International Journal of Food Microbiology 191 (2014) 116–124

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Identification of lactobacilli with inhibitory effect on biofilm formation by pathogenic bacteria on stainless steel surfaces Fatma Ait Ouali a, Imad Al Kassaa b,c, Benoit Cudennec b, Marwan Abdallah b, Farida Bendali a, Djamila Sadoun a, Nour-Eddine Chihib b, Djamel Drider b a

Laboratoire de Microbiologie Appliquée, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia, Algeria Laboratoire Régional de Recherche en Agroalimentaire et Biotechnologies: Institut Charles Viollette, Bâtiment Polytech'Lille, Université Lille 1, Avenue Paul Langevin, Cité Scientifique, 59655 Villeneuve d'Ascq Cedex, France c Centre AZM de Biotechnologie, EDST-Université Libanaise Tripoli-Lebanon, Faculté de santé publique section 3, Université Libanaise, Tripoli, Lebanon b

a r t i c l e

i n f o

Article history: Received 17 March 2014 Received in revised form 8 September 2014 Accepted 14 September 2014 Available online 18 September 2014 Keywords: Biofilm Antagonism Lactic acid bacteria S. aureus Stainless steel Cytotoxicity

a b s t r a c t Two hundred and thirty individual clones of microorganisms were recovered from milk tanks and milking machine surfaces at two distinct farms (Bejaja City, Algeria). Of these clones, 130 were identified as lactic acid bacteria (LAB). In addition Escherichia coli, Salmonella, Staphylococcus aureus and Pseudomonas aeruginosa species were identified in the remaining 100 isolates—spoilage isolate. These isolates were assayed for ability to form biofilms. S. aureus, Lactobacillus brevis strains LB1F2, LB14F1 and LB15F1, and Lactobacillus pentosus strains LB2F2 and LB3F2 were identified as the best biofilm formers. Besides, these LAB isolates were able to produce proteinaceous substances with antagonism against the aforementioned spoilage isolates, when grown in MRS or TSB-YE media. During the screening, L. pentosus LB3F2 exhibited the highest antibacterial activity when grown in TSB-YE medium at 30 °C. Additionally, L. pentosus LB3F2 was able to strongly hamper the adhesion of S. aureus SA3 on abiotic surfaces as polystyrene and stainless steel slides. LAB isolates did not show any hemolytic activity and all of them were sensitive to different families of antibiotic tested. It should be pointed out that LB3F2 isolate was not cytotoxic on the intestinal cells but could stimulate their metabolic activity. This report unveiled the potential of LB1F2, LB14F1, LB15F1, LB2F2, and LB3F2 isolates to be used as natural barrier or competitive exclusion organism in the food processing sector as well as a positive biofilm forming bacteria. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Biofilms are sustainable sources of contaminations that are responsible of food-related illness and important economic losses. This lifestyle confers protection to bacterial cells and decreases the efficiency of cleaning and disinfection procedures. Consequently, the need for the development of new strategies and anti-biofilms agents allowing the prevention and control of biofilm-formation by various pathogens is of major importance in order to reduce the risk of contamination in food sector. The process of bacterial biofilm formation is occurring in four dependent stages (Donlan, 2002). The bacterial adhesion stage is associated with the production of exopolyscharides, DNA and proteins. The initial stage of bacterial adhesion was reported to be reversible because of the weakness of the interactions between bacteria and surfaces, however this stage becomes irreversible as a result of anchoring by appendages and/or production of extracellular polymers mainly exopolysaccharides. Industrial formulations acting on the bacterial

E-mail address: [email protected] (D. Drider).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.011 0168-1605/© 2014 Elsevier B.V. All rights reserved.

adhesion to food contact surfaces such as stainless steel are needed to reduce cross-contamination, food spoilage and transmission of diseases due to biofilms. Stainless steel is largely used in the food industries sector because of its mechanical strength, resistance to corrosion, and longevity (Marques et al., 2007). Staphylococcus aureus is among the most common pathogenic bacteria isolated from different surfaces such as stainless steel in food processing plants (Pastoriza et al., 2002), where it can adhere and forms biofilms (Kunigk and Almeida, 2001; Archer et al., 2011; Brooks and Jefferson, 2012; Abdallah et al., 2014). Foodborne disease caused by S. aureus is typically intoxication due to the ingestion of enterotoxins preformed in food by enterotoxigenic strains (Normanno et al., 2007). The development of concepts and the discovery of novel anti-staphylococcal biofilm agents are expected to be promising issues in food safety. Related to this topic, different metabolites such as oregano essential oil, bacteriophage-derived peptidase, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic staphopains, β-2,2-amino acids derivatives, phenolic compounds and flavonoids have recently been described as potent anti-staphylococcal biofilm agents (Schillaci et al., 2013; Fenton et al., 2013; Luis et al., 2013; Manner et al., 2013). Strategies using biofilms produced by the competitive exclusion microorganisms to

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inactivate foodborne pathogens in the food processing environments are of major importance. Kim et al. (2013) showed the inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms. Zhao et al. (2013) reported the reduction of Listeria monocytogenes in a ready-to-eat poultry processing plant by LAB. Pérez-Ibarreche et al. (2014) reported that lactobacilli with biofilm forming aptitudes were able to control L. monocytogenes biofilms. Lactobacilli is an important part of the natural LAB consortium. They are considered as potentially antagonistic to spoilage and pathogenic bacteria because of their capabilities to produce an array of inhibitory substances such as organic acids, H2O2 and bacteriocins. Lactobacilli have been reported to compete with pathogens in urogenital and intestinal tracts (Reid et al., 1988), and interfere with their adhesion on catheters device (Hawthorn and Reid, 1990; Rodrigues et al., 2004). The use of lactobacilli as anti-biofilm strategies is a promising hope in food safety. Here, two groups of microorganisms were isolated from the industrial samples: LAB and non-LAB. Considering LAB natural and friendly environmental microorganisms, we term the remaining nonLAB as “spoilage,” bearing in mind their possible unfriendly nature and recognizing that some of them may in fact be foodborne pathogens, such as Salmonella. The objectives of this study were to explore novel and atypical ecological niches, isolate LAB with potential applications as hurdles against biofilm forming pathogens and spoilage strains. 2. Materials and methods 2.1. Isolation of lactic acid bacteria and spoilage bacteria One hundred and twenty of samples (materials) were collected from the inner surfaces of milk tanks and milking machines in two distinct farms located at Bejaia city (Algeria). The samples were serially diluted (0.85% m/v NaCl) and plated onto the surface of MRS medium (de Man et al., 1960) (Merck, Germany), and M17 agar (Merck, Germany) plates, respectively. Plates were then aerobically and anaerobically incubated at 30 °C for 24–48 h. Gram-positive and catalase negative isolates were assumed as LAB. Basic microbiological methods were employed for characterization of non-LAB isolates. The presence of E. coli was tested on Eosin Ethylene Bleu agar plates (Merck), Salmonella clones were identified using Hektoen agar plates (Merck), and S. aureus isolates were selected on Chapman and Baird Parker media (Merck), followed by the assay for coagulase and DNase activities. Pseudomonas isolates were identified on nutritive agar (Fluka, Spain) and characterized for their pigmentation. To keep the viability of the newly isolated strains, all of them were periodically streaked in MRS or Nutrient agar (Fluka). All isolates were kept at −80 °C in the presence of 20% glycerol (Sigma, France). 2.2. Assessment of LAB antagonism and preliminary of characterization of the inhibitory substances The antagonism of 130 LAB isolates were tested against the aforementioned “spoilage” isolates using the spot method (Jacobsen et al., 1999) with some modifications. Briefly, 5 μl of overnight MRS cultures of LAB isolates grown in anaerobic jars (GasPak, BBL) to avoid the effect of H2O2, were spotted onto 1.5% MRS agar. Plates were dried for 30 min at room temperature, incubated under anaerobic conditions at 37 °C for 18 h; and overlaid with 10 ml of Brain Heart Infusion (BHI) (Merck) medium containing 0.75% agar seeded with 1% (v/v) of overnight culture of the indicator strain, leading to a final concentration of about 106 cells/ml. The incubation was carried out aerobically at 37 °C for 18 h. The sterile MRS broth was tested as a negative control. To identify the inhibitory substances secreted into the growth medium, LAB isolates presenting antagonism were grown overnight at 30 °C in 20 ml MRS broth. The cell-free supernatant (CFS) obtained by

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centrifugation (8000 ×g, 20 min, 4 °C), filtered with 0.22 μm-pore-size Acrodisc® syringe filters (Pall Gelman Laboratory, USA) was separated into three samples named 1, 2 and 3. Sample 1 was directly tested; sample 2 was adjusted to pH 6.5 with 1 N NaOH (Merck-eurolab, Briare Le Canal, France) to rule out the hypothesis of acid inhibition. Sample 3 was treated with catalase (300 U/ml) (C-3515, Sigma-Aldrich Chemie, Steinheim, Germany) to rule out the hypothesis of inhibition by the H2O2. The antagonistic activities of these three samples were determined at least in triplicate for each LAB isolate using the well diffusion assay (Ennahar et al., 1998). The absence or presence of any inhibitory zone was recorded after 18 h of incubation at 37 °C. The CFS obtained from the antagonistic LAB isolates were treated with proteases such as α-chymotrypsin, proteinase K and papain (Sigma-Aldrich Chemie, Steinheim, Germany). Each CFS was adjusted to pH 6.5 with 1.0 mol/l NaOH (Sigma), filter sterilized (0.22 μm), treated with proteases at 1.0 mg/ml and left for 1 h at 30 °C. The treated and untreated CSF (control samples) were heated at 100 °C for 5 min and then immediately cooled in ice to inactivate the proteases. The residual activity of treated and untreated samples was determined by measuring the diameter of inhibition zones according to the above cited methods. 2.3. Biomolecular typing of the antagonistic LAB isolates The identification of the antagonistic LAB isolates was carried out by the API 50 CHL system (Biomérieux, France), and the 16S rDNA sequence analysis. For the last method, total DNA was extracted from LAB isolates with the Wizard® Genomic DNA purification Kit (Promega Corp., France). The amplification of 16S rDNA was done with primers S1 and S2 (Table 1), and the following PCR program: denaturation at 95 °C/3 min, 29 cycles at 94 °C/40 s, annealing at 55 °C/50 s and extension at 72 °C/1 min, followed up by a final extension cycle at 72 °C/10 min. The PCR amplicons were separated on 0.6% (w/v) agarose gel upon electrophoresis carried out at 100 V for 1 h. The amplicons were purified, cloned into the pGEM®-T Easy Vector System (Promega Corp., France), and transferred to E. coli JM109 strain. The recombinant plasmids containing the 16S rDNA were extracted by GeneJET plasmid Miniprep (Fermentas), and sequenced at Eurofins MWG Operon (Germany). The resulting sequences were assembled into a unique contig with BioEdit sequence alignment software and analyzed using the NCBI-Standard Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov). Alignment with known 16S rDNA sequences in the NCBI database (http://www.ncbi.nlm.nih.gov/BL ASTU) was done with the basic local alignment search tool, an online software. The sequences have been deposited in gene banks and were assigned the following accession numbers: KF923749, KF923750, KF923751, KF923752 and KF923753. LAB isolates with the highest antibacterial activity were characterized at the molecular level using a specific Lactobacilli PCR previously described by Dubernet et al. (2002), and a REP-PCR fingerprinting technique (Al Kassaa et al., 2014). The specific Lactobacilli PCR required the use of primers LbLMA1 and R16-1 (Table 1) and the following PCR program: (i) denaturation at 95 °C/5 min, (ii) 34 cycles of denaturation at 94 °C/1 min, annealing at 55 °C/1 min and extension at 72 °C/1 min, followed by (iii) a final extension at 72 °C/5 min. The 50 μl reaction mixture contained 25 μl of DreamTaq™ Green PCR Master Mix 2X (dNTPs, MgCl2 1.5 mM), reaction buffer DreamTaq™ polymerase (Fermentas), 2 μl of each primer (20 mM) and 5 μl of DNA sample of

Table 1 Primers used in this work. Primers

Sequences 5′-3′

References

Primer S1 Primer S2 LbLMA1 R16-1 (GTG)5

5′-AGAGTTTGATC(A,C)TGGCTCAG-3′) 5′-GG(A,C)TACCTTGTTACGA(T,C)TTC-3′ 5′-CTCAAAACTAAACAAAGTTTC-3′ 5′-CTTGTACACACCGCCCGTCA-3′) 5′-GTGGTGGTGGTGGTG-3′

Dubernet et al., 2002 / Messaoudi et al., 2011 Dubernet et al., 2002 / Messaoudi et al., 2011 Al kassaa et al., 2014

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cellular suspension from a single colony grown overnight and free RNase and DNase water (16 μl). The REP-PCR was carried out using the primer (GTG)5 (Table 1) and the following PCR program: (i) denaturation at 95 °C/7 min, (ii) 29 cycles of denaturation at 94 °C/1 min, annealing at 40 °C/1 min and extension at 72 °C/8 min, followed by (iii) a final extension at 72 °C/16 min. The 50 μl reaction mixture contained 25 μl of DreamTaq™ Green PCR Master Mix 2× (dNTPs, MgCl2 1.5 mM), reaction buffer DreamTaq™ polymerase (Fermentas), 5 μl of each primer (20 mM) and 5 μl of DNA sample of cellular suspension from a single colony grown overnight and free RNase and DNase water (15 μl). The amplicons gathered from the specific Lactobacilli PCR and REP-PCR were separated on 1.2% (w/v) and 0.7% (w/v) agarose gels, respectively. In both cases, the electrophoresis was carried out at 100 V for 1 h and the gels were stained with 0.5 μg/ml gel red. 2.4. Adhesion of the LAB and spoilage isolates to polystyrene tissue culture plates (TCP) The semi-quantitative method of adhesion to polystyrene tissue culture plates described by O'Toole and Kolter (1998) was used with some modifications. Briefly, 100 μl of each culture (LAB isolates, 108 CFU/ml and spoilage isolates, 106 CFU/ml) in TSB-YE (TSB supplemented with 0.6% yeast extract, Difco, France) were added to the wells of sterile 96well polystyrene tissue culture plates (Nunc®, polystyrene, France), and incubated for 24 h at 30 °C in a humid atmosphere created by placing the microplate in a box containing four beakers filled with water. Cultures were decanted and wells were washed twice with sterile distilled water to remove the non adherent cells. The adherent cells in each well were fixed with 200 μl of 99% ethanol (Sigma, France), and after 15 min the plates were emptied and left to dry, then they were stained for 45 min with 200 μl of 0.1% crystal violet (Biochem Chemopharma, Québec, Canada). The stained biofilms were rinsed three times with 200 μl of distilled water and extracted with 200 μl of 33% (v/v) glacial acetic acid (Sigma, France). The content of each well (125 μl) of each well was then transferred to new sterile microplate, and the amount of biofilm was quantified by measuring the OD595nm using a microplate reader (ELX800. Bio-Tek, USA). 2.5. Inhibition of the spoilage isolates by the LAB forming biofilms LAB and spoilage isolates at 108 CFU/ml, and 106 CFU/ml, respectively were grown in TSB-YE, as appropriate medium for biofilm formation, and incubated at 37 °C for 18 h. After which, CFS, obtained by centrifugation (8000 × g, 20 min, 4 °C) were filtered with 0.22 μm-pore-size Acrodisc® syringe filters. The 96-well polystyrene microplates were inoculated with 50 μl of overnight spoilage cultures and 50 μl of the LAB neutralized CFS. The microplates were left stirring for 15 min before to be incubated for 24 h at 37 °C in a humid atmosphere. Afterwards, the microplates were rinsed three times with 200 μl of distilled water. The adherent cells in each well were fixed with 200 μl of 99% ethanol (Sigma, France). After 15 min, the plates were emptied and left to dry, then were stained for 20 min with 200 μl of 0.1% crystal violet (Biochem Chemopharma, Québec, Canada). The stained biofilms were rinsed three times with 200 μl of distilled water and extracted with 200 μl of 33% (v/v) glacial acetic acid (Sigma, France). The content of each well (125 μl) was transferred to new sterile microplate. The amount of biofilm was quantified by measuring the OD595nm using a microplate reader (ELX800. Bio-Tek, USA) as described above. Each experiment was performed in triplicate using independently grown cultures. 2.6. Inhibition of the biofilm formation on stainless steel AISI 304L 2.6.1. Stainless steel cleaning treatment Before carrying the adhesion assays, the stainless steel slides were cleaned according to the procedure described by Abdallah et al. (2014).

They were kept for a night in 70% ethanol (Sigma) for disinfection, and then washed twice with sterile distilled water until complete removal of the ethanol. Cleaning procedure of the stainless steel slides was pursued for 20 min by immersion, under agitation conditions, in 200 ml of an alkaline detergent 1% (v/v) TFD4 solution (initial temperature 20 °C). The slides were rinsed with sterile distilled water followed by five successive immersions of 1 min each in sterile ultra pure water (milliQ water, Millipore) with agitation. Finally, they were dried, individually enveloped and autoclaved at 120 °C for 20 min. 2.6.2. S. aureus SA3 strain culture One milliliter of S. aureus SA3 culture at 105 CFU/ml was seeded in 50 ml of TSB and incubated for 15 h at 37 °C with agitation (160 rpm). The cells pellet recovered by centrifugation (5000 ×g, 20 min), was washed twice with 20 ml of PBS buffer [phosphate buffered saline; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.27 g KH2PO4 per liter; pH 7 corrected with HCl/NaOH, and resuspended in the same buffer. Serial dilutions were made in (0.85% w/v) NaCl (Sigma-Aldrich Chemie, Steinheim, Germany) in order to have an inoculum of 107 CFU/ml. 2.6.3. Kinetic of S. aureus SA3 biofilm development on AISI 304L Twenty AISI 304L stainless steel slides prepared as been described before were used in this study. Two milliliters of S. aureus SA3 suspension at 107 CFU/ml (as described before) were deposited on each slide surface and maintained for 1 h at 37 °C. After the initial adhesion time of 1 h, the bacterial suspension was removed and slides were washed twice with PBS to remove loosely attached cells. The adhered cells were observed using epifluorescence microscopy or detached for cell counting on Tryptic Soy Agar (TSA) plates. The bacterial counts, and microscopic observations were presented as a 0 h of biofilm formation. For the biofilm formation assays, washed slides were placed in sterile Petri plates, the upper face was covered with 2 ml of TSB and slides were incubated, at 37 °C. The biofilm formation was monitored by counting sessile cells, and biofilm observation using epifluorescence microscopy, after 3, 6, and 24 h. The counts of S. aureus SA3 adherent bacteria to the slides surfaces was performed as follow: The TSB medium was removed, the slides were washed twice with PBS phosphate buffer (pH 7), and immersed in 30 ml of the same buffer before to be subjected to sonication for 5 min at 50 kHz. 2.6.4. Epifluorescence microscopy Biofilm cells were stained with a BacLight LIVE/DEAD bacterial viability staining kit according to the manufacturer's instructions (Molecular Probes, Invitrogen, France). In brief, 1.5 μl of each reagent were diluted in 1 ml of physiological water (0.85% m/v NaCl). Then 1 ml of the mixture was gently deposited on the upper face of the slide (i.e. on the face of the biofilm development). After 15 min incubation of the slides in the dark, the staining solution was aspirated and biofilms were observed using an epifluorescence microscope (Nikon Optiphot-2 EFD3, Japan). 2.6.5. Conditioning of slides with the LAB neutralized supernatant Two milliliters of neutralized CFS prepared as above described were deposited on the surface of each AISI 304L slide placed in sterile Petri plates, and maintained for 2 h at 37 °C. This step may lead to the formation of a conditioning film on the AISI surface. In parallel, sterile TSB-YE was used as control for the conditioning step of the stainless steel. After this period, the CFS and the TSB-YE were removed and replaced by 2 ml of S. aureus A3 suspension at 107 CFU/ml prepared as described in Section 2.6.2. At the end of 1 h of incubation time, the buffer containing the non-adherent S. aureus SA3 cells was aspirated and 2 ml of sterile TSB medium were deposited on the surface of each coupon for 0, 3, 6, and 24 h incubation to monitor the installation of S. aureus A3 biofilm. After each time of incubation, the slides were washed twice with 30 ml of PBS buffer (pH 7.0). Finally, the slides were immersed individually in 30 ml phosphate buffer and sonicated. The detached S. aureus

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SA3 cells were enumerated by plating the bacteria on TSA after growth at 37 °C for 24 h. Additional slides were prepared and served to the epifluorescence observation after their staining with live/dead components as described in Section 2.6.4. 2.6.6. Antibiotics susceptibility The susceptibility of the antagonistic LAB isolates was tested against nearly all antibiotic classes encompassing penicillin, cephalosporins, aminosides, tetracycline, macrolides, glycopeptides, and polypeptide (MAST, UK). The bacterial suspensions of about 107 CFU/ml were seeded onto MRS agar plates by the flooding technique. The plates were air dried for 15 min, and then disks impregnated with antibiotics were deposited on the plates. The formation of inhibition zones around the disks was determined after 24 h of incubation at 37 °C. The susceptibility to these antibiotics was determined based on the recommendations of the Antibiogram Committee of the French Microbiology Society. 2.6.7. Crude measure of hemolytic activity To test the crude measure hemolytic activity, fresh cultures of isolates were streaked on blood agar (Columbia base, Biokar Diagnostics), containing 5% (w/v) sheep blood and incubated for 48 h at 30 °C. After this period, blood agar plates were examined for the presence of the hemolytic zones around colonies (Ghrairi et al., 2008). 2.6.8. Cytotoxicity assessment Cytotoxicity of Lactobacillus pentosus LB3F2 against intestinal cells was investigated using Cell Proliferation Kit II (XTT) (Roche Applied Science, USA). This assay was based on the reduction of a tetrazolium salt (XTT) into yellow formazan salt by active mitochondria. STC-1 cell line was a gift gratefully received from Dr. C. Roche (INSERM U865, Lyon, France). This cell line is derived from an endocrine tumor developed in the small intestine of double transgenic mice (Rindi et al., 1990). All chemicals for the cell culture were from PAN-Biotech GmbH (Germany). Cells were routinely grown in 75 cm2 flasks at 37 °C, 5% CO2 atmosphere in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 4.5 g/l glucose, 5% of fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml of streptomycin. When 80% confluence were reached, cells were trypsinized and seeded into 96-well plate at a density of 7000 cells per well in 100 μl of DMEM. After 48 h of incubation at 37 °C, and under 5% CO2 atmosphere, confluent cells were washed twice with PBS. In parallel, L. pentosus LB3F2 cells were harvested by centrifugation (8000 ×g, 10 min, 4 °C) from MRS broth 18 h old cultures and washed twice with 2 ml of minimum medium (DMEM without serum and antibiotics). Absorbance (490nm vs 655nm reference) was measured in a microplate reader spectrophotometer (ELx808, BioTek, USA). For each condition, absorbance of wells without STC-1 cells and with bacteria was subtracted from absorbance of wells containing STC-1 cells and bacteria. Results were expressed as percentage of proliferation. 2.6.9. Statistical analysis All data in this study represented the mean of three experimental replicates. Statistical comparisons among the different results obtained by the different tests were performed by one-way analysis of variance using XL-STAT with Student–Newman–Keuls method (version 2009). Significant differences among adherent cell counts on different stainless steel slides surfaces, untreated, treated with TSB or with the CFS, were determined by one-way analysis of variance using XL-STAT software version 2009 to determine differences among various treatments at a 95% confidence level. 3. Results Of 400 bacterial isolates recovered from the milk tanks and milking machine surfaces at Bejaia city (Algeria), 130 of them were considered as presumptive LAB because of the positive Gram staining and the

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absence of catalase activity, whereas 100 isolates were identified as E. coli (32%), Salmonella sp., (22%), S. aureus (20%) and Pseudomonas aeruginosa (22%) and considered thereof as “spoilage” isolates. The other 170 strains were other than the aforementioned spoilage isolates. Notably, the antagonistic isolates were identified by the API 50 CHL system and 16S rDNA sequence analysis. These two independent methods of identification fully reliable as only three species of five were identified similarly (Table 2). The use of genus-specific lactobacilli PCR proposed by Dubernet et al. (2002) allowed the amplification of 250 bp DNA fragment from total DNA extracted from LAB isolates arguing on their belonging to the Lactobacillus genus (Fig. 1A). The relatedness between LB1F1, LB2F2, LB3F2, LB14F1 and LB15F1 isolates as studied by the REP-PCR (GTG)5 fingerprinting approach and interpreted by the numerical analysis permitted three groups based on a distance cut-off of 70% relatedness (Fig. 1B). The Lactobacillus brevis isolates were not closely related each to other. LB14F1 and LB15F1 (L. brevis), LB2F2 and LB3F2 (L. pentosus) were contained in two distinct groups; whereas LB1F2 (L. brevis) was found in the third group that was more linked to L. brevis (Fig. 1B). Further, the cultivation of the LAB isolates in TSB-YE permitted to show their adhesive and biofilm-forming properties particularly for LB1F2, LB2F2, LB3F2, LB14F1 and LB15F1 isolates. LB2F2 and LB3F2 isolates were classified as strong biofilm producers with an OD595 nm of 0.78 and 0.72, respectively (Fig. 2). Regarding, the spoilage isolates, S. aureus SA3 with an OD 595nm of 0.6 was considered as well as a strong biofilm producer at 37 °C, whereas, the other ones including S. aureus, E. coli, P. aeruginosa and Salmonella sp. showed a moderate (0.22 ≥ OD ≥ 0.12) even a weak potential (0.12 ≥ OD ≥ 0.08) of biofilm formation at 37 °C. Remarkably, the identification of S. aureus SA3 was recently confirmed by pyrosequencing (unpublished results). Statistical analysis revealed significant differences (P b 0.05) of the aptitudes of the biofilm formation for the different LAB isolates, except for LB2F2 and LB3F2 isolates (P N 0.05). These data underline the high discrepancy in terms of the biofilm formation of LAB isolated from the same niche, even between LAB belonging to the same genus and species. LB1F1, LB2F2, LB3F2, LB14F1 and LB15F1 isolates exhibited the highest biofilm-forming properties. The LAB isolates with the highest adhesion properties were tested for their antagonism against the 100 spoilage isolates obtained from the same ecological niche. Although, all the LAB isolates were antagonistic, the potency of their inhibition was exerted in strain dependent manner. The antagonism of this LAB was confirmed by the agar-well diffusion method using the native and neutralized supernatants (Table 3). The best antagonism was observed for LB1F2, LB2F2, LB3F2, LB14F1 and LB15F1 isolates which meanwhile were the best biofilm-forming isolates. Among these five LAB isolates, the highest antagonism was registered for LB3F2 isolate. The experimental conditions allowing optimization of the antibacterial activity of LB3F2 isolate were obtained with the culture supernatant (pH 5.5) after its growth in TSB-YE broth for 15 h at 30 °C. To identify the metabolites responsible of such antagonism, the neutralized CSF (pH 6.5) gathered from LB1F2, LB2F2, LB3F2, LB14F1 and LB15F1 LAB isolates were tested after treatment with various metabolite-specific enzymes. Afterwards, the inhibitory activity was

Table 2 API 50 CH and 16S rDNA sequencing identifications of the selected LAB strains. LAB strains

API 50 CH

16S rDNA

LB1F2 LB2F2 LB3F2 LB14F1 LB15F1

L. L. L. L. L.

L. L. L. L. L.

brevis plantarum plantarum brevis brevis

brevis pentosus pentosus brevis brevis

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Fig. 1. (A) Specific Lactobacilli PCR amplification. Lanes 1 to 5 correspond to amplicons gathered from LB15F1, LB14F1, LB3F2, LB2F2 and LB1F2 isolates respectively. Lane C corresponds to the negative control and lane M to DNA molecular weight markers. The arrow indicates the PCR product of 250 bp usually obtained for LAB of Lactobacillus genus. (B) Pattern of Rep-PCR fingerprinting technique of the selected LAB isolates.

recorded after addition of catalase to the CFS excluding therefore the involvement of hydrogen peroxide. However, the addition of proteases as papain, α-chymotrypsin and proteinase K abolished the antagonism indicating the proteinaceous nature of the secreted inhibitory substances. Additionally, the inhibitory substances were insensitive to the heat treatment at 100 °C for 5 min. All these data let us to conclude that LB1F2, LB2F2, LB3F2, LB14F1 and LB15F1 LAB isolates were able to produce bacteriocins or bacteriocinslike inhibitory substances (BLIS). Further, the CFS from LAB isolates LB1F2, LB14F1, LB15F1, LB2F2 and LB3F2 were able to impede the adhesion and subsequently the biofilm formation of S. aureus SA3 based on the data obtained with the semiquantitative TCP method (Fig. 3). These data were strengthened by the P-value recorded (P b 0.05) for the adhesion levels in the absence and presence of CFS. The conditioning of AISI 304L stainless steel slides for 2 h with the neutralized CFS from L. pentosus LB3F2 permitted a decrease along time of the viable S. aureus SA3 adherent cells arguing clearly the effectiveness of the bacteriocin(s) or BLIS secreted into the growth medium.

The effects of L. pentosus LB3F2 on the intestinal STC-1 cell metabolic activity were assayed using XTT test (Fig. 4). The results showed that after 3.5 h and 4 h of contact LB3F2 was not toxic for STC-1 cells at the concentrations of 105, 106 and 107 CFU/ml. Moreover, after 4 h of contact, LB3F2 at 107 CFU/ml exerted a positive effect on the intestinal cell metabolism activity. The data depicted in Fig. 5 showed the number of S. aureus SA3 cells adhering on the stainless steel slides at 37 °C. This number appeared to increase from 4 to 7 Log CFU/ml after 6 h of contact, and to 8 Log CFU/ml after 24 h of contact. The DNA binding dye Syto-9 labeled viable cells (green) allowed to track the biofilm formation by S. aureus SA3 on the untreated stainless steel slides surfaces after a contact of 0, 3, 6 and 24 h at 37 °C (Fig. 6A, B, C, D). This experiment was conducted on stainless steel slides treated with neutralized supernatants. Untreated slides and TSB treated ones were used as negative controls. The results showed that, after 24 h of biofilm development, S. aureus SA3 adhered much less on the conditioned slides (Fig. 6H) (i.e. treated with native supernatant), than on untreated, and TSB treated, slides (Fig. 6D). In addition, our data underlined that, in the first 6 h of biofilm formation, most of attached cells on conditioned slides were predominantly stained with the propidium iodide (Fig. 6E, G), which underlines the antibiofilm efficacy of such treatment. The hardy S. aureus SA3 adhered much less on the conditioned slides (Fig. 6E) and an important number of cells labeled in red showing not only the inhibition of adherence but also the induction of cell death (Fig. 6E, F, G, H). Notably, while no crude measure of hemolytic activity was observed after 48 h of incubation, the aforementioned isolates appeared to be sensitive to all the antibiotics tested, except for ciprofloxacin, fosfomycin, and vancomycin (Table 4). 4. Discussion

Fig. 2. Biofilm formation of LB2F2, LB3F2, LB15F1, LB1F2 and LB14F1 isolates. OD was used to quantify the biofilm formation. The data are the means of at least three independent experiments. C designates the negative control, which corresponds to sterile TSB-YE. The error bars represent the standard deviations.

It is important to study the interactions between bacteria and the surfaces in a specific food processing environment in order to provide more effective measures for prevention of biofilm formation and for its removal. The aim of this study was to examine the microbial consortium cohabiting in the dairy environment such as the milk tanks and milking machines used in two traditional and distinct farms located at Bejaia City, Algeria. After describing the lactobacilli and spoilage

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121

Table 3 Antagonism of the selected lactobacilli strains before and after pH neutralization of the culture supernatant. The data are the means of at least three independent experiments. The target strains used were isolated from the same ecological niche as the lactobacilli producing inhibitory substances. In each column there are two data (before neutralization “BN” and after neutralizing “AN” of the supernatant). LAB strain

Diameter of the inhibition zones (mm) E. coli

S. aureus

E1

L. L. L. L. L.

pentosus LB3F2 pentosus LB2F2 brevis LB1F2 brevis LB14F1 brevis LB15F1

E2

E3

SA1

P. aeruginosa SA2

SA3

SA4

P1

P2

P3

BN

AN

BN

AN

BN

AN

BN

AN

BN

AN

BN

AN

BN

AN

BN

AN

BN

AN

BN

AN

10 09 10 10 08

05 03 04 04 03

10 10 11 10 09

03 03 04 03 03

11 10 11 09 08

05 04 03 03 03

17 15 12 15 12

08 04 04 05 03

17 16 14 15 15

07 05 04 03 03

19 15 13 13 15

09 05 05 04 03

18 15 14 13 14

06 05 04 03 03

08 09 08 10 09

03 03 03 03 03

09 10 09 10 10

03 04 03 03 03

08 11 10 09 10

02 03 03 04 03

bacteria contained in this consortium, our investigations focused on the lactobacilli because of their potential of applications. This study permitted the recovery of at least 130 LAB isolates among which LB1F2, LB2F2, LB3F2, LB14F1 and LB15F1 were able to form biofilms, and to produce bacteriocins like inhibitory substances that alter the formation of S. aureus SA3 biofilm on stainless steel slides. Bacteriocins are small antimicrobial peptides ribosomally synthesized by Gram positive and Gram negative bacteria (Drider and Rebuffat, 2011) exhibiting thereof safe (Belguesmia et al., 2011), and possibilities of biotechnological applications (Yang et al., 2014). Of particular interest the bacteriocins produced by lactobacilli which are able to influence septoformation, peptidoglycan and protein synthesis, affect cytoplasmic membranes leading to their destabilization and consequently to cell death (Rybal'chenko et al., 2013). Bacteriocins produced by L. pentosus and L. brevis species designed as pentocins and brevicins, respectively are not very abundant in the literature. Pentocin TV35b and pentocin 31-1with antagonism against Gram positive bacteria have been reported (Okkers et al., 1999; Zhang et al., 2009). We hypothesize that BLIS produced by LB15F1, LB2F2 and LB3F2 isolates are different from pentocins TV35b and 31 previously reported because of the discrepancies in their spectra. Further, the potential of the bacteriocin produced by L. brevis species is not very well documented as well. To date only few bacteriocins including brevicin 27 and brevicin 286 have been reported by Benoit et al. (1997) and Coventry et al. (1996) respectively. The level of adhered S. aureus SA3 cells on AISI 304 has increased, within 24 h, from 4.7 to 8 log CFU/ml. In this process, the temperature of 37 °C appeared to play a positive role on the adhesion process. Morton et al. (1998) pointed out the role of the optimum growth temperature on the adhesion process. According to de Oliviera et al. (2010) the initial inoculum size might influence the bacterial adhesion process as well. Related to this last point, Zottola and Sasahara (1994)

suggested earlier that the initial number of bacterial cells for biofilm formation should be 106 to 107 CFU/ml. The anti-adhesive and anti-biofilm-forming properties of lactobacilli have been assessed in different circumstances. Indeed, Abedi et al. (2013) showed the good anti-adhesive properties of Lactobacillus delbrueckii against E. coli in different competitive conditions. Vuotto et al. (2013) reported that L. brevis CD2 prevents the biofilm formation by Prevotella melaninogenica. Based on the data obtained in this study, LB1F2, LB14F1, LB15F1, LB2F2 and LB3F2 isolates are considered as good candidates for applications as natural barriers or competitive-exclusion microorganisms to control staphylococcal biofilm formation. To strengthen this option of application, the antibiotic susceptibility and hemolytic activity of LB1F2, LB14F1, LB15F1, LB2F2 and LB3F2 isolates were investigated. They resulted sensitive to the most antibiotics tested except for fosfomycine, vancomycin and ciprofloxacin. Resistance of Lactobacillus spp. to antimicrobial agents was reported to be species-dependent (Danielsen and Wind, 2003). Related to this, resistance to vancomycin and ciprofloxacin was reported by Vay et al. (2007) and Danielsen and Wind (2003) for the homofermentative lactobacilli group. Temmerman et al. (2003) examined the resistance of bacterial isolates recovered from 55 European probiotic products using the disc diffusion method. According to these authors, 79% of the isolates were resistant to kanamycin, 65% to vancomycin, 26% to tetracycline, 23% to penicillin G, 16% to erythromycin and 11% to chloramphenicol. It should be pointed out that the natural bacterial resistance to antibiotics is not as a major risk to animal or human welfare, contrarily to the acquired resistance which is propagated by plasmids and transposons elements (Gueimonde et al., 2013). Moreover, the isolates LB1F2, LB14F1, 150 Ctrl

Proliferation (% of control)

5

10

b

6

10

125

7

10

a

a

a

a a

a a

100

75

50 3.5

4.0

Time of incubation (h) Fig. 3. Adhesion inhibition of S. aureus SA3 to polystyrene microplates by LAB strains cell free supernatants (CFS): CFS1: LB2F2, CSF2: LB3F2, CSF3: LB15F1, CFS4: LB1F2, CFS5: LB14F1. The positive control (PC) was the untreated S. aureus culture, and the negative control (NC) was the TSB-YE alone. The data are the means of at least three experiments. The error bars represent the standard deviations.

Fig. 4. Effects of L. pentosus LB3F2 on the STC-1 cell mitochondrial activity. XTT's response after 3.5 and 4 h of incubation with LB3F2 at 105, 106 and 107 CFU/ml. Data are the mean ± SD of six values. Means without a common letter are different (p b 0.05) using one way ANOVA with Student–Newman–Keuls method.

122

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10

Table 4 Antibiotic susceptibility of the studied LAB strains. a

log CFU/ml

8 b cd c

g

b d

e

f

6

a

g

4 h

2 0

3

6

24

Time (hours) Fig. 5. Adhesion of S. aureus SA3 cells (Log CFU/ml) at different incubation times, to stainless steel surfaces without (■) and after conditioning with sterile TSB medium ( ) or L. pentosus LB3F2 supernatant ( ) for 2 h. The error bars represent the standard deviations on bacteria counting. The data are the means of at least three independent experiments. Means without a common letter are different (p b 0.05) using one way ANOVA with Student–Newman–Keuls method.

Antibiotic

Concentration LB1F2 LB14F1 LB15F1 LB2F2 LB3F2

SXT:TS:cotrimoxadole (trimethoprim/ sulphamethoxazole) Tetracycline Rifampin Cefepine Cefotaxime Ampicilin Penicillin G Oxacillin Cefoxitin Kanamycin Lincomycin Ciprofloxacin Gentamycin Streptomycin Erythromycin Vancomycin Fosfomycin

25 μg

S

S

S

S

S

30 μg 30 μg 30 μg 30 μg 10 μg 10 UI 5 μg 30 μg 1 μg 5 μg 5 μg 500 μg 500 μg 15 μg 30 μg 50 μg

S S S S S S R S S R R S S S R R

S S S S S S R S S S R S R S R R

S S S S S S R S S S R R S S R S

S S R S S S S S S R R S S S R R

S S R S S S S S S R R S S S R R

R = resistant, S = sensitive.

Fig. 6. Epifluorescence images of S. aureus SA3 adhered cells on slides treated with TSB (A, B, C, D) and neutralized CFS (E, F, G, H). Sessile cells were stained with live/dead BacLight™ bacterial viability kit after 1 h (A, E), 3 h (B, F), 6 h (C, G) and 24 h (D, H).

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LB15F1, LB2F2 and LB3F2 did not show any hemolytic sign. Concretely, this study unveiled the potential of the LB1F2, LB14F1, LB15F1, LB2F2 and LB3F2 isolates to inhibit the growth of both Gram positive and Gram negative bacteria isolated from the same ecological niche. Additionally, the aforementioned LAB isolates were able to strongly interfere on the staphylococcal biofilm formation. Of particular interest LB3F2 that was subjected to additional studies. Indeed, this isolate was not only non cytotoxic on the intestinal STC-1 cells but stimulated their metabolic activity. Regarding this last point, more experiments are needed to confirm the ability of LB3F2 to stimulate proliferation of intestinal cells. To attribute nutraceutical applications to LB3F2 isolate, its ability to reduce pathogen adhesion and associated cytotoxicity against STC-1 cells remain to be done. The presence of foodborne pathogens in milk and in the dairy environment could be originated by several outbreaks including the ingestion of contaminated feed followed by their amplification in bovine hosts and fecal dissemination in the farm environment (Oliver et al., 2005) As for the Brazilian diary farms (Lee et al., 2014), the prevalence and persistence of S. aureus biofilm forming bacteria is of public health concern because unprocessed milk is regularly consumed by the Algerian population. This work highlighted the impact of lactobacilli LB14F1, LB15F1, LB2F2 and LB3F2 isolated from dairy environment on the staphylococcal biofilm formation by altering their adhesion on the steel stainless slides. Overall LB1F2, LB14F1, LB15F1, LB2F2 and LB3F2 offer pertinent academic research and real biotechnological applications. Acknowledgments The authors are grateful for the farmers (Akbou and Tazmalt, Bejaia, Algeria) who permitted the sampling in their farms, and rendering this project possible. The authors are indebted to Dr. Andres Hidalgo (CNIC, Madrid) and Pr. Mike Chikindas (Rutgers University, NJ) for English editing and critical reading of the manuscript. References Abdallah, M., Chataigne, G., Ferreira-Theret, P., Benoliel, C., Drider, D., Dhulster, P., Chihib, N.E., 2014. Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectant. Appl. Microbiol. Biotechnol. 98, 2597–2607. Abedi, D., Feizizadeh, S., Akbari, V., Jafarian-Dehkordi, A., 2013. In vitro anti-bacterial and anti-adherence effects of Lactobacillus delbrueckii subsp bulgaricus on Escherichia coli. Res. Pharm. Sci. 8, 260–268. Al Kassaa, I., Hamze, M., Hober, D., Chihib, N.E., Drider, D., 2014. identification of vaginal lactobacilli with potential probiotic properties isolated from women in North Lebanon. Microb. Ecol. 67, 722–734. Archer, N.K., Mazaitis, M.J., Costerton, J.W., Leid, J.G., Powers, M.E., Shirtliff, M.E., 2011. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence 2, 445–459. Belguesmia, Y., Madi, A., Sperandio, D., Merieau, A., Feuilloley, M., Prévost, H., Drider, D., Connil, N., 2011. Growing insights into the safety of bacteriocins: the case of enterocin S37. Res. Microbiol. 162, 159–163. Benoit, V., Lebrihi, A., Millière, J.B., Lefebvre, G., 1997. Purification and partial amino acid sequence of brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27. Curr. Microbiol. 34, 173–179. Brooks, J.L., Jefferson, K.K., 2012. Staphylococcal biofilms: quest for the magic bullet. Adv. Appl. Microbiol. 81, 63–87. Coventry, M.J., Wan, J., Gordon, J.B., Mawson, R.F., Hickey, M.W., 1996. Production of brevicin 286 by Lactobacillus brevis VB286 and partial characterization. J. Appl. Bacteriol. 80, 91–98. Danielsen, M., Wind, A., 2003. Susceptibility of Lactobacillus spp. to antimicrobial agents. Int. J. Food Microbiol. 82, 1–11. de Man, J.C., Rogosa, M., Sharpe, M.E., 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130–135. de Oliviera, M.M., Brugnera, D.F., Alves, E., Piccoli, R.H., 2010. Biofilm formation by Listeria monocytogenes on stainless steel surface and biotransfer potential. Braz. J. Microbiol. 41, 97–106. Donlan, R.M., 2002. Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8, 881–890. Drider, D., Rebuffat, S., 2011. Prokaryotic Antimicrobial Peptides: From Genes to Applications. Springer, (ISBN-13: 978-144197691). Dubernet, S., Desmasures, N., Guéguen, M., 2002. A PCR-based method for identification of lactobacilli at the genus level. FEMS Microbiol. Lett. 214, 271–275.

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