Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes

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Pleasecitethisarticleinpressas:TodorovSD,etal.CombinedeffectofbacteriocinproducedbyLactobacillusplantarum

www.elsevier.es/ram

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MICROBIOLOGÍA

ORIGINAL

ARTICLE

Combined

effect

of

bacteriocin

produced

by

Lactobacillus

plantarum

ST8SH

and

vancomycin,

propolis

or

EDTA

for

controlling

bioﬁlm

development

by

Listeria

monocytogenes

Svetoslav

D. Todorov

∗

,

Otávio

A.L.

de

Paula,

Anderson

C. Camargo,

Danilo

A. Lopes,

Luís

A. Nero

UniversidadeFederaldeVic¸osa,DepartamentodeVeterinária,CampusUFV,36570-900Vic¸osa,MG,Brazil

Received14September2016;accepted4April2017

KEYWORDS Listeria monocytogenes; Lactobacillus plantarum; Bioﬁlm; Bacteriocin; Propolis; EDTA; Vancomycin; Lacticacidbacteria

Abstract The Listeria monocytogenesstrains selected inthe presentstudy exhibited simi-lar behavior inbiofilm formation,independently ofthetested conditions (bacteriocinfrom L. plantarumST8SH, vancomycin,propolis (anaturalantimicrobialproduct)andEDTA (che-latingagent)),individualorinassociations.Theindividualapplicationofvancomycinhadbetter inhibitoryactivitythanthatofpropolisandEDTA;however,theassociationofthepreviously mentionedantimicrobialagents withbacteriocinsresultedinbetterperformance. However, when wecomparedtheeffectsofvancomycin, propolisandEDTA,we couldclearlyobserve that thecombined applicationofbacteriocin andvancomycin was moreeffective thanthe combination ofbacteriocin andpropolis,andbacteriocin andEDTA.Consideringthecurrent needtoreducetheuseofantimicrobialsandchemicalsubstancesinfoodprocessing,propolis canrepresentanalternativetoimprovetheinhibitoryeffectofbacteriocinsagainstL. mono-cytogenesbiofilmformation,basedontheobtainedresults.Ingeneral,highconcentrationsof bacteriocinproducedbyL.plantarumST8SHweremoreeffectiveinbiofilminhibition,and sim-ilarresultswereobservedforvancomycinandpropolis;however,alltestedEDTAconcentrations hadsimilareffectonbiofilmformation.

∗_{Corresponding}_author.

E-mailaddress:slavi310570@abv.bg(S.D.Todorov).

http://dx.doi.org/10.1016/j.ram.2017.04.011

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Pleasecitethisarticleinpressas:TodorovSD,etal.CombinedeffectofbacteriocinproducedbyLactobacillusplantarum PALABRASCLAVE Listeria monocytogenes; Lactobacillus plantarum; Bioﬁlm; Bacteriocina; Própolis; EDTA; Vancomicina; Bacteriaslácticas

EfectocombinadodelabacteriocinaproducidaporLactobacillusplantarumST8SH

ylavancomicina,elprópolisoelEDTAparacontrolareldesarrollodelbioﬁlm

producidoporListeriamonocytogenes

Resumen Las cepas de Listeria monocytogenes seleccionadas en elpresente estudio pre-sentaroncomportamientossimilaresenlaformacióndebiofilms,independientementedelos tratamientosalasquefueronsometidas(bacteriocinadeLactobacillusplantarumST8SH, van-comicina,própolis(produtonaturalantimicrobiano)yEDTA(agentesquelante)),individualoen combinaciones.Laaplicaciónindividualdevancomicinapresentóunamejoractividad inhibito-riafrentealasaplicacionesindividualesdeprópolisydeEDTA;sinembargo,lacombinaciónde estosagentesantimicrobianosconlasbacteriocinasresultóenunmejordesempeño.Seobservó claramentequelaaplicacióncombinadadebacteriocinayvancomicinafuemásefectivapara controlareldesarrollodebiofilmencomparaciónconlacombinacióndelabacteriocinayel própolisodelacombinacióndelabacteriocinayelEDTA.Considerandolanecesidadactual dereducirel usodesustanciasantimicrobianasy químicasenelprocesamiento de alimen-tosysobrelabasedelosresultadosobtenidos,sepuedeafirmarqueelprópolisrepresenta unaalternativa para mejorar elefecto inhibitorio debacteriocinas contra la formación de biofilmdeL.monocytogenes.Engeneral,altasconcentracionesdelabacteriocinaproducidapor L.plantarumST8SHfueronmáseficacesenlainhibicióndelbiofilm,yseobservaronresultados similaresparala vancomicinayelprópolis;sinembargo,todaslasconcentracionesdeEDTA evaluadastuvieronunefectosimilarenlaformacióndebiofilm.

Introduction

Biofilm formation by pathogenic bacteria is considered a seriousprobleminhealthcarefacilitiesandfoodindustry. Microbialcommunitiesdevelop biofilmsfor self-protection againstunfavorableconditions;thisprocessusuallyinvolves morethanasinglespeciesthatexchangeinformationmainly throughquorumsensing. Manyfactorssuchasthe charac-teristics of the adhering surface, temperature, nutrients, expositiontolight,stressingconditions,amongothers25_,_can interferewithbiofilmformationbymicrobialcommunities.

Listeriamonocytogenesis afoodbornepathogenknown

by itsability toform bioﬁlms under different conditions, resultingineconomiclossestofoodindustrydueto contam-ination of end products and serious risks for consumers5_. Studiesonthemechanismsofbioﬁlmformationbydifferent

L.monocytogenesstrainsindicatedthattheluxSgeneplays

animportantroleinthisprocess;thisgeneisresponsiblefor triggeringthe quorumsensing responsein other microbial species,suchasVibrioharveyi2_._L._{monocytogenes}_mutant strainsharboringtheluxSgeneweredescribed ascapable of formingdense bioﬁlms23_,_and _many _other _speciﬁc pro-teinsareinvolved inthisprocess,suchastheautoinducer moleculeAi-214_._In_addition,_L._{monocytogenes}_can_carry_a varietyofproteinsassociatedtoitspathogenicactivity,such asinternalins,listeriolysinsandphospholipases,which are essentialtoitsvirulencecycle9_.

The control of L. monocytogenes contamination and bioﬁlm formation is a current challenge for food indus-tries. Sterilization by high temperatures and ultraviolet light,disinfectantsandgoodhygienicpracticesarestandard procedures often used individually or in association to

clean potential contamination points in the environment, equipmentandutensils.However,thereis acurrentclaim by consumers to reduce the use of chemical products in hygienic procedures by the food industries, in order to decrease the chances of residual contamination in end products. Natural products are being increasingly used in cleaning procedures, representing alternatives to avoid bioﬁlm formation in food industries.In this context, nat-ural products such as propolis and antimicrobial proteins produced by lacticacid bacteria (LAB) can beconsidered alternative strategiestocontrolL.monocytogenesbioﬁlm formation.

Propoliswasalreadydescribed aspossessing antibacte-rialandantifungalproperties,duetothepresenceofactive compoundssuchasgalangin(aflavonoid)8_and_caffeic_acid (a hydroxycinnamic acid)22_. _Propolis _is _a _resinous _mixture collected from honey bees from tree buds, sap flows, or otherbotanicalsources.Thebiologicalpurposeofpropolis hasbeensuggestedtoberelatedtodifferentbiological con-tributions,includingasasealantforunwantedopenspaces inthehive,andasanantibacterialagent.Sincepropolisisa naturalproduct,itscompositionmayvarydependingonthe hive,thegeographicalarea,theseasonandtheplantsthat arepollinatedbybees;however,ingeneralitiscomposed ofplantresinsandvegetablebalsams,waxes,essentialoils, andpollen8,22,24,28_._LAB_are_well_known_due_to_their_ability_to producebacteriocins, ribosomalsynthesizedpeptidesthat killtargetorganismsgenerallybyincreasingthe permeabil-ityofthecytoplasmicmembrane,andbeinguseddirectlyas asemi-purifiedcompound,orindirectlyviatheapplication ofabacteriocin-producingorganism15_._Bacteriocin_activity can beaffected by thepresence of chemical surfactants,

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suchasEDTA19_,_and_present_a_synergetic_effect_when_applied withsomeantibiotics20,26_.

The present study aimed to evaluate alternative pro-cedurestocontrolbioﬁlm formation byL.monocytogenes

strainsbyusingthebacteriocinsproducedbythe bacterio-cinogenicstrainLactobacillus plantarumST8SHassociated toEDTA,vancomycinandpropolis.

Materials

and

methods

Bacterialstrainsandbacteriocinogenicactivity

Bacteriocin producer strain L. plantarum ST8SH was pre-viously isolated,identiﬁed and the produced bacteriocins were characterized27_. _Pure _culture _containing _bacteriocin producerandothermicroorganismsusedinthisstudywere maintained at −80◦C in MRSbroth and BHI (BD, Franklin Lakes,NJ,USA)supplementedwith30%(v/v)glycerol.

Bacteriocin-containingcellfreesupernatant(BCFS)was prepared asrecommended in ourprevious work: L.

plan-tarumST8SHwasgrowninMRSbrothat37◦Cfor24h.BCFS

wasobtainedaftercentrifugation(10,000×g,10min,4◦C) and pH was corrected to 6.0 with 1M NaOH, and tested againstL.monocytogenesScottA,L.monocytogenesATCC 7644,L.monocytogenes211 andL.monocytogenes506to conﬁrmbacteriocinproduction,accordingtoTodorovetal.27 Bacteriocinactivitywasrecordedasmillimetersofinhibition zones,andarbitraryunits(AU)perml27_.

Determinationoftheminimalinhibitory

concentration(MIC)ofvancomycinandpropolis

L.monocytogenes(ScottA,ATCC7644,211and506)were

growninBHI for 24h at 37◦C. Cellswerewashed2 times withsterile salinesolution(0.85%NaCl)andre-suspended in the original volume of trypticase soy broth (TSB, BD).

Each L. monocytogenes culture (10␮l) was inoculated in

100␮l of TSB (BD) on different wells of a flat-bottom 96-well microtiter plate (NUNC, Thermo Scientific). Van-comycin (Sigma---Aldrich, St. Luis, MS, USA) was diluted two-foldinsterileultrapurewater(MilliQ,Millipore, Biller-ica, MA, USA) and 10␮l of each dilution (from1.0␮g/ml to0.015␮g/ml) wasaddedtoL. monocytogenes.As posi-tive controls, aliquotsof 10␮l of each target strain were alsotransferredtothewellsandaddedwith100␮lofTSB (BD)and 10␮lof sterile water. Theprepared plateswere incubatedat37◦Cfor24handgrowthwasrecordedbased onopticaldensitychanges(OD)at550nmonamicro-plate spectrophotometer(BioTekInstruments,Inc.,Winooski,VT, USA). In a separate experiment, the effect of commer-ciallyavailablepropolis(ApiculturaMilliflor,S.P.Ferros,MG, Brazil)asantimicrobialagentagainstthesameL.

monocyto-genesstrainswasdeterminedinasimilarmanner.

Bioﬁlmformation

FourL.monocytogenesstrains(ScottA,ATCC7644,211and

506)weretestedindividuallytoassesstheirabilitytoform bioﬁlms,consideringtheindividualandcombined

interfer-enceofBCFSfromL.plantarumST8SH,EDTA,propolis,and

vancomycin.Bioﬁlmdevelopmentwasassessedasdescribed by Galvão et al.13 _In _summary, _aliquots _of ₂₀_␮l _of _a _L.

monocytogenescultureweretransferredtowellsofa

ﬂat-bottom96-wellmicrotiterplate,andaddedwith20␮lBCFS

fromL.plantarumST8SH(2.25---12800AU/ml),vancomycin

(Sigma---Aldrich,0.015---1.0␮g/ml),propolis(Apicultura Mil-liﬂor,non-dilutedcommercialproductto128timesdiluted) orEDTA(Sigma---Aldrich,0.15---10mM).AssociationsofBCFS

fromL.plantarumST8SHwithvancomycin,propolisorEDTA

werealsotestedconsideringthesameconcentrations.Then, TSB was added in each well to complete the ﬁnal vol-ume of 140␮l. Prepared microtiter plate were incubated at37◦Cfor24h.Afterincubation,culturemediawere dis-carded,andthewellsofthemicrotiterplatewerewashed withphosphate-bufferedsaline(PBS,pH7.2)threetimesto removenon-attachedcells.CellsofL.monocytogeneswere ﬁxedbytheadditionofmethanol(Sigma---Aldrich),andair dried for 10min. In the nextstage, a crystal violet solu-tion(1%,w/v)wasadded,andafter15mintheplateswere washedwithtapwater.Afterdrying,95%ethanolwasadded toeachwell,andtheabsorbancewasmeasuredafter incu-bationfor30minatroomtemperature (=550nm,BioTek Instruments).Foreachpreparedplateappropriatecontrols wereincluded.

Thesameapproachwasconsideredtochecktheeffect of tested antibacterial substances on previously formed biofilms.TestedL.monocytogenesstrainswereinoculated individually in microtiter plates, asdescribed above,and added with TSB to complete the final volume of 140␮l perwell.After biofilmformation (37◦Cfor 24h), cultures werediscardedandBCFSfromL.plantarumST8SH,EDTA, propolis, and vancomycin were added individually or in association (as described above), beingeach well volume completedwithsterilewateruntil140␮l,andincubatedat 37◦Cfor150min.Then,thewellcontentswerediscarded, whenstainingandabsorbance procedureswereconducted asdescribed above.The same blankandpositive controls describedabove wereconsidered in this assay, aswell as theinterpretationofresults.

DetectionofvirulencemarkersandluxSgene

ThefourL.monocytogenesstrainswerealsotestedforthe

presenceofvirulenceandbioﬁlm-relatedgenes.The pres-enceofgenes inlA,inlB, inlC,inlJ,plcA, hlyA,actA,and

iapwascheckedbyPCRasdescribedbyCamargoetal.4_In addition,aPCRprotocoldescribedbyBonsagliaetal.2_was usedtocheckthepresenceoftheluxSgene.

Results

Vancomycin exhibited antimicrobial activity against the

L.monocytogenesstrainsconsideredinthisstudy,andthe

MIC wasdetermined to be around0.075␮g/ml. Based on theseresults,vancomycinwasconsideredinconcentrations ranging from 1.0 to 0.015␮g/ml in the bioﬁlm inhibi-tionassays described above. Moreover, propolisexhibited inhibitoryactivityagainstL.monocytogenestargetstrains evenwhen diluted16times fromthecommercialproduct used.Basedontheobtainedresults,weselectedarangeof dilutions(lowerandhigherthemMIC) ofpropolisin order

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toexplore theanti-Listeria effectof thisnatural product incombinationwithbacteriocinproducedbyL.plantarum

ST8SHandtoobservepossiblesynergisticeffects.

Inourstudywe haveexploredthecombination of bac-teriocinsproducedbyL.plantarumST8SHandvancomycin, propolisandEDTAinordertoavoidtheformationofbioﬁlms (Fig.1a---d)andinordertoeliminatealreadyformedbioﬁlms (Fig.1a---d)byfourL.monocytogenesstrains.Theobtained resultsdemonstratedthat bacteriocins fromL.plantarum

ST8SH,vancomycin,propolisandEDTAinhibitedbiofilm for-mationby L.monocytogenes (Fig.1a---d).In addition,the obtainedresultsshowedasynergisticeffectofbacteriocins and vancomycin, propolis or EDTA (Fig. 1a---d). However, after biofilm formation, the tested substances presented thepromisingabilitytodestroythealreadyformedbiofilm structures,despitebeingtestedinisolationorinassociation (Fig.1a---d).

Nevertheless, when we compared the effects of van-comycin, propolis and EDTA, we could clearly observe that the combined application of bacteriocins and van-comycin was more effective when compared with the combination of bacteriocin and propolis, and bacteriocin and EDTA (Fig. 1a---d). The individual application of van-comycinexhibitsbetterinhibitoryactivitywhencompared toindividual applications of propolis and EDTA; however, theassociation of thepreviously mentioned antimicrobial agents with bacteriocins resulted in better performance (Fig. 1a---d). Moreover, considering the current need to reduce the use of antimicrobial and chemical substances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against

L.monocytogenesbioﬁlmformation,basedontheobtained

results(Fig.1a---d).Ingeneral,highconcentrationsof bacte-riocinsproducedbyL.plantarumST8SHweremoreeffective in inhibiting biofilm formation, and similar results were observedforvancomycinandpropolis;however,EDTA con-centrationsapparentlydidnotinfluencebiofilm formation results(Fig.1a---d).

Regardingthegeneticmarkersofbioﬁlmformationand virulence, all tested L. monocytogenes strains presented expectedampliﬁcationproducts for inlA,inlC,inlJ, hylA,

actA,iap,plcAandluxSgenes.However,nopositiveresults

forampliﬁcationofinlBwererecorded.

Discussion

Antimicrobialactivityofbacteriocins producedbyL.

plan-tarum ST8SH against high number of L. monocytogenes

strains(includingtheselectedstrainsinthepresentstudy) was previously reported27_. _LAB _bacteriocins _with

anti-Listeriaactivityarefrequentlyreportedandusedtocontrol

L.monocytogenesindifferentfoodproducts1,7_._It_is

impor-tanttohighlightthatbacteriocinsproducedbyL.plantarum

ST8SH exhibit strong anti-Listeria activity27_. _In _addition, Todorovetal.27 _reported_that_L._plantarum _ST8SH_carries genesforproductionofbacteriocinswithhighsimilarityto plantaricin423,apediocinPA-1familybacteriocinwith pos-sibledifferencesinamino-acidsintheC-terminalpartofthe molecule.Speciﬁcallyinthisstrain, theN-terminalofthe ‘‘pediocinbox’’ishighlyconserved,whichcanexplainthe wideactivityagainstListeriaspp.Thisanti-Listeriaactivity

wasconﬁrmedinthepresentstudyagainstthetargetstrains

ofL.monocytogenes(52600AU/ml).Basedontheseresults,

BCFS produced by L. plantarum ST8SHwere used in con-centrations rangingfrom12800to25AU/mlinthebioﬁlm inhibitionassaysdescribedabove.

Vancomycin is a glycopeptide antibiotic inhibiting the synthesis of thebacterial cellwall. Vancomycinexhibited antimicrobialactivityagainsttheL.monocytogenesstrains considered in this study, and the MIC was determined to bearound0.075␮g/ml.Basedontheseresults,vancomycin was considered in concentrations ranging from 1.0 to 0.015␮g/mlinthebioﬁlminhibitionassaysdescribedabove.

L.plantarumhasanaturalhighresistancetovancomycin11_,

whichisduetothepresenceofd-Ala---d-Lactateinits pep-tidoglycan ratherthan in the d-Ala---d-Ala dipeptide. Such resistanceisthusintrinsicinmostLABbecausetheantibiotic target isabsent andnotcomparable tothetransmissible, plasmid-encodedvancomycinresistancefoundin enterococ-cal species10_. _Although _{homofermentative} _lactobacilli _are sensitivetovancomycin,manyL.plantarumstrainspossess intrinsicresistancetoit,duetothepresenceof d-alanine---d-alanine ligase-related enzymes11_. _However,_vancomycin _is frequentlyusedforcontrolofL.monocytogenesinfections, as previously mentioned, vancomycin couldbe applied in combination withbacteriocinogenicL.plantarum inorder tocontrolListeriaspp.contamination.Thisprocesscanbe corruptedbyseveralfactorsrelatedtotheproductionand expressionofbacteriocins,suchasavailabilityofnutrients, temperature,wateractivity,orpresenceofspeciﬁcabiotic andbioticfactors12_.

Propolis exhibited inhibitory activity against L.

mono-cytogenestarget strainsevenwhendiluted16 timesfrom

thecommercialproductused.Applicationofpropolisunder this concentration can be considered safe, since higher concentrations were recommendedfor therapeutic use in pharmaceuticalpreparations.Propolisisaresinousmixture collected from honeybees from tree buds, sap flows, or other botanical sources. The biological purposeof propo-lis has never been fully understood; however, it is used as a sealant for unwanted open spaces in the hive, and also asan antibacterial agent.Since propolisis anatural product, itscompositionmay varyfromhive tohive, and can differ depending on the geographical area, the sea-son andplantsthatarepollinated bybees28_._The _analysis ofpropolisdemonstratedthepresenceofapproximately50 constituents,primarilyresinsandvegetablebalsams(50%), waxes (30%), essential oils (10%), and pollen (5%). Dif-ferent substances were already described as constituents of propolis, and many of them, such as polyprenylated benzophenones,viscidone,naphthoquinoneepoxide, differ-ent acids, chrysin, isoflavonoidsand phenethyl ester8,22,24 presentantimicrobialactivity.Propolisalsocontains persis-tent lipophilic acaricides,a natural pesticide that deters mite infestations. However, the properties of propolis dependontheexactsourcesusedbyeachindividualhive. Scientific studies have shown that some types of

propo-lis have in vitro antibacterial activity21 _and _antifungal

activity3_due_to_the_presence_of_active_constituents_including ﬂavonoids, such asgalangin8_, _and_{hydroxycinnamic} _acids, suchascaffeicacid22_.

Inourstudywehaveexploredthecombinationof bacte-riocinsproduced by L. plantarumST8SH andvancomycin,

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Bacteriocin and EDTA a

b

c

Bacteriocin and EDTA

Bacteriocin and vancomycin

Bacteriocin ST8Sh (AU/ml)

Bacteriocin and vancomycin

Bacteriocin and propolis

Bacteriocin ST8Sh (AU/ml)

Bacteriocin and vancomycin Bacteriocin and propolis

Bef ore After Bef ore After Bef ore After OD 550 nm OD 550 nm OD 550 nm 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 1.0x 0.25x 0.06x 0.015xNo propolis 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0,0 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 1.0x 0.25x 0.06x 0.015xNo propolis 1,0 0,8 0,6 0,4 0,2 0,0 1 0,8 0,6 0,4 0,2 0 1 0,8 0,6 0,4 0,2 0 1 0,8 0,6 0,4 0,2 0 1,0 0,8 0,6 0,4 0,2 0,0 1,0 0,8 0,6 0,4 0,2 0,0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 1.0x 0.25x 0.06x0.015x No propolis 1.0x 0.25x 0.06x0.015x No propolis 1.0x 0.25x 0.06x0.015x No propolis 1.0x 0.25x 0.06x 0.015xNo propolis 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0,00 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00 EDT A (mM), v a ncom ycin ( µ g/mL) or propolis (dilution) EDT A (mM), v ancom ycin ( µ g/mL) or propolis (dilution) EDT A (mM), v ancom ycin ( µ g/mL) or propolis (dilution)

Figure1 (a)EffectofbacteriocinST8SH(producedbyLactobacillusplantarumST8SH),EDTA,vancomycinandpropolisonbioﬁlm formation(pre-andpost-treatment)byListeriamonocytogenesATCC7644.(b)EffectofbacteriocinST8SH(producedby

Lactobacil-lusplantarumST8SH),EDTA,vancomycinandpropolisonbioﬁlmformation(pre-andpost-treatment)byListeriamonocytogenes

ScottA.(c)EffectofbacteriocinST8SH(producedbyLactobacillusplantarumST8SH),EDTA,vancomycinandpropolisonbioﬁlm formation(pre-andpost-treatment)by Listeriamonocytogenes506.(d)EffectofbacteriocinST8SH(producedbyLactobacillus

plantarumST8SH),EDTA,vancomycinandpropolisonbioﬁlmformation(pre-andpost-treatment)byListeriamonocytogenes211.

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Pleasecitethisarticleinpressas:TodorovSD,etal.CombinedeffectofbacteriocinproducedbyLactobacillusplantarum 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 0.06 µg/ml0.015 µg/ml No vamcomycin 0.25 µg/ml 1.0 µg/ml 1 0,8 0,6 0,4 0,2 0 12800 A U/ml 6400 A U/ml 3200 A U/ml 1600 A U/ml 800 A U/ml 400 A U/ml 200 A U/ml 100 A U/ml 50 A U/ml 25 A U/ml No bac No EDTA 0.15 mM 0.62 mM 2.5 mM 10 mM 1 0,8 0,6 0,4 0,2 0 1 0,8 0,6 0,4 0,2 0 1,0 0,8 0,6 0,4 0,2 0,0 1,0 0,8 0,6 0,4 0,2 0,0 1,0 0,8 0,6 0,4 0,2 0,0 1.0x 0.25x 0.06x0.015x No propolis 1.0x 0.25x 0.06x0.015x No propolis Bef ore After OD 550 nm

d

Bacteriocin and vancomycin Bacteriocin and propolis

Bacteriocin ST8Sh (AU/ml) EDT

A (mM), v ancom ycin ( µ g/mL) or propolis (dilution) Figure1 (Continued)

propolis and EDTA, in order to avoid the formation of biofilms(Fig.1a---d)andinordertoeliminatealreadyformed biofilms (Fig. 1a---d) by four L. monocytogenes strains. BiofilmsformedbyListeriaspp.strainsareusuallycomplex and stable systems, being hardly destroyed by bacteri-ocinsandweakantimicrobialagents4_._The_obtained_results demonstratedthatbacteriocins fromL.plantarum ST8SH, vancomycin,propolisandEDTAinhibitbiofilmformationby

L. monocytogenes (Fig. 1a---d). In addition, the obtained

results demonstrated a synergistic effect of bacteriocins andvancomycin, propolis andEDTA (Fig. 1a---d).However, after biofilm formation, the tested substances showed a promising ability to destroy the already formed biofilm structures,despitebeing testedin isolation or in associa-tion(Fig.1a---d).Oneofthewellacceptedmodesofaction ofEDTAisrelatedtothedisruptionofthe lipopolysaccha-ridestructure ofthebacterialmembrane:asaresult,the cellmembranebecomesmorepermeabletoother antimi-crobialagentswhenaninhibitorysynergisticeffectcanbe observed.Usually,EDTAisassociatedtodifferent antimicro-bialsubstancestoimprovetheirinhibitoryactivityagainst gram-negativebacteria,suchasPseudomonasaeruginosa17_. A synergistic effect of EDTA was also recorded in asso-ciation with lysozyme, leading to the degradation of the bacterialpeptidoglycanlayer andresultinginthe produc-tionofspheroplasts,inwhichthecellwallhasbeentotally strippedaway18_. _Khan _et _al.16 _investigated _the _combined application of nisin and EDTA against Gram-negative and Gram-positivebacteria.Camargoetal.4 _demonstrated_the synergisticeffectof bacteriocins andEDTAfor controlling biofilmdevelopmentbyL.monocytogenesstrains,using dif-ferentculturemediaandgrowthtemperatures.

The L. monocytogenes strains selected in the present

study exhibited a similar behavior in bioﬁlm formation, independentlyof the testedconditions (bacteriocins from

L. plantarum ST8SH, vancomycin, propolis and EDTA, as

singleagents or in associations,Fig. 1a---d).These results were expected, since the selected strains exhibited sim-ilar susceptibility to bacteriocins produced L. plantarum

ST8SHand vancomycin. However, whenwe compared the effectsofdifferentappliedantimicrobialagents,wecould clearly observethat the combinedapplication of bacteri-ocinsandvancomycinwasmoreeffective whencompared with the combination of bacteriocin and propolis, and bacteriocin and EDTA (Fig. 1a---d); however, this was not surprising, since both applied antimicrobial agents (van-comycinandbacteriocinproducedbyL.plantarumST8SH) areeffective anti-Listeria antimicrobials. Moreover,asan individualantimicrobialagent,vancomycinpresentsbetter inhibitoryactivitywhencomparedtoindividualapplications ofpropolisorEDTA;however,theassociationofpreviously mentioned antimicrobialagentswithbacteriocinsresulted in better performance (Fig. 1a---d). We will need to con-sider thecurrent need toreduce the useof antimicrobial and chemical substances in food processing, and in this respect, propoliscan represent an alternative toimprove the inhibitory effectof bacteriocins against L.

monocyto-genes bioﬁlm formation, based on the obtained results

(Fig.1a---d).Itis importanttohighlight the factthat high concentrations of bacteriocins produced by L. plantarum

ST8SHweremoreeffective onbiofilm inhibition,and sim-ilar results were observed for vancomycin and propolis; however,EDTAconcentrationsapparentlydidnotinfluence theresults ofbiofilm formation (Fig.1a---d).Changetal.6 observedthattheadditionoflowEDTAconcentrationsatthe startingtimeofbiofilmformationresultedinastrongbiofilm inhibitoryeffect,buttheadditionofEDTAafter8hhadno inhibitoryeffectonbiofilmformation.Moreover,thesafety of using propolis as an alternative for controlling biofilm formation byL.monocytogenesshouldbeaddressed, asa singleanti-Listeriaagentorincombinationwithbacteriocin substances(Fig.1a---d);EDTAapplicationcanbeagood alter-native aswell;however, restrictionsshouldbeconsidered sinceitisachemicalhealingagent.

Regardingthegeneticmarkersofbioﬁlm formationand virulence, all tested L. monocytogenes strains presented expectedampliﬁcation productsfor inlA,inlC, inlJ,hylA,

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forampliﬁcationofinlBwererecorded.Previously,Camargo etal.4_reported_the_presence _of_these_genes _in_more_than

80L.monocytogenesisolatesobtainedfromdifferent

eco-logical niches.In anotherstudy,Camargoetal.4 _reported thehighlyfrequentpresenceofthesegenesindifferentL.

monocytogenes strains. These genes play important roles

in virulencemechanisms.Someof them comprise Listeria

spp.pathogenicityisland-1(LIPI-1)thathasbeenrecognized asessentialtointracellularcolonization,encodingproteins suchasLLO,ActA,PlcA,PlcB,MplandPrfA.Moreover,other additionalgenesdetectedaspositiveforallisolates,arealso essentialforfullvirulencepotential9_._The_control_of_highly virulentL.monocytogenesstrainscanbeconsideredakey pointin safetyissuesby thefoodindustry.However,once weobservedasimilarbehaviorofstrainsinbiofilmcontrol assays,andsimilarresultsofvirulenceandbiofilmgenetic markers,noassociation amongbiofilm formationand viru-lencegenescouldbeaddressed.

Conclusions

Similar susceptibility of bioﬁlm formed by investigated

L.monocytogenesstrainstobacteriocinsproducedL.

plan-tarum ST8SH and vancomycin (as single antimicrobial

agents)wereobservedandpresentsbetterinhibitory activ-ity when compared to individual applications of propolis or EDTA. However, a synergetic effect of combination of bacteriocin and propolis, or bacteriocin and EDTA were recorded.Presentresultsareinterestingapproachfor con-trollingbioﬁlm formationby L.monocytogenes.Moreover, basedontheobtainedresults,consideringthecurrentneed toreducetheuseofantimicrobialandchemicalsubstances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against

L.monocytogenesbioﬁlmformation.Inaddition,itis

neces-sarytoaddressthesafetyofusingpropolisasanalternative for controlling bioﬁlm formation byL. monocytogenes,as singleanti-Listeriaagentorincombinationwithbacteriocin substances.

Conﬂict

of

interests

Theauthorsdeclarethattheyhavenoconﬂictofinterests.

Ethical

responsibilities

Protection of human and animal subjects.The authors declarethatnoexperimentswereperformedonhumansor animalsforthisstudy.

Conﬁdentialityofdata.Theauthorsdeclarethatnopatient dataappearinthisarticle.

Right to privacy and informed consent.The authors declarethatnopatientdataappearinthisarticle.

Acknowledgments

This work has been supported by grants from FAPEMIG, CAPESandCNPq.

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