Antimicrobial effect of copper surfaces on bacteria isolated from poultry meat

(1)

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

Food

Microbiology

Antimicrobial

effect

of

copper

surfaces

on

bacteria

isolated

from

poultry

meat

Angel

Parra

1

_,

_Magaly

_Toro

1

_,

_Ricardo

_Jacob,

_Paola

_Navarrete,

_Miriam

_Troncoso,

Guillermo

Figueroa,

Angélica

Reyes-Jara

∗

LaboratoryofMicrobiologyandProbiotics,InstituteofNutritionandFoodTechnology(INTA),UniversityofChile,Santiago,Chile

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19December2017 Accepted5June2018

Availableonline24August2018 AssociateEditor:ElaineDeMartinis

Keywords: Antimicrobialcopper-alloys Foodbornepathogens Salmonella Listeriamonocytogenes Poultrymeat

a

b

s

t

r

a

c

t

Poultrymeatisafoodproductthatusuallycarrieshighratesofmicrobialcontamination, includingfoodbornepathogens.Thepoultryindustryhasestablisheddifferentsystemsto minimizethesehazards.Inrecentyears,extensiveliteraturehasdemonstratedthe antimi-crobialactivityofdifferentcontactsurfacesmadeofcoppertoeffectivelyreducemicrobial loads.Theaimofthepresentstudywastoevaluatetheantibacterialeffectofcoppersurfaces onthetransmissionoftwofoodbornepathogens–SalmonellaentericaandListeria monocy-togenes–andapoultrynativemicrobiotabacterialspecies–Enterobactercloacae.Wealso evaluatedtheimpactofthepoultrymeatmatrixontheantimicrobialactivityofacopper surface.Ourresultsindicatedthatcoppersurfacesreducedthebacterialloadquickly(<than 4min)whenthemicroorganismswereexposedtopolishedcoppersurfaces.Evenwhen bacteriawereinoculatedoncoppersurfacessoiledwiththeorganicmatrix(washingwater frompoultrycarcasses)andsurvivalratesweresigniﬁcantlyhigher,anantimicrobialeffect wasstillobserved.Survivalratesoftwomicroorganismssimultaneouslyexposedtocopper didnotshowsigniﬁcantdifferences.Wefoundanantimicrobialeffectoverpathogenicand non-pathogenicmicroorganisms.Resultssuggestapotentialroleforcoppersurfacesinthe controlofmicrobiologicalhazardsinthepoultryindustry.

Introduction

Foodborne diseases are an important public health prob-lem resulting in signiﬁcant social and economic burden worldwide.1–3 _Poultry _meat _is _recognized _as _an _important reservoirofpathogenicmicroorganisms,anditisoneofthe food products most frequently associated with foodborne

∗ _{Corresponding}_author_at:_Laboratorio_de_{Microbiología}_y_{Probióticos,}_INTA,_Universidad_de_Chile,_Av._El_Líbano_5524,_Macul,_Santiago,

Chile.

E-mail:areyes@inta.uchile.cl(A.Reyes-Jara).

1 _These_authors_have_contributed_equally_to_this_work.

diseases.4,5 _Illnesses_caused_by_the _consumption_of_poultry andpoultryproductscause annualeconomiclossesofover $2.4billionintheUnitedStates.6_During_poultry_processing, carcassesarehighlysusceptibletomicrobialcontamination. Themajor sourcesofcontamination are thehigh bacterial loads,whichincludefoodborne pathogens,ofthe intestine andcloacalandcontaminationinthefoodprocessingplant

https://doi.org/10.1016/j.bjm.2018.06.008

(2)

environment.7–10 _Moreover, _{environmental} _conditions _such ashighhumidityand bacterialbioﬁlmformationmay con-tributetothepersistenceofpathogenicandnon-pathogenic microorganismsinpoultryprocessingplants.11,12 _Therefore, thepoultryindustrysetsstrictmicrobiologicallimitsand con-trolstoreducetheriskofcontaminationwithpathogensfor increasedshelflife.11,13_Accordingly,_efforts_to_reduce micro-bialcontamination,avoidbioﬁlmformation,andpreventcross contaminationareneeded.

In2008,theEnvironmentProtectionAgencyoftheUnited States (EPA) approved the use of copper alloys as clinical contactsurfacesduetoconﬁrmed antimicrobialproperties. Copperalloyshavesincebeentestedformultipleuses.The antibacterialactivityofcopperdependsontheclosecontact betweenbacteriaandthesurfacereleasingioniccopper.The coppercontactkillingeffectcanbemodiﬁedbydifferent fac-torsincludingtemperature,characteristicsofthecopperalloy, humidity,bacterialspecies,typeofcontactbetweenthe bac-teriaandthesurface,andtheoxidizationstateofthecopper, amongothers.14–16 _Regarding _the_mechanisms_that _explain theantimicrobialeffectofcopper,ithasbeenproposedthat copperions releasedfrom surfaces inducemembrane bac-teriadamagegeneratingalossofmembranepotentialand cytoplasmic content. In addition, reactive oxygen species producedbycopperionsinducegreater damagetocellular structuresandevenDNAdegradation.17,18

Copper surfaceshave been demonstrated to reducethe bacterialloadoffoodbornepathogenssuchasEscherichiacoli O157:H7,19 _Salmonella_enterica,20 _and_Listeria_{monocytogenes.}21 However, studies have only considered direct effects of the microorganisms, without considering the inﬂuence of both the food matrix or the presence of other microor-ganisms. Enterobacter cloacae has been frequently isolated from poultry products, and is considered part of the native microbiota of poultry.22 _The _effect _of _copper sur-faces on this bacterial species, however, has not been evaluated.

Theantimicrobialactivity ofcoppersuggests this metal could be used as a food-processing surface. We hypothe-sizedthatcoppersurfacescouldreducemicrobialloadinthe presenceofthefoodmatrix.Inthisstudy,weevaluatedthe antimicrobialeffect ofcoppersurfaces over twofoodborne pathogensfrequentlyassociatedwithpoultrymeat:S. Enteri-tidisandL.monocytogenes.Ourstudyconsideredtheimpactof thepoultrymeatmatrixandthepresenceofpoultrynative microbiota, represented by E. cloacae, on the antimicrobial activityofcopper.

Materials

and

methods

Bacterialstrains

S. Enteritidis (S410), L. monocytogenes (L452), and E. cloacae (E11) were obtained from our culture collection which were originally isolated from poultry meat. Bacterial identity was conﬁrmed by speciﬁc PCR reactions using primers INVA1-(5-ACAGTGCTCGTTTACGACCTGAAT-3) and INVA2 (5-AGACGACTGGTACTGATCGATAAT-3)23 _and Salm-gyrF (5-GGTGGTTTCCGTAAAAGTA-3) and Salm-gyrR

(5-GAATCGCCTGGTTCTTGC-3)forSalmonellaspp. confirma-tion and primers lmo3F (5-GTCTTGCGCGTTAATCATTT-3) lmo4R(5-ATTTGCTAAAGCGGGAATCT-3)forL.monocytogenes confirmation.E. cloacae identity,representing native micro-biota, was confirmed through biochemical tests: TSI, LIA, MIO,Citrate,PhenylalanineandUrea.Strainswererecovered inovernightculturesinTripticaseSoyAgar(TSA)(OxoidLtd, Basingstoke,Hampshire,England)andinTripticaseSoyBroth (TSB)(OxoidLtd,Basingstoke,Hampshire,England).

Determinationofminimuminhibitoryconcentrationof copper

To characterize strains’ susceptibility to copper, we deter-mined the minimum inhibitory concentration for copper (MIC-Cu),withthesaltcoppersulfateCu2SO4,forthethree

strainsaccordingtothemethodologydescribedinReyes-Jara etal.24

Pre-treatmentofcoppersurfaces

Coppersurfacesusedinthestudywere89%copperand11% tin. Stainlesssteel surfaces were used as controls,and all surfaces were cut as coupons(2.5cm×2.0cm). Previousto exposureassays,thecouponswerepre-treated.Cleansed cop-persurfaces:coppercouponsweretreatedwithethanol70% for 2min, to eliminate microbiological contamination and organicresidues,andrinsedwithdistilledsterilewaterand driedatroomtemperature.Treatedcoppersurfaces:copper couponswere cleanedasdescribed inthe cleansedcopper surface groupandthen placedfor2mininpoultrycarcass rinsewaterwhichhadbeenpreviouslysterilizedbyrepeated freezingandthawing(10times),exposuretoultraviolet radi-ation for 5min (twice), and a ﬁnal ﬁltration step (0.4␮m). After treatment, couponswere dried and placed insterile Petridishesuntilcompletelydry.Allcouponswereusedonly once.

Exposureofbacteriatocoppersurfaces

To determine the antibacterial effect over more than one microorganism simultaneously, S. Enteritidis or L. monocy-togenes were exposed in a mixed culture with E. cloacae, which represented the microbiota present in poultry car-casses. S. Enteritidisand L. monocytogeneswere exposed to copper surfaces (cleansed and treated)asmonocultures or in amixturewith E. cloacae.Anovernight culturefor each bacterium was refreshed with a same sterile medium for adjusting to an OD600nm: 0.05, and grown until it reached

an exponential growth phase (OD600nm: 0.5).Bacteria were

harvested,re-suspendedandadjustedto1×1010_CFU/mL_in

sterilephosphate-bufferedsaline(PBS),andthen40␮Lofthe suspensionweredisposedasadropovercopperand stain-lesssteelcoupons(controlsurface).Totestbacterialmixtures (S.Enteritidis–E.cloacae orL.monocytogenes–E.cloacae),40␮L of each bacterial suspension were mixed and disposed on coupons.InoculatedcouponswereplacedinPetridishesat 25◦Cduring exposuretimesasdescribed byEspíritoSanto etal.25

(3)

0.0 2.0 4.0 6.0 8.0 10.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Log survivorscells(CFU) Time (min)

S. Enterica (R2_0.99); _{S. Enterica+}_E._cloa_{cae (R}2_0.9) S. Enterica control surface.

0.0 2.0 4.0 6.0 8.0 10.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0

Log survivors cells (CFU)

Time (min)

Lm (R2_0.98); _{Lm + E.}_cloa_{cae (R}2_0.99) Lm control surface.

A

B

Fig.1–Inactivationkineticscurvesfor(A)S.EnteritidisS410and(B)ListeriamonocytogenesL452onpolishedcopper

surfaces.ThepathogenswereexposedindividuallyandinamixturewithEnterobactercloacaeE11.Theaverageof3

repetitionsisshown.Controlsurfaceswerestainlesssteelcoupons.Errorbarsdepictstandarderror.

Bacterialcount

Bacteriawererecoveredbyintroducingeachcouponinatube containing3mLofPBSand ten5mmsterileglassesbeads. Thetube wasvigorouslyvortexedfor1min.Then,aliquots weretakentodeterminebacterianumberbyplatecountingin TSAandonXLDagarmedia(OxoidLtd,Basingstoke, Hamp-shire,England)forS.EnteritidisandPalcamagar(OxoidLtd, Basingstoke,Hampshire,England)forL.monocytogenes.All cul-tureswerecarriedoutat37◦C,andexperimentswererepeated atleastthreetimes.

Dataanalysis

Inhibition curves were obtained by plotting total bacterial counts (Log 10 CFU) over time. Since each experimen-tal condition was run in triplicates, three curves were available for each treatment. Inhibition curves were fit to the Log Linear+Shoulder model using the GInaFit v1.7 plugin for Microsoft Excel.26,27 _A _regression _analysis con-firmed the model was the best fit for each dataset (R2_>_0.9).

T(4D)reductionvalues(thetimerequiredtoreduce bac-terialcountsin4logs),calculatedbyGInaFitv1.7,wereused tocompareexperimentalconditions.Statisticalanalyseswere performedusingthesoftwareRStudio.28_First,_a_{Shapiro–Wilk} testwasruntoverifythatdatafollowedanormaldistribution, andthen,athree-wayANOVAwasusedtoanalyzepossible differences amongthe treatments.To determinestatistical signiﬁcance,ap-value≤0.05wasset.

Results

Minimuminhibitoryconcentrationofcopper

TheMIC-Cuwasdeterminedforallthreemicroorganisms iso-lated frompoultrymeat. SalmonellaS410and E. cloacaeE11 showedMIC-Cuvaluesof2mMandL.monocytogenesL452had avalueof4mM.

Table1–ReductiontimeT(4D)forS.Enteritidisand

Listeriamonocytogenesexposedtocoppersurfacesunder

differentexperimentalconditions.

Microorganism Enterobactercloacae T(4D)(min) Polished Treated

S.Enteritidis − 3.8±0.7a ₁₉_±_1.8b

S.Enteritidis + 3.3±0.9a _17.5_±_3.3b

L.monocytogenes − 3.1±0.3a _30.6_±_1.2c

L.monocytogenes + 3.4±1.1a _33.3_±_1.5c

Differentlettersindicatesigniﬁcantdifferencesp-value<0.05.

Antimicrobialactivityofpolishedcoppersurfaces

The antimicrobial effect of copper surfaces over two pathogens wasevaluatedusing cleansed,polishedsurfaces duringdifferenttimecourses.Thesurvivalrateofpathogens exposedtocoppersurfaceswaslowerincleansed,polished coppersurfaceswhencomparedtothecontrol(stainlesssteel coupons)independentfromthepresenceofE.cloacae(Fig.1). Incleansedcoppersurfaces,a4-logreductiontime(T(4D))of SalmonellaandL.monocytogeneswasachievedinapproximately 3min(Table1).

Antimicrobialactivityoftreatedcoppersurfaces

Tomimictheeffectsoffoodresidueoncoppersurfaces,we treatedthesurfaceswithsterilizedcarcassrinsewaterand thenexposedbacteriatoit.Inactivationkineticcurves demon-strated that bacterialload reductiontooklonger intreated surfacesthanoncleansedcoppersurfaces,notonlyin mono-cultures,butalsoinmixturesofthepathogensandE.cloacae (Fig.2).ThetimerequiredtoreachreductionratesofT(4D) in the presenceof organic material (treated surfaces) was signiﬁcantlylongerforbothpathogens(Table1).L. monocyto-genessurvivedgreaterthan30minintreatedcoppersurfaces. WeobservedsigniﬁcantdifferencesinT(4D)reductiontimes between both pathogens under different study conditions (Table 1). L. monocytogenes almost doubled the T(4D) value observedforS.Enteritidisintreatedcoppersurfaces.

(4)

0.0 2.0 4.0 6.0 8.0 10.0 0 10 20 30 40

Time (min)

S. Enterica (R2_0.99); _S._Enterica_{+ E.}_cloa_{cae (R}2_0.9) S.Enterica control surface .

0.0 2.0 4.0 6.0 8.0 10.0 0 10 20 30 40

Time (min)

Lm (R2

0.98); Lm + E. cloacae (R2

0.99) Lm control surface .

A

B

Fig.2–Inactivationkineticscurvesfor(A)S.EnteritidisS410and(B)ListeriamonocytogenesL452ontreatedcoppersurfaces

withpoultrycarcassrinsewater.PathogenswereexposedindividuallyandinamixturewithEnterobactercloacaeE11.The

averageof3repetitionsisrepresentedonthegraph.Errorbarsdepictstandarderror.

AntimicrobialactivityofcoppersurfacesonE.cloacae Weevaluatedtheantimicrobialeffectofpolishedandtreated coppersurfacesonE.cloacae.TheresultsshowedthatE.cloacae inactivationwasfasterthan the othertwomicroorganisms whenexposedtocoppersurfaces(supplementalFig.1).The T(4D)ofE.cloacaeinpolishedsurfaceswaslowerthan1.5min whenexposedindividuallyorinthepresenceofapathogenic bacteria.TheT4(D)increasedto5±0.8minwhenE.cloacaewas exposedtotreatedcoppersurfacesbyitself(supplementalFig. 1).

Discussion

Thehighdemandforpoultrymeatandotherpoultry prod-uctshasledtheindustrytocommercializemillionsoftons eachyear.Asaconsequence,companiesmust control haz-ardsrelatedtotheseproducts,focusingonmicroorganisms thatcancause seriousdiseasesand/oreconomiclosses for theindustry.

The antimicrobial effect of copper surfaces has been previouslystudied.Warnesetal.29 _analyzed_the antimicro-bial effect of a 99.9% copper alloy surface for Salmonella Typhimuriumobservinga7logreductionaftera5-min expo-sure.SimilarresultswereshowedbyEspíritoSantoetal.30_who showeda9logreductionofE.coliafter1minofexposureto surfaceswhichwere99%copper.Inbothcases,bacterialdeath occurred in afew minutes, similarly towhat we observed whenweexposedSalmonellaandListeriatosurfacesthatwere 89% cleansedcopper. Conversely, Wilks et al.21 _exposed _L. monocytogenestohighcoppercontentsurfaces(>90%copper), butthepathogensurvivedforoveranhour.Inanotherstudy,S. entericaandCampylobacterjejunishowedareductionof approx-imately 4log in4h whenexposed tometallic (electrolytic purity)coppersheets.31

In copper exposurestudies, one ofthe most important factorsassociatedwithmicrobialdeathtime,isthemedium selected to suspend the microorganisms under testing.In general, shorter bacterial death times are observed when bacteriaaresuspendedinPBS,anon-nutritivesolution.On

thecontrary,longerdeathtimes(>1h)arereachedwhen bac-teriaare suspendedinculturemedia.Forinstance,Espírito Santoetal.30_added_EDTA_and_{bathocuproine}_disulfonate_to thesuspensionmedia,increasingbacterialdeathtime.Those resultsindicatedthatthepresenceofcopperchelating sub-stancesreducetheantimicrobialactivityofcoppersurfaces. Inourstudy,weobservedlongerbacterialdeathtimeswhen organicmatterfrompoultrycarcasseswereaddedtocopper surfaces before being exposed to bacteria, thus reducing the antimicrobial activity of copper alloys. We decided to use poultry carcassrinse waterover the copper surfaceto mimicconditionsinahypotheticalpoultryprocessingplant. Althoughthereasonforhighersurvivalratesinthepresence oforganicmatterisnotclear,itisthoughtthatcompounds suchasproteinorcarbohydratesmightinteractwithcopper, acting like copper chelating complexes which prevent or delaytheactivityofcopperovercellmembranes.20,32

Interestingly,L.monocytogenesstrainL452showedahigher tolerance tocopper antimicrobialactivity; which isrelated tothehigherMIC-Cuofthis bacteriumincomparisontoS. EnteritidisS410andE.cloacaeE11.30_Also,_when_L. monocyto-geneswasexposedtotreatedcoppersurfaces,asignificantly higherT(4D)wasobservedcomparedtoS.EnteritidisT(4D). Thesedifferencesmayberelatedtothepresenceofspecific Listeriamechanismsrelatedtocopperhomeostasis,suchas: transporters,systems toextra-and intracellular sequestra-tion,enzymaticdetoxificationandcellwall.Whichseemto be more efficient in Gram positive than in Gram negative bacteria.33

Theresultsoftheantimicrobialactivityofcopperoverthe three microorganismsin the study suggest apotential use of copper surfaces forthe control of foodborne pathogens inthepoultryindustry.However,somepotentialdrawbacks needtobeconsidered.Forexample,thetransferenceofcopper fromsurfacestofoodneedstobeaddressed.Faúndezetal.31 reported copper transference valuesunder 2.5mg/100gfor poultrymeatafter50minofexposurewhentestinga99.999% copper alloy(electrolyticcopper). Transferencevalues were similarforpoultrymeat,liver,almond,andseafood.31,34_The alloyusedinthisstudyhadalowerpercentageofcopper,thus wemightexpectlowertransferencelevels.Sincevaluesshown

(5)

byFaúndezetal.31_are_close_to_the_upper_limit_of_the_acceptable dailyintakeofcopperforadults,morestudiesarerequiredto determinethecoppertransferenceofdifferentcopperalloys thatdisplayantimicrobialactivity.

Undersituations where organicmatter waspresent, we identiﬁedlower antimicrobialactivity.This aspectmust be considered when using copper surfaces to control bacte-rial pathogens sincethepresence oforganic matterinthe poultryprocessingplantiscommon,thusreducingthe effec-tivenessofthisantibacterial alternative.Theuse ofcopper surfaces,asanyotherantimicrobialalternativeforthe indus-try,maybeoneofmanytoolstohelpreducethepresenceof pathogensinprocessingplants.Coppersurfacesmayhelpto preventbioﬁlmformation;however,thisstrategyisnotone thatwilleliminatepathogensfromtheenvironment.Theuse ofgoodmanufacturingpracticesandfoodsafetymanagement systemswillremainthe basisforimprovingfood safety in processingplants.

Inconclusion,theantibacterialactivityofcoppersurfaces forbacterialpathogenswasnotaffectedbythepresenceof arepresentativeofthepoultrymicrobiota.Conversely,when bacteriawereexposedtosurfacescontainingorganic mate-rial,survivaltimeswere signiﬁcantlylonger.Theresultsof thisstudysupportnewtrendsthatpromotetheuseof cop-perascontactsurfacesforfoods.Additionally,ourconclusions consider aspectsthat bettersimulate conditionsofdiverse food pathogens in distinct food matrices. We believe that theuseofhighpercentagecopperalloysascontactsurfaces couldhelptoreducethepresenceofpathogensinthepoultry andfoodindustry.Additionalstudiesthatconsiderconditions suchtemperature,thepresenceofotherbacteria,andthose conductedinrealsettingsarenecessary.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ThisworkwassupportedbygrantsfromENLACEENL018/16 andFONDECYT(No.1171575).AuthorsthankMs.EstelaBlanco forediting.

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2018.06.008.

r

e

f

e

r

e

n

c

e

s

1.HaldT,AspinallW,DevleesschauwerB,etal.WorldHealth Organizationestimatesoftherelativecontributionsoffood totheburdenofdiseaseduetoselectedfoodbornehazards: astructuredexpertelicitation.PLOSONE.2016;11(1): e0145839.

2.HavelaarAH,KirkMD,TorgersonPR,etal.WorldHealth Organizationglobalestimatesandregionalcomparisonsof

theburdenoffoodbornediseasein2010.PLoSMed. 2015;12(12):e1001923.

3.NewellDG,KoopmansM,VerhoefL,etal.Food-borne diseases—thechallengesof20yearsagostillpersistwhile newonescontinuetoemerge.IntJFoodMicrobiol.

2010;139:S3–S15,

http://dx.doi.org/10.1016/j.ijfoodmicro.2010.01.021.

4.CDC.SurveillanceforFoodborneDiseaseOutbreaksUnitedStates, 2014:AnnualReport.CDC;2014:24.https://www.cdc.gov/ foodsafety/pdfs/foodborne-outbreaks-annual-report-2014-508.pdf[accessed11.07.17].

5.SilvaF,DominguesFC,NerínC.Trendsinmicrobialcontrol techniquesforpoultryproducts.CritRevFoodSciNutr. 2016:1–19,http://dx.doi.org/10.1080/10408398.2016.1206845. 6.BatzMB,HoffmannS,MorrisJG.RankingtheRisks:The10

Pathogen–FoodCombinationswiththeGreatestBurdenonPublic Health;2011.https://folio.iupui.edu/bitstream/handle/ 10244/1022/72267report.pdf?sequence=1[accessed11.07.17]. 7.JiménezSM,TiburziMC,SalsiMS,PirovaniME,Moguilevsky

MA.Theroleofvisiblefaecalmaterialasavehiclefor genericEscherichiacoli,coliform,andotherenterobacteria contaminatingpoultrycarcassesduringslaughtering.JAppl Microbiol.2003;95(3):451–456,

http://dx.doi.org/10.1046/j.1365-2672.2003.01993.x.

8.GoksoyEO,KirkanS,KokF.Microbiologicalqualityofbroiler carcassesduringprocessingintwoslaughterhousesin Turkey.PoultrySci.2004;83(8):1427–1432,

http://dx.doi.org/10.1093/ps/83.8.1427. 9.SmithDP,CasonJA,BerrangME.Effectoffecal

contaminationandcross-contaminationonnumbersof coliform,Escherichiacoli,Campylobacter,andSalmonellaon immersion-chilledbroilercarcasses.JFoodProtect. 2005;68(7):1340–1345,

http://dx.doi.org/10.4315/0362-028X-68.7.1340.

10.EstebanJI,OportoB,AdurizG,JusteRA,HurtadoA.Asurvey offood-bornepathogensinfree-rangepoultryfarms.IntJ FoodMicrobiol.2008;123(1–2):177–182,

http://dx.doi.org/10.1016/j.ijfoodmicro.2007.12.012.

11.BolderNM.Microbialchallengesofpoultrymeatproduction. World’sPoultrySciJ.2007;63(3):401–411,

http://dx.doi.org/10.1017/S0043933907001535. 12.WangH,YeK,WeiX,CaoJ,XuX,ZhouG.Occurrence,

antimicrobialresistanceandbioﬁlmformationofSalmonella isolatesfromachickenslaughterplantinChina.FoodControl. 2013;33(2):378–384,

http://dx.doi.org/10.1016/j.foodcont.2013.03.030. 13.LoretzM,StephanR,ZweifelC.Antimicrobialactivityof

decontaminationtreatmentsforpoultrycarcasses:a literaturesurvey.FoodControl.2010;21(6):791–804, http://dx.doi.org/10.1016/j.foodcont.2009.11.007.

14.BleichertP,EspíritoSantoC,HanczarukM,MeyerH,GrassG. Inactivationofbacterialandviralbiothreatagentson metalliccoppersurfaces.Biometals.2014;27(6):1179–1189, http://dx.doi.org/10.1007/s10534-014-9781-0.

15.HansM,MathewsS,MücklichF,SoliozM.Physicochemical propertiesofcopperimportantforitsantibacterialactivity anddevelopmentofauniﬁedmodel.Biointerphases. 2016;11(1):18902,http://dx.doi.org/10.1116/1.4935853. 16.VincentM,HartemannP,Engels-DeutschM.Antimicrobial

applicationsofcopper.IntJHygieneEnvironHealth. 2016;219(7):585–591,

http://dx.doi.org/10.1016/j.ijheh.2016.06.003. 17.GrassG,RensingC,SoliozM.Metalliccopperasan

antimicrobialsurface.ApplEnvironMicrobiol. 2011;77(5):1541–1547,

http://dx.doi.org/10.1128/AEM.02766-10.

18.LuoJ,HeinC,MücklichF,SoliozM.Killingofbacteriaby copper,cadmium,andsilversurfacesrevealsrelevant

(6)

physicochemicalparameters.Biointerphases.

2017;12(2):020301,http://dx.doi.org/10.1116/1.4980127. 19.GyawaliR,IbrahimSA,AbuHasfaSH,SmqadriSQ,HaikY.

Antimicrobialactivityofcopperaloneandincombination withlacticacidagainstEscherichiacoliO157:H7inlaboratory mediumandonthesurfaceoflettuceandtomatoes.J Pathogens.2011;2011:650968,

http://dx.doi.org/10.4061/2011/650968.

20.ZhuL,ElguindiJ,RensingC,RavishankarS.Antimicrobial activityofdifferentcopperalloysurfacesagainstcopper resistantandsensitiveSalmonellaenterica.FoodMicrobiol. 2012;30(1):303–310,

http://dx.doi.org/10.1016/j.fm.2011.12.001.

21.WilksSA,MichelsHT,KeevilCW.SurvivalofListeria monocytogenesScottAonmetalsurfaces:implicationsfor cross-contamination.IntJFoodMicrobiol.2006;111(2):93–98, http://dx.doi.org/10.1016/j.ijfoodmicro.2006.04.037. 22.HintonA,IngramKD.Useofoleicacidtoreducethe

populationofthebacterialﬂoraofpoultryskin.JFoodProtect. 2000;63(9):1282–1286,

http://dx.doi.org/10.4315/0362-028X-63.9.1282.

23.ChiuCH,OuJT.RapididentiﬁcationofSalmonellaserovarsin fecesbyspeciﬁcdetectionofvirulencegenes,invAandspvC, byanenrichmentbrothculture-multiplexPCRcombination assay.JClinMicrobiol.1996;34(10):2619–2622.

24.Reyes-JaraA,CorderoN,AguirreJ,TroncosoM,FigueroaG. Antibacterialeffectofcopperonmicroorganismsisolated frombovinemastitis.FrontMicrobiol.2016;7(April):1–10, http://dx.doi.org/10.3389/fmicb.2016.00626.

25.EspíritoSantoC,LamEW,ElowskyCG,etal.Bacterialkilling bydrymetalliccoppersurfaces.ApplEnvironMicrobiol. 2011;77(3):794–802,http://dx.doi.org/10.1128/AEM.01599-10. 26.GeeraerdAH,HerremansCH,VanImpeJF.Structuralmodel

requirementstodescribemicrobialinactivationduringa

mildheattreatment.IntJFoodMicrobiol.2000;59(3):185–209, http://dx.doi.org/10.1016/S0168-1605(00)00362-7.

27.GeeraerdAH,ValdramidisVP,VanImpeJF.GInaFiT,a freewaretooltoassessnon-log-linearmicrobialsurvivor curves.IntJFoodMicrobiol.2005;102(1):95–105.

28.TeamRStudio.IntegratedDevelopmentEnvironmentforR. RStudio;2015.http://www.rstudio.com/.

29.WarnesSL,CavesV,KeevilCW.Mechanismofcoppersurface toxicityinEscherichiacoliO157:H7andSalmonellainvolves immediatemembranedepolarizationfollowedbyslower rateofDNAdestructionwhichdiffersfromthatobservedfor Gram-positivebacteria.EnvironMicrobiol.

2012;14(7):1730–1743,

http://dx.doi.org/10.1111/j.1462-2920.2011.02677.x.

30.EspíritoSantoC,TaudteN,NiesDH,GrassG.Contributionof copperionresistancetosurvivalofEscherichiacolionmetallic coppersurfaces.ApplEnvironMicrobiol.2008;74(4):977–986, http://dx.doi.org/10.1128/AEM.01938-07.

31.FaúndezG,TroncosoM,NavarreteP,FigueroaG. Antimicrobialactivityofcoppersurfacesagainst

suspensionsofSalmonellaentericaandCampylobacterjejuni. BMCMicrobiol.2004;4:19.

32.HansM,ErbeA,MathewsS,ChenY,SoliozM,MücklichF. Roleofcopperoxidesincontactkillingofbacteria.Langmuir. 2013;29(52):16160–16166,http://dx.doi.org/10.1021/la404091z. 33.BondarczukK,Piotrowska-SegetZ.Molecularbasisofactive

copperresistancemechanismsinGram-negativebacteria. CellBiolToxicol.2013;29(6):397–405,

http://dx.doi.org/10.1007/s10565-013-9262-1.

34.OlivaresM,PizarroF,dePabloS,ArayaM,UauyR.Iron,zinc, andcopper:contentsincommonChileanfoodsanddaily intakesinSantiago,Chile.Nutrition.2004;20(2):205–212, http://dx.doi.org/10.1016/j.nut.2003.11.021.