ANTI-LISTERIAL AND ANTI-BIOFILM ACTIVITIES OF POTENTIALPROBIOTIC LACTOBACILLUS STRAINS ISOLATED FROMTUNISIAN TRADITIONAL FERMENTED FOODRIHAB BEN SLAMA1,4*, BOCHRA KOUIDHI1,3*, TAREK ZMANTAR1, KAMEL CHAIEB1,2 and AMINA BAKHROUF1

1Laboratoire d’Analyses, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Faculté de Pharmacie, Monastir, Tunisie2College of Arts and Sciences, Yanbu, Taibah University, Al-Madinah Al-Munawarah, Kingdom of Saudi Arabia3College of Applied Medical Sciences, Yanbu, Taibah University, Al-Madinah Al-Munawarah, Kingdom of Saudi Arabia

4Corresponding author.TEL: +21673461000;FAX: +21673461830;EMAIL: rbs_23@yahoo.fr

*Rihab BEN SLAMA and Bochra KOUIDHIcontributed equally in this manuscript.

Received for Publication August 21, 2012Accepted for Publication November 29, 2012

doi: 10.1111/jfs.12017

ABSTRACT

Three Lactobacilli were isolated from traditional Tunisian fermented food andthen characterized for its ability to inhibit Listeria monocytogenes growth. Antago-nistic effect of Lactobacillus plantarum on four L. monocytogenes strains was testedin soft artisanal cheese. Anti-biofilm activity of Lactobacillus extracts was alsotested.

Our results demonstrate that the selected lactic acid bacteria (LAB) extractexhibited a good antibacterial effect against L. monocytogenes ATCC 19115 withlow MICs values: 8.33 � 2.1, 20 � 1.2 and 23.33 � 1, respectively. L. plantarumalso exhibited a stronger inhibitory effect against L. monocytogenes when grown incheese. Moreover, a potential anti-biofilm effect of the three LAB extracts withBIC50 values ranging from 5% to 15% for L. monocytogenes ATCC 19115 was dem-onstrated. Although LAB extracts were able to eradicate significantly a preformedL. monocytogenes biofilm (P < 0.05). Growth inhibition of preformed biofilm wasmore difficult to achieve. LAB could be used as a bioprotective culture in cheeseripening to prevent Listeria growth.

PRACTICAL APPLICATIONS

A wide range of lactic acid bacteria (LAB) produce bacteriocins, which wereessentially active against the food-borne pathogen Listeria monocytogenes. 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazoliumhydroxide (XTT) reduction method was used to assess the anti-biofilm and anti-listerial activity of LAB extract. XTT reduction assay is dependent on the micro-bial respiratory activity.

Since XTT is used for quantification of the microbial respiratory activity, itwas used to quantify viable mature biofilm and to compare LAB activity againstplanktonic and biofilm cells.

INTRODUCTION

Listeria monocytogenes is a gram-positive bacterium thatinvades the intestinal epithelium, causing a potentiallyfatal disease. It is recognized as one of the most importantfood-borne pathogens (Rasooli et al. 2006) occurring in awide range of foods such as milk, meat products, fish andseafood. L. monocytogenes infection has been estimated tocause 12,500 illnesses annually in the United States (Mead

et al. 1999). The bacterium is capable of growing at refri-geration temperatures and ready-to-eat products (Gandhiand Chikindas 2007). It can resist several environmentalstresses, including pH, temperature and osmolarity (Coleet al. 1990). L. monocytogenes, can be transmitted frommother to fetus/neonates transplacentally or during passagethrough the birth canal (Delgado 2008). Thus, it can com-monly be isolated from production lines, packaging andstorage facilities (Arihara et al. 1993; Castellano et al. 2004).

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Several studies have demonstrated that L. monocytogenes isable to form biofilms (Bonaventura et al. 2008) and produceextracellular polymeric substances (Lindsay and von Holy2006) on various food contacts (Jeong and Frank 1994).

Since biofilms are more resistant to antimicrobial agents(Stewart et al. 2001), the capability of L. monocytogenes toform biofilms on different surfaces poses a major concernfor the food industry (Gandhi and Chikindas 2007; Adriaoet al. 2008). The adhesion of L. monocytogenes to a variety ofsurfaces such as stainless steel, plastic and rubber, stainlesssteel and glass (Mafu et al. 1990) led to the decrease of itssusceptibility to disinfectants (Ronner and Wong 1993).

A wide range of lactic acid bacteria (LAB) produce anti-microbial peptides (bacteriocins) that are mainly activeagainst various pathogen. The best-studied probiotic strainswere Lactobacillus acidophilus, Lactobacillus fermentum,Lactobacillus plantarum, Lactobacillus brevis (De Vrieset al. 2006; Galdeano et al. 2007; Ranadheera et al. 2010).Biopreservation systems, such as LAB cultures and/ortheir bacteriocins, have received increasing attention forthe control of pathogenic and spoilage microorganisms(Vignolo et al. 1998; Bredholt et al. 1999; Zhang andMustapha 1999; Mattila et al. 2003).

A different approach such as the incorporation of viableprotective starter cultures to suppress growth of foodbornepathogens was proposed (Vogel et al. 1993). Differentstudies have shown that LAB and their metabolites reduceL. monocytogenes growth on seafood and meat products(Tahiri et al. 2009; Concha-Meyer et al. 2010) and contrib-ute to the safety of fermented foods (Ross et al. 2002). Theaddition of bacteriocins to cheese, meat and ready-to-eatproducts increase the food safety of such products (Eijsinket al. 2002; Galvez et al. 2008). The ability of a bacteriocin-producing L. plantarum strain to exert an antimicrobialeffect against L. monocytogenes was reported (Eijsink et al.2002; Kouakou et al. 2008). LAB bacteriocins have success-fully been used to control L. monocytogenes in variousfood matrices (Galvez et al. 2008). In addition, it has beenreported that the oral administration of L. delbrueckii var.bulgaricus UFVH2b20 protects germ-free mice againstinfection with L. monocytogenes (dos Santos et al. 2011).

The purpose of the present study was to investigate, forthe first time, the anti-listerial and anti-biofilm effectivenessof Tunisian bacteriocin-producing LAB (L. plantarum) and

the use of appropriate isolates as associated ripening culturefor artisanal cheese.

MATERIALS AND METHODS

Bacterial Strains, Media andCulture Conditions

Preparation of L. monocytogenes Cultures. FourL. monocytogenes strains were used in this study includingL. monocytogenes ATCC 19115 (S1) and three strains (S2, S3and S4) isolated from meat as previously published (Miladiet al. 2008).

Each strain was individually grown in tryptic soy brothwith 0.6% yeast extract (TSB-YE) (Pronadisa, Spain) at 37Cfor 20 h. The cultures were centrifuged at 8,000 rpm for10 min and resuspended in 0.1% peptone. The opticaldensity of each strain was adjusted to 0.5 (108 cfu/ml) at600 nm using a spectrophotometer (Spectro UV-Vis; ModelUVD-2960, Labomed, Inc., CA).

Characterisation of LAB. Three Lactobacillus strains iso-lated from Tunisian traditional fermented food (Table 1): S1from fermented olive; S2 from conserved fish and S3 fromricotta cheese (traditional fermented food made from milk).Theses strains were selected based on their antibacterialinhibitory activity observed using the agar diffusion tests(Bagamboula et al. 2001; Erdemoglu et al. 2003).

Biochemical identification was performed using Api-50CHL system (BioMerieux, Marcy-l’Étoile, France) accord-ing to the manufacturer’s recommendations. The resultswere observed with a microbiological mini-Api automate(bioMerieux). In addition, their enzymatic profile wascharacterized using the Api-ZYM systems (bioMerieux).Molecular confirmation of the selected strains was per-formed by PCR technique using L. plantarum-specificprimers: forward IDL04F: 5′-AGGGTGAAGTCGTAACAAGTAGCC-3′ and reverse IDL62R 5′-CTAGTGGTAACAGTTGATTAAAACTGC-3′, giving a product size about 428 bpas described by Kwon et al. (2004).

Chromosomal DNA was extracted using a Wizard Genomicpurification kit (Promega, Charbonnières-les-Bains, France)according to the manufacturer’s recommendation. PCR wasperformed in a 25 mL reaction volume containing: 20 ng

TABLE 1. ORIGIN, PHENOTYPIC ANDMOLECULAR IDENTIFICATION OF POTENTIALLACTIC ACID BACTERIA STRAINS

API 50 CHL

Molecularconfirmation

Percentage ofidentification

PhenotypiccharacterizationStrains Origin

L170 (Lp1) Fermented olive 98.7 L. plantarum L. plantarumL805 (Lp2) Conserved fish 96.7 L. plantarum L. plantarumL899 (Lp3) Ricotta cheese 99.5 L. plantarum L. plantarum

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of extracted DNA, 5 mL green Go Taq buffer (5¥), 200 mMof each deoxynucleoside triphosphates (dNTP), 0.5 mM ofeach forward and reverse primer, 1 U of GO Taq DNA poly-merase (Promega). PCR products (5 mL) were analyzedon 1% agarose gel in 1X Tris-borate-EDTA buffer (TBE)pH 8.3, and visualized under ultraviolet transillumina-tion, photographed Syngene apparatus (Bio-Rad, Marnes-la-Coquette, France), and their sizes were determined with100 bp molecular size marker.

Lactobacillus Extract Preparation. Lactobacillus strainswere first cultivated in MRS broth (Difco, Lawrence, KS) for24 h at 37C. Then the culture was centrifuged (12,000 rpmfor 10 min at 4C). The supernatant was collected, sterilizedthrough a 0.22 mm pore size filter and frozen at -20C forlater use.

Minimal InhibitoryConcentration Determination

The broth microdilution method was used to determinethe minimum inhibitory concentration (MIC) of the LABextract according to the Clinical and Laboratory StandardsInstitute guidelines (CLSI 2006).

Sterile LAB extract were properly prepared and trans-ferred to each microplate well containing Muller Hintonbroth (MH) in order to obtain a twofold serial dilution ofthe original extract to yield final concentrations rangingfrom 5% to 90% of the extract in MH broth.

The inocula (10 mL) of L. monocytogenes S1, S2, S3 andS4, respectively containing 5¥ 105 cfu were added to eachwell. A number of wells were reserved in each plate to testfor sterility control (no inoculum added), and the inoculaviability (no LAB extract added). Plates were incubated at37C for 24 h. After incubation, bacterial growth was evalu-ated by the presence of turbidity and a pellet on the wellbottom. MIC value was defined as the lowest concentrationof LAB extract that had no macroscopically visible growth.

Minimum BactericidalConcentration Determination

The minimum bactericidal concentration (MBC) values ofLAB extract was determined as follow: 10 mL of each wellmedium with no visible growth was poured on MullerHinton plates and incubated at 37C during 24 h. Finally,the number of surviving organisms was recorded. MBC wasdefined as the lowest concentration at which 99% of thebacteria were killed (Magina et al. 2009).

Biofilm Formation Assay

Production of L. monocytogenes biofilms were determinedusing a semi-quantitative adherence assay on U-bottomed

96-well tissue culture plates as described previously (Chris-tensen et al. 1982) with some modifications. An overnightculture of L. monocytogenes grown in TSB-YE (Bio-Rad) at37C was diluted to 1:100 in TSB-YE supplement with 2%(w/v) glucose as reported elsewhere (Rachid et al. 2000).

A total of 200 ml of these cell suspensions was transferredin a U-bottomed 96-well microtiter plate. Wells with sterileTSB alone served as controls. After their incubation for 24 hat 37C, the culture was removed, and the plates were washedthree times with 200 ml of phosphate-buffered saline (PBS)(7 mM Na2HPO4, 3 mM NaH2PO4 and 130 mM NaCl atpH 7.4) to remove non-adherent cells and then dried in aninverted position. Adherent cells were fixed with 95% ethanoland were stained with 100 ml of 1% (w/v) crystal violet solu-tion for 5 min. Then unbound crystal violet was removed,and the wells were washed three times with 200 ml of steriledistilled water. The water was then cleared and the microtiterplate was air dried for 2 h. The optical density (OD) of eachwell was measured at 570 nm using an automated Multiskanreader (Gio De Vitae, Rome, Italy). Each strain was testedin triplicate. Biofilm formation was categorized as highlypositive (OD570�1), low-grade positive (0.1 � OD570 < 1), ornegative (OD570 < 0.1) (Chaieb et al. 2007).

Antagonistic Effect of Lactobacillus onListeria Strains Incubated in Cheese

Round curd of soft cheese (approximately 250 g) was pur-chased from a local processing plant just after fabricationand then cut into small cubes (1 g � 0.1). Cheese cubeswere sterilized by UV irradiation during 15 min. To evaluatethe anti-listerial potential of LAB in situ on soft cheese,ripening experiments of model cheeses were conducted aspreviously described by (Ennahar et al. 1998).

L. monocytogenes grown in TSB-YE and L. plantarum inMRS. Each cheese curd was sprayed with homogenate solu-tion of LAB strain (105 UFC/ml) and 1 ml of pathogenicbacteria solution (105 UFC/ml).

Sampling and analyses were carried out after 0, 2, 4, 8,16 and 20 h as previously described (Ennahar et al. 1998).During the incubation period, slices were periodicallyhomogenized in 9 mL of peptone buffer saline, seriallydiluted, spread on Palcam agar plates and incubated for 24 hat 37C. Number of L. monocytogenes colonies was countedto evaluate the inhibitory potential of LAB strains.

Anti-biofilm Activity of Lactobacillus Extract

Inhibition of Cell Attachment. LAB extracts were testedfor their potential anti-biofilm activity. Serial dilution tech-niques were employed to determine the biofilm inhibitionconcentration (Hanafy and Hatem 1991). Assays were per-formed in sterile flat-bottomed 96-well polystyrene plates.

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Listeria strains were grown in 5 ml TSB-YE at 37C for24 h. An aliquot of 10 ml (105 UFC) was dispensed into eachwell of 96-well plates (Nunc, Roskilde, Denmark) contain-ing LAB-Extract (5–90%) prepared in TSB-YE supple-mented with 2% glucose (w/v). The wells containing onlyTSB-YE/glucose (2% w/v) were served as control. Thenplates were incubated at 37C for 24 h to allow cell attach-ment and biofilm development.

Following incubation, the modified crystal violetassay was performed to assess biofilm biomass (Djordjevicet al. 2002), and the results were expressed as percen-tage of biofilm inhibition concentration (BIC): BIC =[(ODnegative control - ODExperimental)/ODnegative control] ¥ 100

Reduction of Listeria Biofilms Growth and Devel-opment. Biofilms were allowed to perform for 24 h priorto the addition of the LAB extract. Biofilm formation wasachieved as previously described. The plates were incubatedat 37C for 24 h to allow cell attachment.

Following the incubation period, media were poured outfrom each well and washed with sterile phosphate-bufferedsaline. LAB extracts (200 ml) prepared at 5–90% in TSB-YEwere serially added to each well. The plates were furtherincubated for 24 h before the crystal violet staining was per-formed (Chaieb et al. 2011). Following incubation, biofilmbiomass was assessed, and the results were expressed aspercentage of biofilm eradication concentration (BEC):BEC = [(ODnegative control - ODExperimental)/ODnegative control] ¥ 100

Statistical Analyses

The statistical analysis was performed on SPSS v.17.0 statis-tics software. The statistical differences and significancewere assessed by ANOVA test. P < 0.05 was consideredsignificant.

RESULTS

Biochemical Characterization andMolecular Identification of PotentialProbiotic Lactobacillus

The three selected Lactobacillus strains were characterizedbiochemically and identified as L. plantarum.

After electrophoresis, a molecular method usingspecific L. plantarum primers has given a product size ofabout 428 bp as presented in Fig. 1, which confirmed thebiochemical identification.

Analysis of enzymatic characters using Api ZYM galleriesshows that the tested strains are able to produce leucine,valine and cystine arylamidase, acid phosphatase, naphthol-As-Bi-phosphohydrolase b-galactosidase and b-glucosidase(Table 2); whereas they are not able to assimilate alkaline,

esterase (C4), lipase (C14), trypsin, a-chymotrypsin andb-glicuronidase, a-mannosidase and a-fucosidase. Somedifferences in their enzymatic profile were also shown.

In Vitro Anti-listerial Activity ofLactobacillus Extract

As presented in Table 3, LAB extracts displayed varyingdegrees of activity against L. monocytogenes growth with lowminimum inhibition and bactericidal concentration values(MIC, MBC) ranging from 5% to 23%.

FIG. 1. AGAROSE GEL ELECTROPHORESES OF PCR PRODUCTSFROM POLYMERASE CHAIN REACTION ASSAYS. LANE 1, 100 BP DNALADDER; LINE 2, NEGATIVE CONTROL; LINES 3–5, L. PLANTARUMSTRAINS: LP1, LP2 AND LP3, RESPECTIVELY

TABLE 2. ENZYMATIC PROFILE OF LACTIC ACID BACTERIA ISOLATES(API ZYM SYSTEM)

Enzyme

Strain

Lp1 Lp2 Lp3

Control - - -Alkaline phosphatase - - -Esterase (C4) - - -Esterase lipase (C8) - + -Lipase (C14) - - -Leucine arylamidase + + +Valine arylamidase + + +Cystine arylamidase + - +Trypsin - - -a-Chymotrypsin - - -Acid phosphatase + + +Naphtol-AS-BI-phosphohydrolase + + +a-Galactosidase - + -b-Galactosidase + + +b-Glucuronidase - - -a-Glucosidase + + -b-Glucosidase + - +N-Acetyl-b-glucosaminidase + - +a-Mannosidase - - -a-Fucisidase - - -

+ Positive character, - negative character.

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The lowest MICs50 values were obtained with L. plan-tarum Lp1 (8.33%) against the reference strain. In additionL. plantarum Lp3 displayed a good MIC50 (13.33) againstL. monocytogenes S2 and S3. We noted also that MBCsvalue were generally at least two times higher than MICs(Table 3).

Antagonistic Effect of L. plantarum onthe Abatement Rate of L. monocytogenesin Cheese

In Fig. 2, we presented the variation of L. monocytogenesstrains incubated into cheese slices (inoculated withL. plantarum) and control (cheese without L. plantarum).

In the Lactobacillus-free cheese, L. monocytogenes rapidlyinitiated growth with counts of approximately 107 cfu/g andof approximately 108 cfu/g after 16 h.

Conversely, in the inoculated cheese, limited increases inL. monocytogenes counts were recorded during the cheeseincubation.

Anti-biofilm Activity of Lactobacillus Extract

Inhibition of Cell Attachment. Prevention of biofilmformation by LAB extract was tested on four positiveL. monocytogenes strains. Results were expressed asminimum biofilm inhibition concentration (BIC50 andBIC90, respectively). As presented in Table 4, the use of LAB

TABLE 3. ANTIBACTERIAL ACTIVITY OF LACTOBACILLUS EXTRACT AGAINST PATHOGENIC LISTERIA

Strains

Antimicrobial susceptibility (LAB extract)

Lp1 (%) Lp2 (%) Lp3 (%)

MIC MBC MIC MBC MIC MBC

L. monocytogene ATCC 19115(S1) 8.33 � 2.1 20 � 2 20 � 1.2 50 � 1.9 23.33 � 1 36.66 � 1L. monocytogene (S2) 23.33 � 1.6 30 � 1.2 13.33 � 0.8 56.66 � 0.76 13.33 � 1.6 23.33 � 2.01L. monocytogene (S3) 23.33 � 0.98 33.33 � 0.9 16.66 � 0.65 43.33 � 0.87 13.33 � 0.87 20 � 0.8L. monocytogene (S4) 16.66 � 0.78 23.33 � 1 20 � 1.03 63.33 � 1.05 16.66 � 0.76 30 � 0.98

LP, Lactobacillus plantarum; MIC, Minimum inhibitory concentration; MBC, Minimum bactericidal concentration.

FIG. 2. SURVIVAL OF L. MONOCYTOGENES DURING INCUBATION IN CHEESE CURDS INOCULATED WITH LACTIC ACID BACTERIA. A, B, C AND D:EXPERIENCES WITH L. MONOCYTOGENES STRAINS S1, S2, S3 AND S4, RESPECTIVELY

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extract to reduce L. monocytogenes attachment to poly-styrene was successful.

The LAB extract exhibited a good anti-biofilms activityagainst the tested strains. At sub-inhibitory concentration, theLAB extract showed at least 50% reduction in cell attachmentas mean of biofilms inhibition percentage (Table 4). TheLAB extract showed various effects on the development ofL. monocytogenes biofilms with BIC50 values ranging from 5%to 15% for the four tested strains. The lowest BIC50 valueswere given with the reference strains 5%, 10% and 15% withL. plantarum Lp1, Lp2 and Lp3, respectively.

We noted also that none of the extracts was able toinhibit completely (CMIs90 < 100% for all tested strains).

Eradication of Preformed Biofilm. The extractsshowed variable effects on the development of a preformedbiofilm. LAB extracts displayed varying degrees of activityagainst L. monocytogenes preformed biofilm with BEC50

values ranging from 5% to 20%. The range of BEC50 valuesin percentage were respectively (5–10%) for L. monocytoge-nes S1, S2 and S4 and (10–20%) for S3. However, BEC90

values were higher. The BEC90 values in percentage were(70, 60, 60), (80, 70, 70), (70, 70, 70), (90, 80, 80) forL. monocytogenes S1, S2, S3 and S4, respectively (Table 4)

DISCUSSION

L. monocytogenes has become a growing concern for thefood industry, which occurs in a wide range of foods(Gandhi and Chikindas 2007). The addition of LAB as“natural” food preservatives to food products such as cheesehas been described (Eijsink et al. 2002; Galvez et al. 2008).LAB and their products have been well documentedfor their antimicrobial activity against L. monocytogenes(Pleasants et al. 2001; Amezquita and Brashears 2002).

Based on our present results, LAB extract exhibited agood antibacterial effect against the tested L. monocytogenesstrains, with low MICs values ranging from 8.33 � 2.1 to23.33 � 0.98 (Table 3).

Wide ranges of LAB produce anti-microbial peptides thatexert an antimicrobial effect against gram-positive patho-gens (Eijsink et al. 2002). Nevertheless, L. monocytogeneshad been considered as a difficult organism to inhibit usingLactobacillus species (Koo et al. 2012).

As presented in Fig. 2, LAB efficiently reduces L. monocy-togenes growth in cheese. Statistical analysis revealed a sig-nificant difference between growth rates of pathogenicbacteria cultured in cheese curds before or after LABsupplementation (P < 0.05).

The number of viable cells of L. monocytogenes ATCC19115 decline from 107 UFC to 106 UFC after 2 h of incuba-tion with L. plantarum cells. However, Listeria showedincrease growth while cultured alone in the cheese curds.Similar results were shown for the other strains. This resultcorrelates with Nielsen et al. (2010), who reported thatL. plantarum extract have a potent growth inhibitory effectagainst L. monocytogenes.

L. monocytogenes ATCC 19115 showed decreased growthrate, and a stable stationary phase could be observed after16 h of incubation period (Fig. 2). The behavior ofL. monocytogenes grown simultaneously with L. plantarumwas somewhat different. Tested L. plantarum strains havedifferent effect on L. monocytogenes (Tables 3 and 4); thiscould be due to the differences of its active enzymes(Table 2) influenced by their original preculture conditions(olive, cheese, fish) (Table 1).

The growth of L. monocytogenes in association withL. plantarum strains was delayed from the beginning of thesimultaneous growth with the presence of a lag phase.The stationary phase started after about 12 h of incubationwhen grown with L. plantarum. These results demonstratedthat L. plantarum exhibited a stronger inhibitory effectagainst L. monocytogenes. This effect may be due to itshigher sensitivity to low pH and the bacteriocin-like inhibi-tory substance acting additively to lactic acid accumulation.

L. monocytogenes ATCC 19115 entered also death phaseafter 20 h of incubation with L. plantarum. Based on ourresults, it appears that LAB could be used as a bioprotective

TABLE 4. ANTI-BIOFILM EFFECT OF LAB EXTRACT AGAINST FOUR BIOFILM POSITIVE LISTERIA STRAINS

Strains

Inhibition of biofilm development (%) Biofilm eradication (%)

Lp1 Lp2 Lp3 Lp1 Lp2 Lp3

BIC50* BIC90† BIC50 BIC90 BIC50 BIC90 BEC50‡ BEC90§ BEC50 BEC90 BEC50 BEC90

L. monocytogene ATCC19115 (S1) 5 80 10 70 15 70 5 70 10 60 <5 60L. monocytogene (S2) 15 90 15 80 10 80 10 80 10 70 5 70L. monocytogene (S3) 10 70 20 80 30 <90 10 70 15 70 20 70L. monocytogene (S4) 5 70 15 <90 10 80 5 90 10 80 5 80

* Minimum biofilm inhibition concentration of LAB extract that showed 50% inhibition on the biofilm formation.† Minimum biofilm inhibition concentration of LAB extract that showed 90% inhibition on the biofilm formation.‡ Minimum biofilm eradication concentration of LAB extract that showed 50% biofilm eradication.§ Minimum biofilm eradication concentration of LAB extract that showed 90% biofilm eradication.

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culture in cheese ripening to prevent Listeria growth.Further studies will be necessary to verify the ability ofLAB, especially L. plantarum, to produce antimicrobialsubstances while growing in cheese.

In order to find a natural source able to inhibit andprevent biofilm formation in food and food industry, wetested the effect of LAB extract on four biofilm-positiveL. monocytogenes strains.

L. monocytogenes strains were shown to be able to adhereand to form biofilm on different abiotic materials (BenSlama et al. 2012). Our results showed a potential anti-biofilm effect of the three LAB extract with low BIC50

values. BIC50 were 5%, 10% and 15% for L. monocytogenesATCC 19115 and L. monocytogenes S4 and (15%, 15%,10%), (10%, 20%, 30%) for S2, S3, respectively.

Although most LAB extract were able to eradicate pre-formed biofilm. The inhibition of growth in a preformedbiofilm was more difficult to achieve. BEC50 ranged from5% to 20%, with BEC90 having still higher values. Thereduced anti-biofilm activity toward a preformed biofilm isevidence that cells in a biofilm are more resistant to anti-microbial agents compared to free-floating cells (Frank andKoffi 1990; Krysinski et al. 1992). Isolation and identifica-tion of the constituents that exhibit anti-biofilm propertiesmight be essential to include these as alternatives in thecontrol of biofilms.

On the basis of the observations made in this exploratorystudy, it can be concluded that the antagonistic activity ofLAB against pathogens is a complex interaction. Additionalstudies are needed to determine the efficacy of these strainsin controlling Listeria when different nutrients and envi-ronmental conditions are present, especially in the foodprocessing plant environment. Our initial studies are veryencouraging and indicate that at least one species of theLAB that we have isolated and characterized is a promisingcandidate for controlling L. monocytogenes biofilms in food-processing facilities.”

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