Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite

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

Food

Microbiology

Bioﬁlm

formation

by

Staphylococcus

aureus

and

Salmonella

spp.

under

mono

and

dual-species

conditions

and

their

sensitivity

to

cetrimonium

bromide,

peracetic

acid

and

sodium

hypochlorite

Maricarmen

I ˜niguez-Moreno,

Melesio

Gutiérrez-Lomelí,

Pedro

Javier

Guerrero-Medina,

María

Guadalupe

Avila-Novoa

∗

UniversidaddeGuadalajara,CentroUniversitariodelaCiénega,DepartamentodeCienciasMédicasydelaVida,Ocotlán,Jalisco,Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26January2017 Accepted9August2017 Availableonline13October2017 AssociateEditor:SolangeI. Mussatto Keywords: Bioﬁlms Salmonellaspp. Staphylococcusaureus Disinfectants Sensitivity

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b

s

t

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a

c

t

TheaimofthisstudywasevaluatedthebiofilmformationbyStaphylococcusaureus4Eand Salmonellaspp.undermonoanddual-speciesbiofilms,ontostainlesssteel316(SS)and pol-ypropyleneB(PP),andtheirsensitivitytocetrimoniumbromide,peraceticacidandsodium hypochlorite.ThebiofilmsweredevelopedbyimmersionofthesurfacesinTSBby10dat 37◦C.Theresultsshowedthatinmonospeciesbiofilmsthetypeofsurfacenotaffectedthe cellulardensity(p>0.05).However,indual-speciesbiofilmsonPPtheadhesionofSalmonella spp.wasfavored,7.61±0.13Log10CFU/cm2,comparedwithmonospeciesbiofilmsontothe

samesurface,5.91±0.44Log10CFU/cm2(p<0.05).Themonoanddual-speciesbioﬁlmswere

subjectedtodisinfectiontreatments;andthemosteffectivedisinfectantwasperaceticacid (3500ppm),reducingbymorethan5Log10CFU/cm2,whiletheleasteffectivewas

cetrimo-niumbromide.Inaddition, S.aureus4EandSalmonellaspp.weremoreresistant tothe disinfectantsinmonothanindual-speciesbioﬁlms(p<0.05).Therefore,theinterspecies interactionsbetweenS.aureus4EandSalmonellaspp.hadanegativeeffectonthe antimi-crobialresistanceofeachmicroorganism,comparedwiththemonospeciesbioﬁlms.

licenses/by-nc-nd/4.0/).

Introduction

Salmonellaisazoonoticbacteria,whichisoneofthemost sig-niﬁcantentericfoodbornebacterialpathogens1_that_caused

lossesestimatedat3.3billiondollarsperyearintheUnites States.2_Extra-animal_survival_is_an_important_parameter_for

∗ _{Corresponding}_author_at:_Av._Universidad_1115,_Col._Linda_Vista,_47820,_Ocotlán,_Jalisco,_Mexico.

E-mail:avila.novoa@cuci.udg.mx(M.G.Avila-Novoa).

the environmental dissemination ofsalmonellae, with the ability ofthesebacteria tosurvive inthe food chaintobe largely dueto theirabilitytosense and adaptto adiverse rangeofadverseenvironmentalconditions.1_Salmonella_is_able

to adhere and form bioﬁlms on a wide range of surfaces, includingmetal,plasticandrubber,duetotheiraggregative ﬁmbriae andlipopolysaccharides; it isalsoabletoproduce

https://doi.org/10.1016/j.bjm.2017.08.002

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brazilian journal of microbiology49(2018)310–319

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cellulose, leading to bacterial cells being immersed in a hydrophobic network.3 _On _the _other _hand, _{Staphylococcus} aureus is a ubiquitous bacterial species commonly found on the skin and hair, as well asin the noses and throats ofpeople andanimals.4,5 _S._aureus_can_produce_heat-stable

enterotoxins,whichcause13–40%ofpoisoningsincountries liketheUnitedStates,CanadaandJapan.6–8 _The_ability_of_S. aureustodevelopbioﬁlmsisstronglylinkedtotheproduction ofpolysaccharideintracellularadhesion(PIA),andadhesins calledMSCRAMM(microbialsurfacecomponentsrecognizing adhesivemoleculesmatrix)onthesurfaceofthe microorgan-ism,whichhavebeenimplicatedasmajorfactorsinbioﬁlm formationbytheica-independentpathway.9,10

The bioﬁlms are the dominant lifestyle of bacteria in all environments, either natural or man-made (e.g. food processing).11_Bioﬁlms_can_develop_on_a_wide_variety_of

sur-faces, including those used within the food industry. The bioﬁlms have been implicatedin food spoilage, foodborne diseases and damagetoprocessingequipment inthe food industry,including the meat industry, brewing, dairy, ﬁsh-eries, and other industries.12–14 _In _the _food _industry, _the

presenceofmicroorganisms,inorganicandorganicdebrison thesurfacesfavoredbioﬁlmformation.15_Therefore,_without_a

suitablecleaningtreatmentfollowedbytheapplicationof dis-infectants,themicroorganismscancolonizeand persiston foodcontactandnon-foodcontactsurfaces.Thus,thefood might be contaminated bycontact with the contaminated surface.16–18 Fewstudies havefocusedon theevaluationof resistancetodisinfectantsbymultispeciesbioﬁlms;mostof thesestudiesdidnotincludeanassessmentoftheresistance fromeachmicroorganisminthesingle-speciesmodels; there-fore,itisimpossibletojudgewhethertheinterspecieseffect affectstheindividualresistanceofeachspeciesinthe multi-speciescommunities.14,19–21_Even_when_these_communities_in

theenvironmentaremainlymulti-specie;andtheinteractions betweenthemicroorganismscanaffectthebioﬁlmstructure andfunction.22_In_addition,_the_bioﬁlm_formation_by_bacterial

pathogensisimportantduetoitsapotentialrisk,the antimi-crobialresistanceandbacterialpersistenceincrease.14,23,24_For

thisreason,theaimofthisstudywasevaluatethebioﬁlm for-mationofS.aureus4EandSalmonellaspp.undermonoand dual-speciesbioﬁlms,ontostainlesssteel316and polypropyl-eneB,andtheirsensitivitytocetrimoniumbromide,peracetic acidandsodiumhypochlorite.

Materials

and

methods

Bacterialstrains

ThebacterialstrainsusedwereS.aureus4Eisolatedfrom a stainlesssteeltablefromthedairyindustry.Thestrainwas conﬁrmedby23SrDNAaccordingtoStraubetal.25_In_addition,

thepresenceoficaABCDoperon26_and_gen_of_bap_protein_were

determined,27_which_are_important_factors_for_ica_dependent

andindependentbioﬁlmformation,respectively.8_Salmonella

spp. were isolated from the meat industry, and was con-firmedbyPCRusingtheprimerpairST11andST15,specific forSalmonellaspp.28 _As_positive_controls_for_biofilm

forma-tion,S.aureusATCC259236,29_and_Salmonella_Enteritidis_ATCC

1307630 _were _used. _Before _utilization, _the _{microorganisms}

wereincubatedintrypticsoybroth(TSB;Bioxon,LePontde Claix,France)for24hat37◦Ctogiveaﬁnalconcentrationof 108_CFU/mL.

BioﬁlmformationofS.aureusandSalmonellaspp. Contactsurfaces

StainlessSteel(SS,AISI 316,0.7×0.8×0.1cm)and polypro-pyleneBcoupons(PP,0.8×2×0.1cm)werecleanedaccording to the method described by Rossoni and Gaylarde (2000), modiﬁedbyMarquesetal.(2007).Brieﬂy,thesurfaceswere immersedinpureacetone(Fermont) for1h toremoveany debris andgrease,immersedinneutraldetergent (30mL/L, providebyCIP&GROUP)for1h,rinsedwithsteriledistilled water,cleanedwithethanol(70%,Hycel),driedfor2hat60◦C, andsterilizedinautoclave(121◦Cfor15min).

Developmentofmonoanddual-speciesbioﬁlmsand quantiﬁcation

For the biofilm formation, each coupon was individually introduced into a glass test containing 5mL of TSB. The monospecies biofilms were inoculated with 50␮L of cul-turesincubatedat37◦Cfor24hofthecorrespondingstrain and the dual-species biofilms were inoculated with 25␮L of each bacterial suspension, afterthat, the biofilms were incubated at 37◦C for 10 d.32,33 _Finally, _after _the

incuba-tion period,the couponswere removedfrom the glasstest usingsterileforces,andrinsedtwotimesbypipetting2mL ofDulbecco’sphosphatebufferedsaline(PBS;Sigma-Aldrich) inordertoremovethelooselyattachedcells.14_Each_coupon

was introducedindividuallyinto aglass testwith10mLof caseinpeptone(BD,Bioxon,BectonDickinson)(1g/L),andthe biofilmswereremovedbysonication(1min,SonicorModel SC-100thoperating50–60Hz).Serialdilutionsand conventional plating ontrypticsoyagar (TSA;BectonDickinson,Le Pont deClaix,France)formonospeciesbiofilmsandTSAwith lac-tose(10g/L,Sigma–Aldrich)andphenolred(0.025g/L,Hycel) for dual-species biofilms were used to estimate the num-berofmicroorganismsinthebiofilm.ThePetridisheswere incubated at 37◦C for 24h.34 _The _colonies _of_S. _aureus_4E

wereyellowduetolactosefermentationandSalmonellaspp. colonieswerecolorless.

Biocideresistanceassays

Thecouponswiththebioﬁlmswereremovedfromthe cul-ture media described above, and immersed individuallyin 2mLaqueoussolutionsofdisinfectants:i)cetrimonium bro-mide (CB; Sigma–Aldrich)at 100and 200ppm, ii) peracetic acid(PAA;Sigma–Aldrich)at10and3500ppmandiii)sodium hypochlorite (NaClO)at100and 200ppm; the disinfectants werepreparedinsteriledistilledwater.TheCBandPAAwere appliedat25◦Cand50◦C,whileNaClOwasassessedat25◦C and37◦C;thethreedisinfectantswereevaluatedattwotimes ofexposure(10and15min).Aftertheexposureperiod,each couponwastransferredinto1.5mLor3mLofneutralizer solu-tion (SS and PP,respectively): sodium thiosulfate1molL−1 forNaClO,LetheenbrothforCB,andGibsonneutralizer(3g soy lecithin,30mLTween-80, 5gsodium thiosulfate,1g

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l-histidineand10mLPBSperliterofdistilledwaterpH7.2)for PAA.16_After₃₀_min_of_contact_with_the_neutralizer,_the

surviv-ingcellswereestimatedbysonicationandcountingplateas describedabove.34

Epiﬂuorescentmicroscopy

BeforeandafterdisinfectionstreatmentallcouponsofSSand PPwerestainedwith5(6)-carboxyfluoresceindiacetate(CFDA, 10␮g/mL),and driedin alevel II cabinet;the CFDA excess wasrinsedwithSDS.ThecouponswereobservedinaNikon EclipseE400EpifluorescentMicroscopeusing100×oil immer-sionlensandfilterBA515B-2aat450–490nm;atleasteighteen fieldswereobserved.35_With_this_technique,_it_is_possible_to

observethepresenceofmetabolicallyactivelivingcells,the CFDAisalipophilicsubstratethatismoderatelypermeableto mostcellmembranes.35_Once_inside_the_cell,_the_diacetate_is

hydrolyzedbyintracellularnon-speciﬁcesterases,producing carboxy-ﬂuorescein(CF),whichisretainedbylivecellswith anintactplasmamembrane.Hence,theconversiontoCFby thecellsindicatestheintegrityoftheplasmamembraneand theesteraseactivity.35

Statisticalanalysis

Alloftheexperimentswereperformedintriplicate;the statis-ticalanalysiswascarriedoutusingANOVA,andthevariances wereexaminedbyaleastsigniﬁcantdifference(LDS)testin thesoftwareStatgraphicsCenturionXV(Statpoint Technolo-gies,Inc.,Warrenton,USA).

Results

Bioﬁlmformationandquantiﬁcation

The cell density in the biofilm and cellular viability were evaluated using standard plate counting and epifluo-rescent microscopy. The biofilm formation by S. aureus 4E at 10 d of incubation at 37◦C reached 6.26±0.15 and 6.17±0.15Log10CFU/cm2 on PP and SS,

respec-tively (p>0.05). On the other hand the cellular density recovered from Salmonella spp. monospecies bioﬁlms was 6.24±0.56 and 5.91±0.44Log10CFU/cm2 from SS

and PP (p>0.05), respectively. Hence the type of surface had not effect on biofilm development by S. aureus 4E and Salmonella spp. under monospecies conditions and both microorganisms shown the same ability to develop biofilms (p>0.05). In dual-species biofilms, Salmonella spp. was present in approximately 1Log10CFU/cm2 more

that S. aureus 4E (p<0.05) in both surfaces. Onto SS the cellular density enhanced 6.65±0.06Log10CFU/cm2, of

which 5.60±0.28Log10CFU/cm2 was S. aureus 4E and

6.61±0.10Log10CFU/cm2 Salmonella spp. in this case the

S. aureus 4E decreased in comparison with monospecies bioﬁlms(p<0.05)whileSalmonellaspp.maintainedtheir den-sity(p>0.05).Incontrast,onPP7.67±0.10Log10CFU/cm2were

recovered, ofwhich 6.77±0.32Log10CFU/cm2 wasS. aureus

4Eand7.61±0.13Log10CFU/cm2ofSalmonellaspp.;therefore

both microorganisms increased their cellular densities in

comparisonwithmonospeciesbiofilmsinthesamesurface (p<0.05).Hence,indual-speciesbiofilmsonPPboth microor-ganisms werefavoredthan intheothers modelsevaluated (p<0.05). Through epifluorescent microscopy, the biofilms were observed.Inthe micrographsofthe negativecontrols SS(Fig.1A)andPP(Fig.1E),onlythesurfacetopographywas observedwithout bacterialcells,inmono and dual-species biofilms of S. aureus 4E and Salmonella spp. metabolically activecellswereobservedonbothsurfaces(Fig.1).

Disinfectantstest

Effectivenessofperaceticacid

The disinfectants were evaluated on monospecies biofilms of S. aureus 4E develop and Salmonella spp. monospecies biofilms and dual-speciesbiofilms developedin TSB,at 10 dofincubation, becausetheseconditionsguaranteedmore than 5Log10CFU/cm2.UsingthePAAat3500ppmat10and

15minoftimeexposureand25and50◦C,itwasnotpossible torecoverbacterialcellsinanyofthestudiedmodels(Table1). Through epifluorescencemicroscopy,itwasnotpossibleto observe viablecellsinthecoupons,withthethreebiofilms evaluated,subjectedtodisinfectionwithPAAat3500ppmat 25◦Cfor15min(Fig.3A–F);however,inthesurfaceswithS. aureus4Ebiofilms, thepresenceofdeadcellswasobserved (Fig.3AandB).Hence,PAA3500ppmwasabletokillS.aureus 4Ecellsinthebiofilmbutwasnotabletoremovethemfrom theSSandPPsurfaces.

On the other hand, with PAA at 10ppm on S. aureus 4E monospecies bioﬁlms, the reductions ﬂuctuated from 3 to 6.5Log10CFU/cm2 on SS and 1–5Log10CFU/cm2 onto

PP surfaces,depending ofthe temperatureand time expo-sure. In Salmonella spp. monospecies bioﬁlms on PP, this was just reduced 2.5–3.5Log10CFU/cm2. Instead, the PAA

at 10ppm showed the same effect as PAA at 3500ppm, on dual-species biofilms on both surfaces and Salmonella spp.monospeciesbiofilmsonSS(p>0.05).Fromdual-species biofilms treated with PAA at 10ppm, Salmonella spp. were recoveredfrom SS(1.12±0.45Log10CFU/cm2_),_and _S._aureus

4E from PP coupons (1.20±0.35Log10CFU/cm2). By

epiﬂuo-rescentmicroscopy,metabolicallyactivecellswereobserved afterthetreatment,exceptinthebioﬁlmsofSalmonellaspp. ontoSSevenwhen1–1.13Log10CFU/cm2wasrecoveredinthe

standardplatecounting.Theresultsobtaineddemonstrated thatPAAhasabettereffectivenesswhenitisusedonSS sur-faces; atlow concentrations,their effectisfavored athigh temperatures, but their effectivenessdependsthe microor-ganismsinthebioﬁlm.

Effectivenessofsodiumhypochlorite

In the case of NaClO, in all of the conditions evaluated, Salmonellaspp.inmonospeciesbiofilmsontoPPcouponswere abated (Table1);thisagreedwiththeresultsobtainedfrom epifluorescentmicroscopy,inwhichcellswerenotobserved (Fig.2I–L).Indual-speciesbiofilms,theresultsforboth con-centrationsofNaClO (100and200ppm)ineach surfaceare similar,withagreatereffectat200ppminbothsurfaces; how-ever,inalltreatments,theabatementachievedwasmorethan 5Log10CFU/cm2(Table1).

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Fig.1–Epifluorescencephotomicrographsofmonoanddual-speciesbiofilmsofS.aureus4EandSalmonellaspp.Biofilms weredeveloponSS(B–D)andPP(F–H)by10dofincubation.(A)and(E)representnegativecontrolsofSSandPPrespectively, (B)S.aureus4E,(C)Salmonellaspp.,and(D)dual-speciesbiofilmsonSS,(F)S.aureus4E,(G)Salmonellaspp.and(H)

dual-speciesbioﬁlmsontoPP.Thewhitebarscaleindicates10␮m.

Fig.2–Epiﬂuorescencephotomicrographsofmonoanddual-speciesbioﬁlmsofS.aureus4EandSalmonellasppafter disinfection.Thedisinfectiontreatmentswereappliedatdifferenttemperatureandtimeexposure:(A–F)50◦Cfor10min; (G–L)37◦Cfor10min;and(M–R)50◦Cfor10minThewhitebarscaleindicates10␮m.

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 310–319

Table1–ComparisonoftheeffectivenessofdifferenttreatmentsinthethreetypesofbioﬁlmsonSSandPPsurfaces.

Disinfectant Concentration (ppm)

Temperature(◦C) Timeexposure (min)

Log10CFU/cm2_reduce_on_SS_±_DSa _Log10_CFU/cm2_reduce_on_PP_±_DSa

S.aureus4E monospecies biofilms Salmonellaspp. monospecies biofilms Dual-species biofilms S.aureus4E monospecies biofilms Salmonellaspp. monospecies biofilms Dual-species biofilms PAA 10 25 10 3.22±0.97ABb_ac _5.86_±_0.30_ABb _ND _3.10_±_0.13 DEFGHa 3.10±0.69ABa ND

10 50 10 5.06±0.51EFa 5.70±0.41ABCa ND 2.04±0.06ABCb 2.73±0.34BDEc 6.14±0.45ABa

10 25 15 3.33±0.10ABa 5.88±0.23Ab 4.86±0.35Ac 5.24±0.25Jc 2.69±0.37BDEd ND

10 50 15 5.26±0.23EFa 5.88±0.25Ab ND 1.67±0.07ABc 3.43±0.42ACd ND

3500 25 10 ND ND ND ND ND ND

NaClO 100 25 10 1.97±0.09Ca 4.91±0.39BCDEb 5.07±0.49ABb 1.65±0.67ABa ND 6.22±0.50ABc

100 37 10 3.71±0.38ABDa 4.57±0.36DEFb 5.52±0.17Bc 4.59±0.77IJabcd ND 5.78±0.97ABbcd

100 25 15 3.29±0.34ABa 4.99±0.81CDEFb ND 2.33±0.88 ABCDEa ND ND 100 37 15 2.95±1.60ABCa 4.97±0.30 ABCDbc 5.62±0.45Bb 3.38±0.67FGHa ND 6.67±0.17Ad 200 25 10 3.25±0.04ABa 4.40±0.23Fb ND 3.24±0.64EFGHa ND ND

200 37 10 3.39±0.87ABa 4.56±0.17DEFb 5.64±0.18Bc 3.73±0.38GHIa ND ND

200 25 15 5.34±0.33Fa 5.27±0.18ABCa ND 3.90±0.57HIb ND ND 200 37 15 3.87±0.53BDa 5.27±0.51ABCb ND 5.33±0.17Jb ND ND CB 100 25 10 1.92±0.13Ca 2.10±0.20Ia 5.38±0.18ABb 1.45±0.17Ac 2.20±0.12EFa 4.76±0.73CDb 100 50 10 2.69±0.98ACa 4.62±0.17DEFb 5.54±0.45Bc 2.35±0.72 ABCDEa 2.41±0.10DEFa 4.45±0.49Db 100 25 15 2.12±0.24Ca 3.48±1.15GHb ND 2.88±0.95 CDEFGabc 2.50±0.39DEFa 4.32±0.42Dc

100 50 15 3.23±0.18ABa 4.82±0.30CDEFb ND 3.83±0.62GHIab 2.81±0.21BDc 3.94±0.36Db

200 25 10 4.50±0.36DEa 4.52±0.08EFa ND 2.62±0.18

BCDEFb

2.96±0.14ABDb 4.49±0.29Da

200 50 10 3.68±0.70ABDa 2.37±0.05HIb ND 3.76±0.14GHIa 2.00±0.07Fc 5.61±0.65BCd

200 25 15 2.87±0.88ABCab 3.43±0.69Gbc ND 2.18±0.15 ABCDa 2.54±0.24DEFa 4.61±0.84Dc 200 50 15 3.50±0.42ABDa 4.76±0.26Ib ND 2.49±0.57 BCDEFc 3.98±0.25Ca ND

a _Mean_of_three_test_of_population_reduction_after_disinfection_in_Log10_±_standard_deviation. b _Values_in_the_same_column_with_the_same_capital_letter_are_not_{signiﬁcantly}_different_(p_>_0.05). c _Values_in_the_same_row_with_the_same_lowercase_letter_are_not_{signiﬁcantly}_different_(p_>_0.05).

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4.5 3.5 2.5 0.5 1 4 2 3 1.5 0 15 10 10 15 15 15 15 15 15 10 100 100 10 100 100 200 10 10 10 10 10 10 10 10 10 25 25 37 37 25 50 25 25 37 37 25 50 25 50 25 50 25 50

PAA NaCIO CB PAA NaCIO CB

SS PP

Treatment applied

Time exposure (min) Temperature (°C) Concentration (ppm) Disinfectant Surface Log 10 UFC/cm 2

Fig.3–Meanofcellsrecoveredfromdual-speciesbioﬁlmsafterdisinfection.Thebarsrepresentsthemeanofthreetests afterdisinfection:S.aureus4E( )andSalmonellaspp.().Thetreatmentsinwhichonesjustonebarappeared,indicates thatjustonemicroorganismwererecovered.

However,forother models,the NaClO effectivenesswas favored at 200ppm and 15min exposure (Table 1). Never-theless, the effect ofNaClO under these conditions varied betweenmodels,inS.aureus4Emonospeciesbioﬁlmsonboth surfacesSSandPP,onlyonetreatmentwasabletoreducemore than5Log10CFU/cm2,whichwasappliedat25◦Cfor15minOn

PPsurfaces,theNaClOappliedat200ppm,for15minat37◦C, reducedbymorethan5Log10CFU/cm2(Table1);nonetheless,

metabolicallyactivecellswereobservedafterthetreatment (Fig.2H).UsingNaClO,therewasnon-apparentresistanceof eithermicroorganism usedin dual-speciesbiofilms; it was possible to recover both in some cases (Fig. 3). With the NaClOat100ppm,activecellswereobservedinSalmonellaspp. monospeciesbiofilmsonSS,butnotonPP;thesameoccursin dual-speciesbiofilms.

Effectivenessofcetrimoniumbromide

CB in this study was the least effective disinfectant com-pared with PAA and NaClO. In monospecies bioﬁlms, the reductions ranged between 2 and 4Log10CFU/cm2, so the

disinfectant was affected more by the application temper-ature (50◦C, p<0.05) and time exposure (15min; p<0.05), ratherthantheconcentration(100or200ppm;p>0.05). More-over,inmonospeciesbioﬁlms,metabolicallyactivecellswere observed after treatment with CB (Fig. 2M–P). However, in dual-speciesbioﬁlms onPPcoupons,reductionsmorethan 5Log10CFU/cm2wereachievedusingCBat200ppm,at50◦C

(Table1).Finally,indual-speciesbioﬁlmsonSS,all

microor-ganismswereabated,usingCBunderanytemperatureand concentration; in these conditions, no cells were observed by microscopy (Fig. 2Q). From dual-species bioﬁlms on PP couponstreatedwithCB,bothmicroorganismswere recov-ered,althoughSalmonellaspp.predominated(Fig.2R),while fromdual-speciesbioﬁlmsonSS,onlycellsofS.aureus4Ewere recoveredaftertreatment(Fig.3).

Ingeneral,S.aureus4Eseemstobemoreresistantthan Salmonellaspp.totheantimicrobialactionofalltested disin-fectants.TheresistanceofSalmonellaspp.wasdependenton thesurface;Salmonellaspp.monospeciesbiofilmsweremore sensitive toPAAat10ppm onPPthan SS, but showed the sameresistanceonbothsurfacestoNaClOandCB.In addi-tion,S.aureus4EandSalmonellaspp.weremoresensitiveto alldisinfectantsindual-speciesconditions;however,thecell densityofSalmonellaspp.wasfavoredinthisgrowth condi-tion.Hence,interspeciesinteractionshaveasignificanteffect ontheantimicrobialresistanceofeachspecieswiththe disin-fectants.Nevertheless,dual-speciesbiofilmsonSSweremore sensitivetoCBthananyothermodel.

Discussion

Biofilmformationhasbecomeaproblemofgreatimportance in a wide range offood industries; including the brewery, dairy,fish andmeatindustry.16,36,37 _The_biofilms_represents

a persistent source of microbial contamination. These are associatedwithfoodspoilageandfoodbornediseases,which resultinhugeeconomiclossesandthreatenfoodsafety; pre-ventingthisisthedailypriorityintheglobalmarket.38_The

bioﬁlmresistancecanbeduetodifferentfactors,rangingfrom inhibitingthediffusionofthedisinfectingagentby extracel-lularpolymericsubstances(EPS),physiologicalheterogeneity causedbythegradientsofnutrientsand oxygengenerated withinthebioﬁlm.17_Therefore,_the_cleaning_and_disinfection

processesarethemostimportantactivitiestobioﬁlmscontrol inthefoodindustry.16

SeveralstudieshaveevaluatedtheabilityofSalmonellaand S.aureustodevelopbioﬁlms.3,6,14,39–42_In_this_study,_S._aureus_4E

inmonospeciesbioﬁlmsonbothsurfacesshownsimilar cel-lulardensity,thismaybeduetoS.aureushavingthesame ability to adhereto hydrophilic or hydrophobic surfaces.42

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InSalmonellaspp.monospeciesbioﬁlms,nodifferenceswere observedbetweencellulardensitiesonbothtypesofsurface; however,othersstudiesdiffer.TheadhesionofSalmonella Wel-tevredenwasevaluatedonthreesurfacesat48h,andshowed increasedadhesiononplastic,followedbycementand stain-lesssteel(7.53, 6.20and 5.48Log10CFU/cm2, respectively).40

Thedifference observed can be attributedto the fact that thebacterialcountsinourstudywereperformedinmature bioﬁlms, when the cell population maintained stable lev-els, while the cells were reversibly adhered after 48h of incubation.33

Indual-speciesbioﬁlmsSalmonellaspp.was foundtobe 10 timesmore oftenthan S. aureus4E,this can berelated tothe with the high rate of growth ofSalmonella; and the predominantmicroorganismsinthebioﬁlmsarethosewith ahighergrowth rate, althoughthesecannevercompletely excludethemicroorganismswithslowgrowth.43_Several

stud-ieshavereportedthatbioﬁlm productionofSalmonellaspp. may be promoted by the presence of other bacteria.4,44–46

However, Knowles et al. (2005) evaluated the biofilm for-mation by these microorganisms on SS at 25◦C for 12 d; the resultsshowed thatbiofilm was constitutedmainly by S. aureus (∼99%, 7–8logCFU/section), while Salmonella was present in lower amounts (6logCFU/section). In line with this,Gkana etal. (2017)evaluatedthe biofilmformation in monoand dual-speciesconditions SalmonellaTyphimurium and S. aureusand found that in dual-species biofilms the cells of each microorganism deceased in comparison with monospeciesbiofilms(p<0.05). Thedifferentresultscanbe relatedwiththefactofthecelldensityofeach microorgan-ismcouldbedependentonstrainabilitytobiofilmformingin co-culture48_;_in_addition_it_has_been_explained_that_Salmonella

requireoxygentoproducebioﬁlmincontrastwithS.aureus.49

In addition, the ability of six environmental strains of S. aureus to develop mono and dual-species bioﬁlms with L. monocytogenesisolatedfromrabbitoutbreakwereevaluated, and the results showed that the effect ofL. monocytogenes onthepopulationofS.aureuswasstrain-dependent.TheS. aureuspopulationincreased,decreasedor wasnotaffected inthepresenceofL.monocytogenesindual-speciesin com-parisonwithmonospeciesbioﬁlms,whiletheL.monocytogenes populationwasnotaffected.50_These_data_show_that

interac-tionsbetweenthemicroorganismscanstimulateordiminish thecolonizationofabioticsurfaces;nonetheless,the interac-tionsaremainlyinfluencedbytheenvironmentalconditions. Inanotherstudy,differentinteractionswerefoundtooccur betweenpathogenic microorganisms, inthe biofilm forma-tion by Listeria monocytogenes and Salmonella under mono and dual-species conditions on SS;both species exhibited similarbiofilmcounts(105_CFU/cm2_),_independent_of_culture

conditions.14

Additionally, theeffect ofthreedisinfectants commonly usedinthefoodindustryonmonoanddual-speciesbioﬁlms ofS. aureus 4Eand Salmonella spp. were evaluated, due to themajorityofstudiesfocusingonlyonmonospecies mod-elsanddual-speciesbioﬁlmsnotoftenbeingstudied.Inthe currentinvestigation,PAAwasthemosteffectivedisinfectant; thisdisinfectantcausedalinearlossofcellviability, demon-stratingthathigherresistancecouldnotbeduetolimitations ofpenetrationbytheEPS.51_The_PAA_at₁₀_ppm_on_S._aureus

4Emonospeciesbioﬁlmsreducedmorethan2Log10CFU/cm2

onSScomparedwithPP,whileSalmonellaspp.monospecies bioﬁlmsweremoresensitiveonPPthanonSS.Inthisline, itwasreportedthatSalmonellabioﬁlmsweremoresensitive todisinfectantswhentheyweredevelopedonstainlesssteel comparedtothoseformedonplastic.40_Due_to_the_stainless

steelbeinghydrophilic,whilePPisahydrophobicsurface,for thesereasons,thePAAinaqueoussolutionhaslesscontact withthesurfaceofPPduetothesurfacetensionofthe solu-tion.

Regarding thetreatmentswithsodiumhypochlorite, the concentration (100and200ppm)had asignificant effectin reducingthebacterialcellsonbothtypesofsurface.However, RossoniandGaylarde(2000)foundthattheconcentrationof sodium hypochlorite(100and 200ppm) didnothavea sig-nificanteffectonS.aureuscelladherencetostainlesssteel, after10minofexposuretothedisinfectantatroom temper-ature.Thedifferencebetweentheseresultscouldbedueto theconcentrationhavinganeffectonmaturebiofilms com-pared with cells in the adhesion process. Mature biofilms are moreresistantduetovariousfactorssuchashigh con-centration EPS. The EPS limits the spread of disinfectant intothebiofilm,withpotentialinteractionsexistingbetween antimicrobialsandbiofilmcomponents;therefore,thestate of slow growth of microorganisms in biofilms increases its resistance to disinfectants.17,52,53 _In _the _Salmonella _spp.

monospecies bioﬁlms treated with an aqueoussolution of sodium hypochlorite, atotal abatementin bioﬁlmsformed on PPsurfaceswas achieved,whereas those formedon SS wererecoveredtomorethan1Log10CFU/cm2,withsixofthe

eighttreatmentsapplied.Josephetal.(2001)treatedSalmonella Weltevredenmonospecies bioﬁlmsonplasticsurfaceswith hypochloriteat200ppmofCl2,for10and15min,and

recov-ered4and3Log10CFU/cm2,respectively,whileafter10min,

3 Log10 CFU/cm2 was recovered from SS surfaces, but no

microorganismwasdetectedafter15minThedifferencecould beattributedtoJosephetal.(2001)havinganinitialpopulation of2Log10CFU/cm2moreonPPthanonSS,whileinthisstudy

obtainednodifferencesbetweenthecountsofSalmonellaspp. onSSandPP.

Instead,theCBhasalowereffectthanPAAonS.aureus 4E monospecies biofilms; in Salmonella spp. monospecies biofilms, theeffectwas smallerthanPAA,evenforsodium hypochloriteonbothsurfaces,whileindual-speciesbiofilms, theCBwaslesseffectivewhenthebiofilmwasdevelopedon PP.TheresistanceofS.aureustoquaternaryammonium com-pound (QAC)is relatedto theQAC effluxsystem, whichis responsible fortheresistanceofQACandcationicbiocides inplanktoniccells.54_Through_this_system,_bacterial_cells_are

able tofree themselves of toxic molecules, allowing them to survive in the presence of these substances.51 _In

addi-tion,theroleofthethree-dimensionalstructureofS.aureus bioﬁlms inQACresistance islimitedand the physiological changes in the bioﬁlm cells are morehighly implicatedin their resistance.55 _In_the _case_of_Salmonella,

Mangalappalli-Illathuetal.56_reported_that_resistance_of_the_sessile_cells_of S.entericaserotypeEnteritidisATCC4931onglasssurfacesto benzalkoniumchloridewasduetothephenotypicadaptation todevelopbioﬁlm.AnotherimportantobservationisQACon S.epidermidis,whichcausedmembranepermeabilizationthat

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317

startedontheperipheryofcellclusters,thenmigratedsteadily inward;therefore,penetrationtothecenterofthebioﬁlmis 60timeslongerthanthetimeestimatedfordiffusiveaccessin theabsenceofsorption.57_Moreover,_the_positive_charge_and

hydrophobicnatureofQACexplainthedelayedpenetration.53

Besides,inPseudomonasaeruginosabioﬁlms,thebacterial resis-tanceincreasedwiththeC-chainlengthofQAC55_;_these_data

canbe relatedtothe low effectivenessofCBinour study, insomuchastheCBhasaC-chainof16C.58

Inthepresentstudy,biofilmmonospecies,treatedwithPAA at10ppm,hadgreaterreductionsonSSthanonPPcouponsin mostinthecases(p<0.05).Duetotheeffectivenessofthe dis-infectantcanbeinginfluencedbytheadhesionsurface;and thisresistanceofbiofilmscanonlybeobservedwithcertain disinfectants.41,59_Therefore,_the_efficiency_of_{disinfectants}_to

thebiofilmsmayvaryconsiderablydependingondisinfectant characteristics,thenatureofthesurfacewherethebiofilm is developed, the type of microorganisms in the biofilm, and other factors suchastemperature and time exposure. Thedual-species biofilms were more sensitive to disinfec-tants than monospecies biofilms. Conversely, the majority ofstudies havedemonstratedthatmulti-species are gener-allyequalormoreresistanttodisinfectionthanmonospecies biofilms.20,48,60–62 _{Unfortunately,}_the_resistance_mechanisms

involvedremainunclear.51_However,_some_possible_ways_are:

chemicalinteractionsbetweenthemicroorganismsproduce amoreviscousmatrixandthespatialubicationof microor-ganismswithinthebioﬁlm.62,63Theinteractionbetweenthe studiedstrainshasanegativeeffectontheirresistance;these dataprovideanewperspectiveonthebehaviorindual-species bioﬁlms.

Itisactuallydifficult touse epifluorescencemicroscopy as a counting technique for cells in biofilms, because the bacterialcells ina biofilm usuallydevelop inlayers. How-ever,itispossibletoobservethepresenceofsurvivingcells afterdecontaminationtreatments,eveninthosetreatments whereit wasnotpossibletoachieveexpressioninthe cul-turemedium,eitherbythedetectionlimitofthetechniqueor bythemetabolicstateofthebacteria.Ontheotherhand,in thosetreatmentswherebacterialcellswererecoveredthrough countingtechnique,butnotwereobservedbymicroscopy,this mightberelatedtothelowpresenceofbacterialcellsperunit area.Additionally,theaerobicplatecountiscommonlyused todetectviablecells;therefore,itdoesnotallowthe quan-tificationofsub-lethallydamagedcellsornon-cultivablebut metabolicallyactivecells.60,64–66_For_these,_it_is_important_to

usemorethanonetechniqueforbioﬁlmstudyasthe microor-ganismsinthebioﬁlmsareindifferentstates:metabolically active,metabolicallyinactiveanddeadcells.67,68_The

discrep-ancybetweenourresultsandthoseofotherstudies14,31,40,47

canbeattributedtoseveralfactors,includingexperimental conditionsandsurfaceproperties.Inthecaseofsurface prop-erties,thetopographyaffectsbacterialadhesion,particularly ifthesurfacehaschannelsthatprotectbacteriafromtheeffect ofdisinfectants.15Anotherreasonthatexplainsthe discrep-ancyisthattheeffectoftheadhesionsurfaceontheresistance ofbioﬁlmsmightdisinfectant-dependent.

Inconclusion,theresistancetoantimicrobialcompounds of microorganisms such as S. aureus 4E and Salmonella spp. in bioﬁlms depends of diverse factors such as type

of surface and interspecies interactions. In this study, the surface did not affect the cellular density in monospecies biofilms,butPPsurfacesfavoreddedual-speciesbiofilms.In dual-speciesbiofilms,Salmonellaspp.predominated,however both microorganisms were more sensitive to disinfectants thaninmonospeciesbiofilms.ThePAAwasthemost effec-tivedisinfectant,whiletheleasteffectivewasCB;however, thedisinfectant effectivenesswasdependentmainlyofthe microorganismsinthebiofilmandthesurfaceonwhichthe biofilmwasdeveloped.

Conﬂict

of

interest

Noconﬂictofinterestdeclared.

Acknowledgments

WethanktheNational CouncilonScienceand Technology ofMexicoforthescholarshipgrantedtoMaricarmenI ˜niguez Moreno.

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