Modulation of drug resistance and biofilm formation of Staphylococcus aureus isolated from the oral cavity of Tunisian children

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Modulation

of

drug

resistance

and

biofilm

formation

of

Staphylococcus

aureus

isolated

from

the

oral

cavity

of

Tunisian

children

Tarek

Zmantar

_,

_Rihab

_Ben

_Slama

_,

_Kais

_Fdhila

_,

_Bochra

_Kouidhi

_,

_Amina

_Bakhrouf

_,

Kamel

Chaieb

a_{FacultyofPharmacy,LaboratoryofAnalysis,TreatmentandValorizationofPollutantsoftheEnvironmentandProducts,} MonastirUniversity,Tunisia

b_{CollegeofAppliedMedicalSciences,MedicalLaboratoryTechnologyDepartment,YanbualBahr,TaibahUniversity,AlMadinahAl} Monawarah,SaudiArabia

c_{CollegeofSciences,BiologyDepartment,YanbualBahr,TaibahUniversity,AlMadinahAlMonawarah,SaudiArabia}

a

r

t

i

c

l

e

i

n

f

o

Received24March2016

Accepted26October2016

Availableonline1December2016

Keywords: Antibacterial Staphylococcusaureus Biofilm Antibiotics Synergetic Lactobacillus

a

b

s

t

r

a

c

t

Objectives: Thisstudyaimstoinvestigatetheantimicrobialandtheanti-biofilmactivitiesof

Lactobacillusplantarumextract(LPE)againstapaneloforalStaphylococcusaureus(n=9)andS. aureusATCC25923.TheinvitroabilityofLPEtomodulatebacterialresistancetotetracycline,

benzalchoniumchloride,andchlorhexidineweretestedalso.

Methods:Theminimuminhibitoryconcentrations(MICs)andtheminimalbactericidal

con-centrations ofLactobacillus plantarum extract, tetracycline,benzalchoniumchloride and

clohrhexidineweredeterminedinabsenceandinpresenceofasub-MICdosesofLPE(1/2

MIC).Inaddition,theLPEpotentialtoinhibitbiofilmformationwasassessedbymicrotiter

plateandatomicforcemicroscopyassays.StatisticalanalysiswasperformedonSPSSv.

17.0softwareusingFriedmantestandWilcoxonsignedrankstest.Thesetestswereusedto

assessinter-groupdifference(p<0.05).

Results:OurresultsrevealedthatLPEexhibitedasignificantantimicrobialandanti-biofilm

activitiesagainstthetestedstrains.AsynergisticeffectofLPEsanddrugsusceptibilitywas

observedwitha2–8-foldreduction.

Conclusion: LPEmaybeconsideredtohaveresistance-modifyingactivity.Amoredetailed

investigationisnecessarytodeterminetheactivecompoundresponsiblefortherapeutic

anddisinfectantmodulation.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan

openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/

by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:chaiebmo@yahoo.fr(K.Chaieb).

http://dx.doi.org/10.1016/j.bjid.2016.10.009

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

28

Introduction

Staphylococci are important causes of infections

associ-ated with various devices. Staphylococcus aureus is one of

the most frequent human pathogen associated to

medi-cal implant.1 _{It has been isolated from parotitis,}2 _gingival

pockets,3 _{periodontitis,}4 _{carious lesions,}5 _{and gingivitis.}6

Orthodonticandother oralappliances mayactasreservoir

ofresistantopportunisticS.aureus.7

S.aureushas the capacity toadhereto various medical

devices and formbiofilm.8 _{Biofilm isassociated to the ica}

geneencodingforpolysaccharideintercellularadhesion(PIA)9

whichleadstoincreasedbacterialdrugresistancecompared

toplanktoniccells.10,11

S.aureusis ableto acquireresistanceto antibioticsand

to antiseptic agents via efflux pumps (TetK, msrA

trans-porters) systems, which export certain tetracyclines and

macrolidesmoleculesoutside thecells.Ontheother hand,

some multidrug resistance (MDR) proteins like NorA and

QacAconferresistancetoawiderangeofstructurally

unre-lated antiseptics.12 _{Inclinical practice,chlorhexidine (CHX)}

andquaternaryammoniumcompounds(QAC)arethemost

frequently used disinfectants to reduce and prevent the

spreadofpathogens.Multidrugresistancepumpshavebeen

recognized as mediators of a number of commonly used

ammoniumcompoundsanddetergents.13_{However,theuseof}

thesedisinfectantsinhospitalsmaycontributetotheriseof

disinfectant-resistantbacteria14,15_{duetoQAC-resistantgenes}

(qacA,qacB,andqacC),whichhavebeenidentifiedinseveral

staphylococcalspecies.16–18

Inthisstudy,theantibacterialactivityofLactobacillus

plan-tarumextract(LPE)wasinvestigated.Inaddition,theabilityof

LPEtomodulatethesusceptibilityofS.aureus,isolatedfrom

theoralcavityofTunisianchildren,totetracycline(TET),

ben-zalchoniumchloride(BC)andclohrhexidine(CHX)wastested.

Secondly,theeffectsofdisinfectantandantibioticassociated

with LPE were tested for biofilm inhibition of S. aureus to

polystyreneandglassusingmicrotiterplateandatomicforce

microscopyassays,respectively.

Material

and

methods

AntibacterialactivityofL.plantarum

L. plantarum strain was isolated from Tunisian traditional

fermented milk (ricotta cheese), identified with a

con-ventional method using Api-50 CHL system (BioMerieux,

Marcy-l’Étoile,France)andbypolymerasechainreaction(PCR)

AntibacterialactivityofLPEwastestedagainstninestrains

(Table 1) isolated from the oralcavity ofTunisian children and S. aureus ATCC 25923 using the broth microdilution

method.20,21_{TheLPEwastestedaloneorincombinationwith}

antibiotics.

Microorganisms

TheS.aureusstrains(n=9) usedinthisstudywere isolated

fromtheoralcavityofTunisianchildrenfromthedentalclinic

ofdentistry(Monastir,CenterofTunisia).

Thecriteriaforinclusionwere:noantibiotictreatmentfour

weekspriortosampling,nouseofmouthrinsesoranyother

preventivemeasurethatmightinvolve exposureto

antimi-crobialagents,andnosystemicdisease.Asterileswabwas

usedforsamplecollectionfromtheoralcavityofeachpatient.

Afterincubation(24hat37◦C),swabswereplatedonblood

agarplatessupplementedwith5%sheepblood(24hat37◦C)

andbacterialidentificationwasachievedusingconventional

methods.

Minimalinhibitoryconcentrationdetermination

Thebrothmicrodilutionmethodwasusedtodeterminethe

minimuminhibitoryconcentration(MIC)ofLPE(5–90%,v/v),

BC(Acrosorganics,USA)(0–1024␮g/mL),CHX(Sigma–Aldrich,

USA) (0–1024␮g/mL) and tetracycline (TET) (0–1024␮g/mL)

accordingtotheClinicalandLaboratoryStandardsInstitute

(2006)guidelines.22

After24hofincubation,bacterialgrowthwasevaluatedby

thepresenceofturbidityandapelletonthewellbottom.MIC

wasdefinedasthelowestconcentrationofthecompoundthat

hadnomacroscopicallyvisiblegrowth.Allexperimentswere

carriedoutthreetimes.

Minimalbactericidalconcentrationdetermination(MBC)

TodeterminetheMBCvalues,10␮Lofeachwellmedium,with

novisiblegrowthwasremovedandinoculatedinMuller

Hin-tonagarplates.MBCwasdefinedasthelowestconcentration

atwhich99%ofthebacteriawerekilled.Eachexperimentwas

repeatedatleasttwice.23

ModulationofS.aureussusceptibilitytoBC,CHXandTET byLPE

To determine the potentialeffect ofLPE to modulate drug

resistanceofS.aureus,MICsofBC,CHXandTET(rangingfrom

0.5to2048␮g/mL)weredeterminedaloneandcombinedwith

b r a z j i n f e c t d i s . 2 0 1 7; 2 1(1) :27–34

29

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

S1 20 60 8 16 4(2) 8(2) 256 512 128(2) 512 8 16 4(2) 8(2) S2 10 60 4 8 4 8 128 512 128 512 4 16 1(4) 16 S3 40 40 32 64 16(2) 64 128 256 32(4) 256 32 64 16(2) 64 S4 20 30 2 16 2 16 64 128 64 64(2) 1 8 1 8(2) S5 20 70 16 32 8(2) 32 128 512 64 512 32 32 8(4) 16(2) S6 50 80 16 64 4(4) 32(2) 64 512 8(8) 256(2) 16 32 16 32 S7 10 30 8 16 4(2) 16 128 128 32(4) 64(2) 8 16 8 16 S8 20 20 8 16 4(2) 16 256 256 32(8) 256 16 32 2(8) 32 S9 20 30 128 256 128 128(2) 128 256 128 128(2) 64 128 32(2) 128 S.aureusATCC25923 20 20 16 32 4(4) 16(2) 32 64 16(2) 16(4) 4 8 2(2) 4(2)

LPE,Lactobacillusplantarumextract;BC,benzalchoniumchloride;CHX,chlorhexidine;TET,tetracycline;MIC,minimalinhibitoryconcentration;MBC,minimalbactericideconcentration. a _{Foldreductionsaregiveninparentheses.}

30

198␮Lofthemediumsupplementedwith2%glucose(w/v)

containingLPE.Plateswereincubatedinaerobicconditions

at37◦Cfor24h.Theanti-biofilmactivity ofTET,CHX,and

BCrangingfrom2to1024␮g/mLweredeterminedagainstS.

aureusstrainswithorwithoutsub-MICoftheLPE(1/2MIC,v/v),

usingthemicrotiterplatesassay.Afterincubation,theplate

waswashedwithphosphatebuffersaline,stainedwith100␮L

of1%(w/v)crystalvioletandincubatedatroomtemperature

for15min.Then,theabsorbanceat590nmwasdetermined

usingamicroplatereader(D.E.E.Dreader,Bio-Radinstrument).

Themean absorbance(OD590nm)ofthe samplewas

deter-mined, and the percentageof inhibitionobtained foreach

concentrationofLPE anddrugswasdeterminedbythe

fol-lowingformula:



(ODgrowthcontrol−ODsample)

ODgrowthcontrol



×100

EvaluationofLPEbiofilminhibitionbyatomicforce microscopy(AFM)

ToevaluatebiofilminhibitionpotencyofLPE,S.aureusATCC

25923wasgrowninTryptonesoybroth(TSB)at37◦Cfor24h

andthendilutedintotheTSBmedium(105_cells/mL)

supple-mentedwith2%glucose(w/v).Foreachconcentrationofthe

testeddrugs(2–1024␮g/mL)andLPE(5–90%),10mLof

bacte-rial suspensionwas incubated ina6-well plate containing

roundglasscoverslipsfor24hat37◦C.Afteradhesion,the

non-adheringbacteriawereremovedbyrinsingthesubstrate

fourtimeswithsterilePBS.Thenthesubstratewasairdried

andobservedusingAFM.AllAFMimageswerethenprocessed

toanalyzethebiofilmformationbehavior.

Statisticalanalysis

Datawere analyzedusingSPSS v.17.0software.The

Fried-mantest,followed bythe Wilcoxonsignedrankstestwere

usedtoassessinter-groupdifferences.Inaddition,statistical

significancewassetatp-value<0.05.

Results

AntimicrobialactivityofLPE

AspresentedinTable1,LPEdemonstratedselective

antimi-crobialproperties.SevenS.aureusstrains(S3,S4,S5,S6,S1,

ModulationofdrugresistancebyLPE

Data presentedinTable1showedthatthepresenceofLPE

at1/2-MIC(v/v)incombinationwithBCresultedina4-fold

reduction of MICs and MBCs of S. aureusATCC 25923 and

oneoralstrain(S6).ConcerningthesynergisticeffectofCHX

and LPE, there was an important diminution of MIC and

MBC ofCHX(2–8-fold potentiation) insixstrains (Table1).

Furthermore, a 2-8-fold reduction of MIC (␮g/mL) of TET

againstsevenstrainswhencombinedwithLPEwasnoticed

(Table1).

BiofilminhibitionbyLPE

BiofilmformationoforalS.aureusstrainswasevaluatedin

96microtiterwellsplatewithLPE(rangingfrom 5%to90%,

v/v).Resultswere expressedasminimumbiofilminhibition

concentration(BIC50andBIC90).AspresentedinTable2,BIC90

ofLPEwasreachedat54%LPEsupplementation,fortwooral

strains(S4andS7).While,BIC50rangedfrom28to77%(v/v)

LPEsupplementation(Table2).

Inaddition,BCshowedvariouseffectsonthedevelopment

of S.aureus biofilmswith BIC50 valuesranging from 12 to

112␮g/mLforthetestedstrains.Moreover,thecombination

ofBCand1/2MICLPEresultedinareductionofBIC50against

eightstrains(Table2).InthesamewayBIC90wasalsoreduced

withthecombinationofBCandLPE.

Concerningthe effectofCHXalone and incombination

withLPE,BIC50valuesrangedfrom34–355␮g/mLforCHXfor

thetestedstrains(Table2)whereasthecombinationofCHX

and1/2MICofLPE%(v/v)exhibitedareductionofBIC50and

BIC90againstallthetestedstrains(Table2).

OurresultsrevealedthatTETmayreduceS.aureus

attach-menttopolystyrene(BIC90reach30␮g/mLforS4).Inaddition,

thecombinationofTETand1/2MICofLPEexhibiteda

reduc-tionofBIC50andBIC90againstninestrains(Table2).

EvaluationofLPEbiofilminhibitionbyAFM

AFMrevealedthatthetestedstrainswerestronglybiofilm

pos-itiveandhadthepotencytoadheretoglasssurface(Fig.1A).

Ontheotherhand,LPEdecreasedsignificantlytheadherence

to glass(Fig.1D). Inaddition, the combination ofLEP with

TET,BCand CHXdecreasedsignificantlythebiofilm

forma-tion (Fig.1B–E).AspresentedinFig.1F,almosttotalbiofilm

inhibitionwasobservedwith16␮g/mLofBCcombinedwith

1/2MICofLPE%(v/v).

b r a z j i n f e c t d i s . 2 0 1 7; 2 1(1) :27–34

31

Strains Inhibitionofbiofilmdevelopment(%)

LPE(%v/v) BC(␮g/mL) BC (␮g/mL)+1/2 MIC(LPE%, v/v) CHX(␮g/mL) CHX (␮g/mL)+1/2 MIC(LPE%, v/v) TET(␮g/mL) TET (␮g/mL)+1/2 MIC(LPE%, v/v)

BIC50 BIC90 BIC50 BIC90 BIC50 BIC90 BIC50 BIC90 BIC50 BIC90 BIC50 BIC90 BIC50 BIC90

S1 32 84 16 84 16 80 50 510 32 62 59 220 10 190 S2 63 81 29 77 15 63 34 90 20 111 10 514 2 126 S3 77 93 30 90 14 60 355 532 126 321 130 321 83 319 S4 28 54 12 64 9 8 60 520 35 132 18 30 16 30 S5 31 92 20 90 8 85 277 556 19 120 29 421 11 216 S6 54 84 30 71 30 67 132 455 60 321 16 120 16 120 S7 31 54 21 16 7 5 66 432 40 509 27 37 11 23 S8 48 75 42 62 16 35 126 300 54 129 31 65 19 44 S9 29 51 112 200 22 14 133 266 65 113 34 73 17 51 S.aureus ATCC25923 38 82 24 1015 12 27 46 64 26 72 16 82 9 46

LEP,Lactobacillusplantarumextract;BIC50,minimumbiofilminhibitionconcentrationthatshowed50%inhibition;BIC90,minimumbiofilminhibitionconcentrationthatshowed90%inhibition;BC, benzalchoniumchloride;CHX,chlorhexidine;TET,tetracycline.

microorganismsonsurfaces.Similarresultshavebeenfound

concerningtheantibacterialactivityofLPEagainstVibriosp.29

andListeria.monocytogenes.30_{Evenwithsub-inhibitory}

concen-trations(1/2 MIC)LPE showed apotentialrole tomodulate

susceptibilityofthetestedS.aureus.SinceBC,TET,andCHX

resistanceismodulatedbyeffluxpumpsystems,wesuggest

thatLPEmaypotentiate,partially,theroleofthedisinfectant

byblocking therespectiveeffluxpump.Theeffluxpumpis

theoneofmajorantibioticresistancemechanismsutilized

bybacterialcellstodevelopresistance.Thus,thisresistance

mechanismeliminatesseveralclassesofantibioticssuchas

fluoroquinolones,TET,andothercompoundssuchasBCand

CHX.

We alsonoted that the MIC of BC (2–4-fold reduction),

CHX(2–8-fold reduction),and TET(2–8-foldreduction) was

reducedwhen themediumwas supplementedwithLPE at

asub-inhibitoryconcentration(Table1).Theresultsshowed

astatistically significant difference between the

antibacte-rialeffectsofdrugswithandwithoutLPE supplementation

(p<0.05).Thus,thisfindingconfirmsourhypothesisthatthe

abilitytomodifysusceptibilitytestedinthisstudy(Table1)is

relatedtocomponentsthathaveapotentialroletoinhibitS.

aureuseffluxpump.

Biofilmformationisanimportantvirulencefactoroforal

bacteria.5_{OurresultsdemonstratethatLPEexhibitedagood}

anti-bioflm activity for all tested strains (Table 2). Similar

resultswerefoundconcerningtheanti-biofilmactivityofLPE

againstL.monoctogenes.30_{Previousstudieshaveshownthat}

the cells in abiofilm were more resistant to antimicrobial

agentscomparedtofree-floatingcells.31,32_{Ontheotherhand,}

theanti-biofilm activity ofdisinfectants andantibiotic (BC,

CHXandTET)topolystyreneandglasswasenhancedwhen

themediumwassupplementedwithLPEatdifferent

percent-ages(Table 2and Fig.1). Thestatisticalanalysis showed a

correlationbetween the percentageinhibitionofbiofilm of

differentdrugsaloneorcombinedwithasub-MICofLPE%

(1/2MIC,v/v)(p<0.05).AsshowninFig.1,AFMdemonstrates

thatS.aureusbiofilmwerestronglyaffectedbythe

supplemen-tationofLPE.Theidentificationofanti-biofilmconstituents

willbeessentialtobeincludedasalternativesinthecontrol

ofbacterialbiofilms.

TheMICsmodifyingabilityofLPEtestedinthisstudymay

berelatedtocomponentsthathaveapotentialroletoinhibit

S.aureuseffluxpump.Furtherstudiesarerequiredtoassess

theircompositionandpotentialclinicalrelevance.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

r

e

f

e

r

e

n

c

e

s

1. HuangH-L,ChangY-Y,LaiM-C,LinC-R,LaiC-H,ShiehT-M. AntibacterialTaN-Agcoatingsontitaniumdentalimplants. SurfCoatTechnol.2010;205:1636–41.

2. SmithA,JacksonM,BaggJ.TheecologyofStaphylococcus speciesintheoralcavity.JMedMicrobiol.2001;50:940–6.

3.ColomboAPV,TelesRP,TorresMC,etal.Subgingival microbiotaofBraziliansubjectswithuntreatedchronic periodontitis.JPeriodontol.2002;73:360–9.

4.SoutoR,AndradeAFBD,UzedaM,ColomboAPV.Prevalence ofnon-oralpathogenicbacteriainsubgingivalbiofilmof subjectswithchronicperiodontitis.BrazJMicrobiol. 2006;37:208–15.

5.KouidhiB,ZmantarT,HentatiH,BakhroufA.Cellsurface hydrophobicity,biofilmformation,adhesivespropertiesand moleculardetectionofadhesinsgenesinStaphylococcusaureus associatedtodentalcaries.MicrobPathog.2010;49:14–22.

6.KoukosG,SakellariD,ArsenakisM,TsalikisL,SliniT, KonstantinidisA.PrevalenceofStaphylococcusaureusand methicillinresistantStaphylococcusaureus(MRSA)intheoral cavity.ArchOralBiol.2015;60:1410–5.

7.MerghniA,NejmaMB,DallelI,etal.Highpotentialof adhesiontobioticandabioticsurfacesbyopportunistic Staphylococcusaureusstrainsisolatedfromorthodontic appliances.MicrobPathog.2016;91:61–7.

8.ProctorRA.Towardanunderstandingofbiomaterial infections:acomplexinterplaybetweenthehostand bacteria.JLabClinMed.2000;135:14–5.

9.MckenneyD,PouliotKL,WangY,etal.Broadlyprotective vaccineforStaphylococcusaureusbasedonanin

vivo-expressedantigen.Science.1999;284:1523–7.

10.CarpentierB,CerfO.Biofilmsandtheirconsequences,with particularreferencetohygieneinthefoodindustry.JAppl Bacteriol.1993;75:499–511.

11.CostertonJW,LewandowskiZ,CaldwellDE,KorberDR, Lappin-ScottHM.Microbialbiofilms.AnnuRevMicrobiol. 1995;49:711–45.

12.MarshallNJ,PiddockLJ.Antibacterialeffluxsystems. Microbiologia.1997;13:285–300.

13.LittlejohnTG,PaulsenIT,GillespieMT,etal.Substrate specificityandenergeticsofantisepticanddisinfectant resistanceinStaphylococcusaureus.FEMSMicrobiolLett. 1992;74:259–65.

14.ReverdyME,BesM,BrunY,FleuretteJ.Evolutionofresistance toantibioticsandantisepticsofhospitalStaphylococcusaureus strainsisolatedfrom1980to1991.PatholBiol(Paris). 1993;41:897–904.

15.RussellAD.Dobiocidesselectforantibioticresistance.J PharmPharmacol.2000;52:227–33.

16.AnthonisenI-L,SundeM,SteinumT,SidhuM,SørumH. OrganizationoftheantisepticresistancegeneqacAand Tn552-related␤-lactamasegenesinmultidrug-resistant Staphylococcushaemolyticusstrainsofanimalandhuman origins.AntimicrobAgentsChemother.2002;46:3606–12.

17.BjorlandJ,SteinumT,KvitleB,WaageS,SundeM,HeirE. Widespreaddistributionofdisinfectantresistancegenes amongstaphylococciofbovineandcaprineorigininNorway. JClinMicrobiol.2005;43:4363–8.

18.ZmantarT,KouidhiB,MiladiH,BakhroufA.Detectionof macrolideanddisinfectantresistancegenesinclinical Staphylococcusaureusandcoagulase-negativestaphylococci. BMCResNotes.2011;4:453.

19.KwonHS,YangEH,YeonSW,KangBH,KimTY.Rapid identificationofprobioticLactobacillusspeciesbymultiplex PCRusingspecies-specificprimersbasedontheregion extendingfrom16SrRNAthrough23SrRNA.FEMSMicrobiol Lett.2004;239:267–75.

20.BagamboulaCF,UyttendaeleM,DebevereJ.Inhibitoryeffects ofspicesandherbstowardsShigellasonneiandS.flexneri. MededRijksunivGentFakLandbouwkdToegepBiolWet. 2001;66:523–30.

21.ErdemogluN,KupeliE,YesiladaE.Anti-inflammatoryand antinociceptiveactivityassessmentofplantsusedasremedy inTurkishfolkmedicine.JEthnopharmacol.2003;89:123–9.

34

22.CLSIPerformancestandardsforantimicrobialsusceptibility testing.ClinicalandLaboratoryStandardsInstitute.Wayne, PA:ClinicalandLaboratoryStandardsInstitute;2006. SupplementM100-S16.

23.MaginaM,DalmarcoE,WisniewskiA,etal.Chemical compositionandantibacterialactivityofessentialoilsof Eugeniaspecies.JNatMed.2009;63:345–50.

24.KouidhiB,ZmantarT,JrahH,etal.Antibacterialand resistance-modifyingactivitiesofthymoquinoneagainst oralpathogens.AnnClinMicrobiolAntimicrob.2011;10: 29.

25.MerrittJH,KadouriDE,O’tooleGA.Growingandanalyzing staticbiofilms.CurrProtocMicrobiol.2005[chapter1]: Unit1B1.

26.EijsinkVG,AxelssonL,DiepDB,HavarsteinLS,HoloH,NesIF. ProductionofclassIIbacteriocinsbylacticacidbacteria;an exampleofbiologicalwarfareandcommunication.Antonie VanLeeuwenhoek.2002;81:639–54.

27.KongS,DavisonAJ.TheroleofinteractionsbetweenO2,H2O2, •_OHe-andO

2-infreeradicaldamagetobiologicalsystems.

ArchBiochemBiophys.1980;204:18–29.

28.GarneauS,MartinNI,VederasJC.Two-peptidebacteriocins producedbylacticacidbacteria.Biochimie.2002;84:577–92.

29.LamariF,SadokK,BakhroufA,GatesoupeF-J.Selectionof lacticacidbacteriaascandidateprobioticsandinvivoteston Artemianauplii.AquacultInt.2014;22:699–709.

30.BenSlamaR,KouidhiB,ZmantarT,ChaiebK,BakhroufA. Anti-listerialandanti-biofilmactivitiesofpotentialprobiotic LactobacillusstrainsisolatedfromTunisiantraditional fermentedfood.JFoodSaf.2013;33:8–16.

31.FrankJF,KoffiRA.Surface-adherentgrowthofListeria monocytogenesisassociatedwithincreasedresistanceto surfactantsanitizersandheat.JFoodProtect.1990;53:550–4.

32.KrysinskiE,BrownL,MarchiselloT.Effectofcleanersand sanitizersonListeriamonocytogenesattachedtoproduct contactsurfaces.JFoodProtect.1992;55:246–51.