkers 1.23 1.25 0.838 0.16 - 9.03

Use of tetracyclines 0.83 0.98 0.881 0.08 - 8.37

Farm size 11.49 13.53 0.038 1.14 - 115.54

CFU 2.22 2.26 0.434 0.30 - 16.40

Somatic cells count 1.52 1.54 0.680 0.20 - 11.18

CFU: colony forming units; OR: odds ratio; SE: standard error; p value: significance level.

milkandtoexploretheriskfactorsassociatedtoitspresence.

Recently, studies of the characteristics andprevalence ofESBL-producing Enterobacteriaceaein animals have increased in different countriesworldwide (Smet et al., 2010; Ohnishi et al.,2013;Timofteet al., 2014;Skočkováet al, 2015;Sudarwanto et al., 2015;Odenthal et al., 2016).However,relativelyfewstudieshavebeenconductedin Colombia on antibiotic resistance in food-producinganimalsandtheirproducts(VanegasLópezet al.,2012;JiménezVelásquezet al.,2013;Donado-Godoyet al.,2015)comparedtoothercountriesandtoourknowledge, this is thefirst studyconductedto determine the occurrence of ESBL-producingEnterobacteriaceaeinbulk-tankmilkinColombia.

ThefrequencyofESBL-producing Enterobacteriaceaereportedinthisstudy(3.3%)agreeswithotherstudiesconductedinbulk-tankmilksamplesinSwitzerland,Brazil, India, theCzechRepublic, Indonesia, andGermany, which reported prevalences rangingbetween0and9%(Geseret al.,2012;Nóbregaet al., 2013;Rasheed et al., 2014; Skočková et al.,2015;Sudarwantoet al.,2015;Odenthalet al.,2016).

Although the reported occurrences are low, theyrepresentasignificantfindingthatcancontributetoclarifying the role that animals and their productshaveinthedisseminationofmulti-resistantbacteriainfoodsofanimalorigin.

The low frequency of ESBL-producing Enterobacteriaceae in bulk-tankmilk could be aresultofthehighhygienestandardsrequiredbymilkprocessingcompaniesinAntioquiatopurchaserawmilk.Milk payment inColombia is dependent onquality parameters such asCFU.Milk processingcompanies pay milk to producers as follows:CFU counts between 0 and 175,000CFU/mL areconsideredasmilkofverygoodqualityandreceiveabonuspayment,CFUcountsbetween175,001and200,000CFU/mLreceiveneitherabonuspaymentnor a discount on the price ofmilk, and counts>200,001CFU/mLreceivediscountonthepriceofmilk (Ministerio deAgricultura, 2012). Improvingthe general hygiene in all stages of productionand thereby reducing themicrobial load of foodproductswillalsoreducetheantimicrobialresistanceload (Wegener, 2012). It is also possible that thelowoccurrenceofESBLs in these farms isdue tolittle use of cephamycins and carbapenems since

95



the selection pressure that drivesESBL evolutionhas usually been attributed to the intensive use ofoxyimino-cephalosporins,mainly third-generationcephalosporins(Gniadkowski,2001).

Our isolates contrast with the predominantisolates in dairy farms fromother areaswhereE. coliandK. pneumoniaehavebeenmostfrequentlyreported (Locatelli et al., 2010;Dahmen et al.,2013;Ohnishiet al.,2013;Sudarwantoet al.,2015;Odenthalet al., 2016).However, the relevance ofotherbacteriaoftheEnterobacteriaceaefamilycouldbe underestimated because studies havemainlyfocusedonE. coliandK. pneumoniae (Smetet al.,2010;Schmidet al.,2013;Sudarwantoet al.,2015;Odenthalet al.,2016).Anyway,thediscoveryofthesebacteria(Enterobacter sp.andSerratia sp.)inmilkisstrikingbecauseusuallySerratia fonticolaisfoundinawidearrayofenvironments,includingdrinkingwater,soil,andsewage(Aljorayidet al.,2016)whileEnterobacter strains tend to colonize hospitalizedpatients, particularly those treatedwith antibiotics,andhavebeenassociatedwith infectionsofburns,wounds,respiratoryandurinarytract(Puerta-GarcíaandMateos-Rodríguez,2010).

Although resistance to different antibioticswas detected, both by the disc diffusionmethodin agar (Kirby-Bauer) and by themicrodilutionbrothmethod, sensitivity remained to carbapenemantibiotics.Carbapenemsare themostpotentbeta-lactam antibiotics because of theirwide spectrum(Bush,2013),andthesebacteriaarestillsensitivetothisfamilyofantibiotics,whichmakesabatteryofeffectiveantibioticsavailablefortreatinginfectionscausedinhumansandanimals.However,carbapenemsrequireresponsibleusetopreventtheemergenceofresistance.

The susceptibility patterns shownbyboth testswere similar.Discrepancies between results canbebasedon thepatterncomparisonstrategyof theVitek® automated expert system,which comparesthe observed phenotypewith descriptions basedon knowledge constructed from data obtained inscientificpublicationscomplementedbyinternaldataof eachhealth system (Gerst, 2000); if the systemdetectsadiscrepancybetweenthatobservedforanantibioticand thedistributionofMICs for the rest

of the antibiotics, it has the ability tomodify theresult,thusprovidingrapid,automaticandsystematicvalidationofeachofthesensitivityresultswhileatthesametimeminimizinghumanerror(Sanderset al., 2001;Schwaberet al., 2006;bioMérieux,2015).

GiventhatproductionofmostESBLsisencodedby plasmids, co-resistance to other groups ofantibiotics is common (Rybaket al., 2004;Xian-Zhiet al.,2007;Geseret al.,2012).Theresistanceshowntogentamicinanderythromycinby100%ofisolates possibly indicates the presence of a geneencodingresistancetoantibioticsofaminoglycosidesandmacrolidesfamiliessimultaneously.Notably,therangeofantibioticsforwhichresistancewasacquiredislargeandsufficientlyworrisomebecauseitshowstheseverityoftheemergenceofantibioticresistancein different pathogens. For example, an infectioncausedbya strainofE. coli suchas that found inourstudycouldrepresentalargerproblembecausetherapeuticoptionsareclearlylimited.

Recently,rapidgrowthinthenumberofpositiveisolateswithCTX-Menzymes has been observed(Woodford,2010).TheCTX-Menzymesconstitutea rapidly growing family ofESBLs enzymeswithsignificant clinical impact (Zhao andHu, 2013).Enterobacteriaceae isolates that are positive forCTX-Mhavebeenfoundinfood-producinganimalsand their products (Carattoli, 2008; Smet et al.,2010; Schmid et al., 2013;Randall et al., 2014).Theenzymesfoundinourisolatesagreewiththosefrequently reportedworldwide. The CTX-M-96(CTX-M-12aalternativename)isoneof109variantsofCTX-Menzymes that have been identified andassignedintheLaheydatabase(ZhaoandHu,2013).TheCTX-M-96was reported in K. pneumoniae ashostorganismandcontainedintheGenBank®withthe codeAJ704396 (NCBI2005).TheCTX-M-96waspreviouslyreportedinK. pneumoniaeinChileandArgentina(NCBI,2015;NCBI2005).Onestudypublished this enzyme as a simulationmodel forevaluatingactivitytowardsoxyiminocephalosporins(Ghiglioneet al.,2015).However,withitsalternatename,theblageneCTX-M-12awasdetectedfirstinColombiainfourisolates:twoE. cloacaeandintwoK. pneumoniaeisolates;oneoftheE. cloacaeisolatesandoneoftheK. pneumoniaeisolateswereassociatedwithnosocomialinfectionswhiletheotheronewas

96



isolatedfromthecommunity(Mantillaet al.,2009).Alaterstudyrevealedthat18outof33ESBLpositivestrains isolated from three Colombian hospitalsharboredgroup1blaCTX-MandanalysisofDNAsequencesrevealedthepresenceof blaCTX-M-12ainthreeofthem(Ruizet al.,2011).Theseresultsalertonthepoliciesconcerningantibioticuse,distribution,sellingandmanagementandotherfactorsthatmaybeassociatedwiththespreadofmultiresistantorganismscausingoutbreaks inhospitals and thecommunity,including the agricultural sector.The presence ofthesamegeneatthehospitallevelandattheanimalproductionlevelgivesrisetoconductfuturestudiestoelucidatetherelationshipbetweenbothenvironmentsandbehaviorofantibioticresistanceinColombia.ItisimportanttoconductmoremolecularepidemiologicalstudiesofbacterialresistancethatpermitidentifyinggeneticdeterminantsandvariantsoftheESBLenzymefamiliesthatmaybeassociatedwithchangesinthespectrumandthatdecreaseantimicrobialactivityinaparticularenvironment.

Relative to risk factors associated with thepresence of ESBL-producing Enterobacteriaceae,theriskfactorsfoundinourstudydifferfromthosereportedinpreviousstudiesinEnglandandnorthofWales, Switzerland and Israel (Snowet al., 2012;Reistet al., 2013;Adleret al., 2015) consideringthat independent variables are different betweenstudies. In thesestudies, the following factors:useofthirdandfourthgenerationcephalosporinsinthelast12months,operatingasanopenfarm,infrequentcleaning of equipment for feeding of calves andstorage of fecalmatter inmanure tanks, animalsoriginating from farmswith primary productiontypedairy, animals originating from farmswithmore thanone animalmovementperdayper100,lackofacoolingsystem,increasedcrowdednessandlackofmanurecleaning,antimicrobialprophylaxisandincreasedfrequencyofveterinarianvisits,wereassociatedwith the presence of ESBL-producing Enterobacteriaceaeincattle(Snowet al.,2012;Reistet al.,2013;Adleret al.,2015).

Theriskfactordairy farmsizeassociatedwiththepresenceofESBL-producing Enterobacteriaceaeinourstudycouldbeexplainedbythefactthatalargernumberofmilkingcowscouldrepresentanincreasein the circulation and occurrence of diseases in a

farm,whichwouldfinally lead to the frequentuseof antibiotics (Kreausukon,2011)and/or increasedcontact betweenmilkers and cows that are sickorbeingtreated.Similarly,thelargernumberofcowsinmilking could lead to non-compliance of thestringent routinemilking standards between cows(Ruiz-Romero,2015)

The fact thatmost of the farms had reportedperformingmilking routine following technicalrecommendationsfavorsthesanitarysituationinthemunicipality because if there is nomonitoring ofgoodmilkingpractices,therewouldbemorecasesofmastitisandthereforeagreaterusageofantibioticswith the possibility of developing or acquiringresistance(OliverandMurinda,2012).Inthesameway, situations inwhich the choice and provisionofantibioticsisinthehandsofapersonotherthanthe veterinarianmay cause variations in bacterialsensitivityalongwithrecurrenceofthepresentationofmicroorganismsassociatedwithdifferentpathologiesasaconsequenceofnotnormallyperformingisolationorevaluationbyantibiogramofthecausalagentofinfection(Betancourtet al.,2003).

Inthisstudy,feedingcalveswithwastemilk(milkof cowswith antibiotic treatment)—although notfoundasariskfactor—continues tobeafrequentactivity in dairy farmsof themunicipality, despitecurrent knowledgeof the association between thispractice and increased resistance to antimicrobialsincalves (Langfordet al.,2003;Austet al.,2013;Bruntonet al.,2014).

Theuse of bulk-tankmilk samples could servetomonitortrendsinresistancetoantibioticsindairyfarms (Bergeet al., 2007).Although the presenceof ESBL-producing Enterobacteriaceaewas nothigh in this study, theuseof antibiotics shouldbecontrolled,sanitarymeasuresshouldbereinforced,andthephenomenonofantibioticresistanceinfood-producinganimalsshouldcontinuetobeinvestigated(FDA, 2012). Timely identification of ESBL-producingEnterobacteriaceaestrains isessential inthemonitoringofthedevelopmentofantimicrobialresistance and in the implementation of infectioncontrolmeasuresfortheprotectionofpublichealth(WHO,2001).

97



Acknowledgments

This studywas fundedby theFondodeApoyoalPrimerProyectodeProfesoresoftheUniversidaddeAntioquia (Comité para el Desarrollo dela Investigación—CODI).Authors thank theLaboratorio deMicrobiologíaVeterinaria of theFacultad de CienciasAgrarias (UdeA),MaríaLuisaVélezAgudelo (joven investigadoraCODI,2013), theEstrategiadeSostenibilidad2013-2014,CENTAURO, theSecretaríadeAsistenciaTécnicaAgropecuariayDesarrolloComunitariodeEntrerríos,andparticipatingdairyherds.

Conflicts of interest

The authors declare they have no conflicts ofinterestwith regard to thework presented in thisreport.

ReferencesAdlerA,SturlesiN,FallachN,Zilberman-BarzilaiD,HusseinO,BlumSE,KlementE,SchwaberMJ,CarmeliY.Prevalence,risk factors and transmission dynamics of ESBL-producingEnterobacteriaceae:anationalsurveyofcattlefarms inIsrael,2013.JClinMicrobiol2015;53(11):3515-3521.

AljorayidA,ViauR,CastellinoL,JumpR.Serratia fonticola,pathogenorbystander?Acaseseriesandreviewoftheliterature.IDCases2016;5:6-8.

AmadoNY, FajardoHD,RamírezRuedaRY,GonzálezGI.PrevalenciadebetalactamasasdeespectroextendidoenbacilosgramnegativosdeunainstitucióndesaluddeTunja(Colombia)enelaño2013.SaludSocUptc2014;1(2):54-60.

Archivo Secretaría deAsistencia TécnicaAgropecuaria yDesarrolloComunitarioMunicipio deEntrerríos,Antioquia,Colombia.Ciclodevacunacióncontrafiebreaftosa,FederaciónColombianadeGanaderos(FEDEGAN)2013-2.

AustV,KnappsteinK,KunzHJ,KasparH,WallmannJ,KaskeM.Feedinguntreatedandpasteurizedwastemilkandbulkmilktocalves:effectsoncalfperformance,healthstatusandantibioticresistanceoffaecalbacteria.JAnimPhysiolAnimNutr(Berl)2013;97(6):1091-1103.

BatchelorM,HopkinsK,Threlfall EJ, Clifton-Hadley FA,StallwoodAD,DaviesRH,LiebanaE. blaCTX-Mgenes inclinicalSalmonellaisolatesrecoveredfromhumansinEnglandandWales from1992 to2003.AntimicrobAgentsChemother2005;49:1319-1322.

BlancoVM,MayaJuanJ,CorreaAdriana,PerenguezMarcela,Muñoz Juan S,Motoa Gabriel, Pallares Christian, RossoFernando,MattaLorena,CelisYamile,GarzónMartha,VillegasMaríaV.Prevalenciayfactoresderiesgopara infeccionesdeltractourinariodeinicioenlacomunidadcausadasporEscherichia coli productor de betalactamasas de espectro extendido enColombia.EnfermInfeccMicrobiolClin2016;34:559-565.

BergeAC,ChampagneSC,FingerRM,SischoWM.Theuseofbulktankmilksamplestomonitortrendsinantimicrobialresistanceondairyfarms.FoodbornePathogDis2007;4(4):397-407.

BetancourtO,ScarpaC,VillagránK.EstudioderesistenciadecepasdeStaphylococcus aureus aisladasdemastitissubclínicabovinafrenteacincoantibióticosentressectoresdelaIXregióndeChile.RevistaCientíficaFCV-LUZ2003,13(5):413-417.

Biomerieux, 2015.Una combinación ganadora para detectarresistencias.[Accessdate:January13th,2017).URL:http://www.biomerieux.com.ar/servlet/srt/bio/argentina/dynPage?doc=ARG_CLN_PRD_G_PRD_CLN_37.

BruntonLA,ReevesHE,SnowLC, Jones JR.A longitudinalfieldtrialtheimpactoffeedingwastemilkcontainingantibioticresiduesontheprevalenceofESBL-producingEscherichia coliincalves.PrevVetMed2014;117(2):403-412.

BushK. Proliferation and significance of clinically relevantβ-lactamases.AnnNYAcadSci2013;1277:84-90.

BushK.Bench-to-bedside review: the roleofβ-lactamases inantibiotic-resistantGram-negative infections.CritCare 2010;14(3):224.

CarattoliA.Animal reservoirs for extended spectrum beta-lactamaseproducers.ClinMicrobiolInfect2008;14(1):117-123.

ClinicalandLaboratoryStandardsInstitute(CLSI).Performancestandards for antimicrobial susceptibility testing; nineteenthinformationalsupplementM100-S18.Wayne,PA:ClinicalandLaboratoryStandardsInstitute;2008.



Dahmen S, Métayer V, Gay E, Madec JY, Haenni M.Characterization of ESBL-carrying plasmids and clones ofEnterobacteriaceae causing cattle mastitis in France.VetMicrobiol2013;162:793-799.

DonadoGodoyP,CastellanosR,LeónM,ArevaloA,ClavijoV,BernalJ,LeónD,TafurMA,ByrneBA,SmithWA,PerezGutierrezE.The establishment of theColombian integratedprogramforantimicrobialresistancesurveillance(COIPARS):apilotprojectonpoultryfarms,slaughterhousesandretailmarket.ZoonosesandPublicHealth2015;62(1):56-58.

98



EuropeanFoodSafetyAuthority(EFSA).Scientificopiniononthepublichealthrisksofbacterialstrainsproducingextended-spectrumbeta-lactamasesand/orAmpCbeta-lactamasesinfoodandfood-producinganimals.EFSAJ2011;9:2322.

FerensWA,HovdeCJ.Escherichia coli O157:H7: animalreservoirandsourcesofhumaninfection.FoodbornePathogDis2011;8(4):465-487.

Food andDrugAdministration (FDA).Center forVeterinaryMedicine.Thejudicioususeofmedicallyimportantantimicrobialdrug in food-producing animals.Guidance for industry.April13th,2012.

González L, Cortés JA. Revisión sistemática de lafarmacorresistencia en enterobacterias de aislamientoshospitalariosenColombia.Biomédica2014;34(2):180-197.

Gerst J. Detección de la resistencia a los antibióticos conlos sistemas vitek y vitek 2 de biomerieux. In: Resistenciaantimicrobiana en lasAméricas:magnitud del problemay sucontención.OrganizaciónPanamericanadelaSalud.2000.

GeserN,StephanR,HächlerH.Occurrenceandcharacteristicsof extended-spectrum beta-lactamase (ESBL) producingEnterobacteriaceaeinfoodproducinganimals,mincedmeatandrawmilk.BMCVetRes2012;8:12.

GhiglioneB,RodríguezMM,HermanR,CurtoL,DropaM,BouillenneF,KerffF,GalleniM,CharlierP,GutkindG,SauvageE,PowerP.Structuralandkineticinsightsintothe“ceftazidimase”behavior of the extended-spectrum β-lactamaseCTX-M-96.Biochem2015;54(32):5072-5082.

GniadkowskiM. Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLs) and ESBL-producingmicroorganisms.ClinMicrobiolInfect2001;7(11):597-608.

Gobernación deAntioquia, 2013. Economía.Municipio deEntrerríos. [Access date:December 1st, 2016].URL: http://entrerrios-antioquia.gov.co/nuestromunicipio.shtml?apc=mfVereda-1-&m=f#economia

Gobernación deAntioquia, 2007. Perfil subregional norteantioqueño. [Access date:December 21st, 2016].URL:http://www.antioquia.gov.co/antioquiav1/organismos/planeacion/descargas/perfiles/Perfil%20Subregional_Norte.pdf

JiménezVelásquezS,TorresHigueraLD,RodríguezBautistaJL, García Castro FE, Patiño Burbano RE. Identificaciónde aislamientosmeticilino resistentes de Staphylococcus sp provenientes de hatos lecheros del Departamento deCundinamarca.RevColombCiencPecu2013;26(Supl).

KreausukonK.Usageof antimicrobials on 60dairy farms inNorthernGermanyandcharacterizationofmethicillin-resistantStaphylococcus aureus (MRSA) and extended spectrumbetalactamases producingEscherichia coli (ESBLs-producingE. coli)isolatedfrombulktankmilksamples.PhDDissFreie,UniversitätBerlin,Berlin,Germany.2011.

LangfordF,BurstynU,FisherL,ShelfordJ,WearyD.Feedingwastemilktocalvesandantibioticresistance.21stAnnualWesternCanadianDairySeminar,RedDeer,ABMarch11-14,2003.

LealAL, Cortés JA,Arias G, OvalleMV, Saavedra SY,BuitragoG,Escobar JA.Emergencia de fenotipos resistentesa cefalosporinas de tercera generación enEnterobacteriaceaecausantesdeinfeccióndeltractourinariodeiniciocomunitarioenhospitalesdeColombia.EnfermInfeccMicrobiolClin2013;31(5):298-303.

LeedomJ.Comparingthefoodsafetyrecordofpasteurizedandrawmilkproducts.[Accessdate:December21st,2016].URL:http://www.foodpoisonjournal.com/food-poisoning-information/comparing-the-food-safety-record-of-pasteurized-and-raw-milk-products/#.V7Yrfyh97IW

LejeuneJT,RajalaSchultzPJ.Foodsafety:unpasteurizedmilk:acontinuedpublichealththreat.ClinInfectDis2009;48(1):93-100.

LeversteinvanHallMA,DierikxCM,CohenStuartJ,VoetsGM,vandenMunckhofMP,vanEssen-ZandbergenA.Dutchpatients,retail chickenmeat and poultry share the sameESBLgenes,plasmidsandstrains.ClinMicrobiolInfect2011;17(6):873-880.

LocatelliC,ScaccabarozziL,PisoniG.CTX-M1ESBL-ProducingKlebsiella pneumoniaesubsppneumoniaeIsolatedfromCasesofBovineMastitis.ClinMicrobiol2010;48(10):3822-3823.

MachadoAlbaJE,GonzálezSantosDM.Dispensingantibioticsto outpatients in aColombian population.Rev salud pública2009;11(5):734-744.

MantillaAnaya JR,BarretoHernándezE,RegueroRezaMT,VelandiaRodríguezDA. Identifying cefotaximase genes inEnterobacteriaceaehospitalisolatesbyPCR-SSCP.RevColombBiotecnol2009;11(2):57-65.

Mesa RJ, Blanc V, BlanchAR, Cortés P, González JJ,LavillaS,MiróE,MuniesaM,SacoM,TórtolaMT,MirelisB, Coll P, LlagosteraM, Prats G, Navarro FJ. Extended-spectrumb-lactamase-producingEnterobacteriaceaeindifferentenvironments (humans, food, animal farms and sewage).AntimicrobChemothe2006;58:211-215.

Ministerio deAgricultura yDesarrolloRural deColombia.Resolución000017:Sistemadepagodelechecrudaalproductor.Bogotá,Colombia.2012.

MinisteriodelaProtecciónSocial.Decreto1880del27demayode2011.Bogotá,Colombia.

MinisteriodelaProtecciónSocial.Decreto616del28defebrerode2006.Bogotá,Colombia.

NCBI(NationalCenterforBiotechnologyInformation)(2005).CTX-M-12a enzymeKlebsiella pneumoniae. GenBank:CAG28417.1.[Accessdate:December1st,2016].URL:http://www.ncbi.nlm.nih.gov/protein/47109376.

NCBI(NationalCenterforBiotechnologyInformation)(2015).Klebsiella pneumoniaestrainK-837beta-lactamaseCTX-M-96(blaCTX-M)gene,blaCTX-M-96allele,completecds.GenBank:KR811026.1.[Accessdate:April20th,2016].URL:http://www.ncbi.nlm.nih.gov/nucleotide/940376853?report=genbank&log$=nucltop&blast_rank=1&RID=C1WP1YKG01R

99



NóbregaDB,GuiduceMVS,GuimarãesFF,RibolDF.Molecularepidemiologyandextended-spectrumβ-lactamasesproductionofKlebsiella pneumoniaeisolatedfromthreedairyherds.PesqVetBras2013;33(7):855-859.

Odenthal S,Akineden Ö, Usleber E. Extended-spectrumbetalactamase (ESBL)-produzierende EnterobacteriaceaeinAnlieferungsmiclh aus hessischenMilchviehbetrieben.In: 54.Arbeitstagung derDeutscheVeterinärmedizinischeGesellschaft (DVG)Arbeitsgebiet Lebensmittelhygiene,Garmisch-Partenkirchen(Alemania),Septimebre24-27de2013.

Odenthal S,Akineden Ö, Usleber E. Extended spectrumβ-lactamase producingEnterobacteriaceae in bulk tankmilkfromGermandairyfarms.IntJFoodMicrobiol2016;5:238:278.

OhnishiAT,OkataniH,EsakiK,HaradaT,SawadaM,MurakamiK,MarumoY,KatoR,SatoK,ShimuraN,Hatanaka,TakahashiT.HerdprevalenceofEnterobacteriaceaeproducingCTX-MtypeandCMY-2b-lactamases among Japanesedairy farms. JAppMicrobiol2013;115:288-289.

OhnishiM,OkataniAT,HaradaK, SawadaT,MarumoK,MurakamiM,SatoR,EsakiH,ShimuraK,KatoH,UchidaN,TakahashidT.GeneticcharacteristicsofCTX-M-typeextended-spectrum-β-lactamase (ESBL)-producingEnterobacteriaceaeinvolvedinmastitiscasesonJapanesedairyfarms,2007to2011.JClinMicrobiol2013;51(9):3117-3122.

Oliver SP,Murinda SE.Antimicrobial resistance ofmastitispathogens.VetClinNorthAmFoodAnimPract2012;28(2):165-185.

PatersonDL,BonomoRA.Extended-spectrumbeta-lactamases:aclinicalupdate.ClinMicrobiolRev2005;18:657-686.

PitoutJDD,ThomsonKS,HansonND,EhrhardtAF,MolandES,SandersCC.B-lactamasesresponsibleforresistancetoexpandedspectrumcephalosporinsinKlebsiella pneumoniae,Escherichia coli,andProteus mirabilis isolates recovered inSouthAfrica.AntimicrobAgentsChemother1998;42:1350-1354.

PuertaGarcíaA,MateosRodríguezF.Enterobacterias.Medicine2010;10(51):3426-3431.

RandallL,HeinrichK,HortonR,BruntonL,SharmanM,BaileyHorneV,SharmaM,McLarenI,ColdhamN,TealeC,JonesJ.DetectionofantibioticresiduesandassociationofcefquinomeresidueswiththeoccurrenceofExtended-Spectrumβ-Lactamase(ESBL)-producingbacteria inwastemilk samples fromdairyfarmsinEnglandandWalesin2011.ResVetSci2014;96:15-24.

RasheedMU,ThajuddinN,AhamedP,TeklemariamZ, JamilK.Antimicrobial drug resistance in strains of escherichia coliisolatedfromfoodsources.RevInstMedTropSaoPaulo2014;56(4):341-346.

ReistM,GeserN,HachlerH,ScharrerS,StephanR.ESBL-producingEnterobacteriaceae:occurrence,riskfactorsforfecalcarriageandstraintraitsintheSwissslaughtercattlepopulationyoungerthan2yearssampledatabattoirlevel.PLOSONE2013;8(8):71725.

Ruiz Romero RA.Mastitis bacteriana en ganado bovino:etiología y técnicas de diagnóstico en el laboratorio. [Accessdate:November22nd,2015].URL:Disponibleen:http://www.ammveb.net/articulos/Mastitis_bacteriana.pdf.

RuizSJ,MontealegreMC,RuizGarbajosaP,CorreaA,BriceñoDF,MartinezE,RossoF,MuñozM,QuinnJP,CantonR,VillegasMV.FirstcharacterizationofCTX-M-15-producingEscherichia coliST131andST405clonescausingcommunity-onsetinfectionsinSouthAmerica.JClinMicrobiol2011;49(5):1993-1996.

RandallL,HeinrichK,HortonR,BruntonL,SharmanM,Bailey-HorneV,SharmaM,McLarenI,ColdhamN,TealeC,JonesJ.Detectionofantibioticresiduesandassociationofcefquinomeresidueswiththeoccurrenceofextended-spectrumb-lactamase(ESBL)-producingbacteria inwastemilk samples fromdairyfarmsinEnglandandWalesin2011.ResVetSci2014;96:15-24.

RybakMJ, PharmD.Resistance to antimicrobial agents: anupdate.SupPharmacotherapy2004;24(12).

SánchezL,RíosR,MáttarS.Detectionofextendedspectrumbeta-lactamases (ESBL) inEscherichia coli andKlebsiella pneumoniaeisolatedinaclinicinVillavicencio,Colombia. Infect2008;12(3):193-200.

SandersCC,PeyretM,MolandES,CavalieriSJ,ShubertC,ThomsonKS,BoeufgrasJM,SandersWE.PotentialimpactoftheVITEK2systemandtheadvancedexpertsystemontheclinicallaboratoryofauniversity-basedhospital.JClinMicrobiol2001;39:2379-2385.

SchmidA,HörmansdorferS,MesselhäusserU,KäsbohrerA,Sauter-LouisC,MansfeldcR.Prevalenceofextended-spectrum-lactamase-producingEscherichia colionbavariandairyandbeefcattlefarms.AppEnvironmentMicrobiol2013;79(9):3027-3032.

SchwaberMJ,NavonVeneziaS,ChmelnitskyI,LeavittA,SchwartzD,CarmeliY.UtilityoftheVITEK2advancedexpertsystemforidentification of extended-spectrumβ-lactamase production inEnterobacterspp.JClinMicrobiol2006;44(1):241-243.

SkočkováA,BogdanovičováK,Koláčková I,KarpíškováR.Antimicrobial-resistant and extended-spectrum β-lactamase-producingEscherichia coli in rawcow’smilk. JFoodProtect2015;2(234):72-77(6).

SmetA,MartelA,PersoonsD,DewulfJ,HeyndrickxM,HermanL,Haesebrouck F, Butaye P. Broad-spectrum b-lactamasesamongEnterobacteriaceaeofanimalorigin:molecularaspects,mobility and impact on public health. FEMSMicrobiolRev2010;34:295-316.

SnowLC,WarnerRG,CheneyT,WearingH,StokesM,HarrisK,TealeCJ,ColdhamNG.Riskfactorsassociatedwithextendedspectrumbeta-lactamaseE. coli (CTX-M) on dairy farms inNorthWest England andNorthWales. PrevVetMed 2012;106(3,4):225-234.

StataCorp.2011.Stata:Release12.StatisticalSoftwareRelease12ed.StataCorpLP,CollegeStation,Texas.

100



SudarwantoM,AkinedenÖ,OdenthalS,GrossM,UsleberE.Extended-spectrumb-Lactamase (ESBL)-producingKlebsiella pneumoniae inbulk tankmilk fromdairy farms in Indonesia.FoodbornePathDis2015;12(7):585-590.

TimofteD,MaciucaIE,EvansNJ,WilliamsH,WattretA,FickJC,WilliamsNJ.Detection andmolecular characterizationofEscherichia coliCTX-M-15andKlebsiella pneumoniaeSHV-12β-lactamasesfrombovinemastitisisolatesintheUnitedKingdom.AntimicrobAgentsChemother2014;58(2):789-794.

ToroC.Protocolodetomademuestrasdeleche:laboratoriodecalidadeinocuidaddelechecruda.FacultaddeCienciasAgrarias,UniversidaddeAntioquia,Colombia.Versión1julio10de2012.

Vacca CP,NiñoCY, Reveiz L. Restricción de la venta deantibióticos en farmacias de Bogotá, Colombia: estudiodescriptivo.RevPanamSaludPública2011;30(6):586-591.

VanegasLópezMC,MorenoJE,RamosRuedaV,ChiriviJS,GarzónA,ArévaloSA,MartínezMF,GardeazábalPA,BaqueroC.Methicillin-resistantStaphylococcus aureus(MRSA)isolatedfromColombianfoods.BIO2012;2:61-67.

VerraesC,VanBoxstaelS,VanMeervenneE,VanCoillieE,ButayeP,CatryB,deSchaetzenMA,VanHuffelX,ImberechtsH,DierickK,DaubeG,SaegermanC,DeBlockJ,DewulfJ,HermanL.Antimicrobialresistanceinthefoodchain:areview.IntJEnvironResPublicHealth2013;10(7):2643-2669.

VillegasMV,CorreaA,PerezF,ZuluagaT,RadiceM,GutkindG,CasellasJM,AyalaJ,LolansK,QuinnJohnP.CTX-M-12β-Lactamase in aKlebsiella pneumoniae Clinical Isolate inColombia.AntimicrobAgentsChemother2004;48(2):629-631.

VillegasMV;GuzmánBlancoM;SifuentesOsornio J,RossiF. Increasing prevalence of extended-spectrum-betalactamaseamongGram-negativebacilli inLatinAmerica–2008updatefrom the study formonitoring antimicrobial resistance trends(SMART).BrazJInfectDis2011;15(1):34-39.

WalshC,FanningS.Antimicrobialresistanceinfoodbornepathogens--acauseforconcernCurrDrugTargets2008;9(9):808-815.

WegenerHC.Antibioticresistance—linkinghumanandanimalhealth. In: Institute ofMedicine (US). Improving food safetythroughaonehealthapproach:workshopsummary.Washington(DC):NationalAcademiesPress(US);2012.A15.[Accessdate:December1st,2016].URL:http://www.ncbi.nlm.nih.gov/books/NBK114485/

WoodfordN,FaganEJ,EllingtonMJ.MultiplexPCRforrapiddetection of genes encodingCTX-M extended spectrumB-lactamases.JAntimicrobChemother2006;57:154-155.

WoodfordN. Rapid characterization of beta lactamases bymultiplexPCR.MethodsMolBiol2010;642:181-192.

WooldridgeM. Evidencefor thecirculationofantimicrobial-resistant strains and genes in nature and especially betweenhumansandanimals.RevSciTech2008;31(1):231-247.

WorldHealthOrganization–WHO(2001):GlobalStrategyforContainment ofAntimicrobialResistance.RevPanamSaludPúblicavol.10n.4WashingtonOct.2001Xian-ZhiL,ManishaM,ShivaG,LateefA.Β- lactamresistanceandβ- lactamasesinbacteriaofanimalorigin.VetMicrobiol2007;121:197-214.

ZhaoWH,Hu ZQ. Epidemiology and genetics of CTX-Mextended-spectrum β-lactamases inGram-negative bacteria.CriticalRevMicrobiol2013;39(1):79-101.

Zeinhoma,MohamedMA,Abdel-Latefb,GihanK.Publichealthriskofsomemilkbornepathogens.Beni-SuefUniversityJBasicAppScienc2014;3(3):209-215.

85 Original Articles Original articles -...

Documents

Transcript of 85 Original Articles Original articles -...