WORKPACKAGE 2.5: Potential risks associated with strategies DELIVERABLE 2.5.6: Data on how bacterial interactions contribute to (i) biofilm formation ability of individual strains, and (ii) their resistance to sanitizers PILLAR 2: Control and intervention strategies along the fork-to-farm chain to ensure beef safety Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens Vienna, March 2010

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Biofilm formation & implications in beef industry In the meat industry, biofilms of both spoilage and pathogenic bacteria may be related to serious problems of food contamination (lowered shelf-life of products, disease transmission) In the majority of natural & industrial environments, monospecies biofilms are relatively rare Conversely, microorganisms are associated with surfaces in complex multispecies communities Bacterial interactions are believed to influence the biofilm forming capacity of individual strains, as well as their antimicrobial resistance

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Main objectives / tasks 1. Investigate attachment to and biofilm forming ability on model abiotic surfaces of some food-relevant bacteria in monoculture and in mixed-culture 1. Evaluate disinfection efficiency of some commercial disinfectants against mono & mixed-culture biofilms

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D bacterial strains* screened for biofilm formation These belonged to bacterial species which are typically found in complex food industrial ecosystems o Listeria monocytogenes (11 strains) o Salmonella enterica (8 strains) o Staphylococcus aureus (3 strains) o Pseudomonas sp. (6 species/strains) o Lactobacillus sakei (11 strains) representatives of pathogens representatives of spoilage bacteria representatives of useful technological bacteria * All tested strains had been previously identified by 16S rRNA analysis and separated by PFGE P. fluorescens, P. fragi, P. aeruginosa, P. phsychrophilla, P. gessardii, Pseudomonas sp.

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Isolation origin of strains All strains were provided by the microorganisms collection of Laboratory of Microbiology and Biotechnology of Foods* (Department of Food Science and Technology, AUA) & had been previously isolated from different sources * Code used FMCC_B, Food Microbiology Culture Collection_Bacteria

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Surfaces used Two surfaces of different physicochemical properties were used as abiotic substrates for biofilm development: 1. Polystyrene (PS) - 96-well microplates 2. Stainless steel (SS) - rectangular coupons of 3 x 1 x 0.1 cm, type AISI material commonly used for the manufacture of food- processing equipment

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Biofilm formation assay using PS microplates Commonly applied method for easy screening biofilm formation by different strains (many repetitions) Stain biofilm cells with crystal violet, dissolving bound dye by ethanol/acetone & quantification with absorbance measurements (A 575nm ) microplate reader PS microplate with stained biofilm cells

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Temperature: 15 o C Growth media: TSB and 1/10 dTSB Initially, bacteria were left to adhere on PS microplates for 3 h (at 15 o C). For this bacterial suspension of ca cfu/ml in ¼ Ringer solution was used Loosely attached cells were then removed by rinsing (with ¼ Ringer) Growth media were added, followed by incubation under static conditions (except for Pseudomonas sp.) for 48 h Growth media were renewed at 24 h Biofilm formation assay using PS microplates

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Results: biofilm formation on PS microplates

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Results: biofilm formation on PS microplates For most L. monocytogenes strains no significant differences were observed on biofilm formation between the 2 growth media All S. enterica strains (except FMCC_B-62) formed more biofilm (p < 0.05) when cultured in 1/10 TSB compared to TSB The 3 Staph. aureus and the 11 L. sakei strains were poor biofilm producers in both nutritional conditions Pseudomonas fluorescens (FMCC_B-29), Pseudomonas aeruginosa (FMCC_B-26) and Pseudomonas gessardii (FMCC_B- 46) formed high amount of biofilm in both growth media. On the contrary, the other 3 Pseudomonas species produced low biofilm

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Sterile SS coupons were fully immersed in bacterial suspensions of ca cfu/ml in ¼ Ringer solution for 3 h at 15 o C (ATTACHMENT STEP) Loosely attached cells were removed by rinsing (with ¼ Ringer) Coupons were then incubated in TSB at 15 o C for 6 days (144 h) (BIOFILM FORMATION STEP) Growth medium was renewed every 48 h Biofilm formation assay using SS coupons SS coupons in TS broth

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Quantification of biofilm formation on SS coupons Method based on detaching attached biofilm cells by “bead vortexing” followed by quantification by “agar plating”

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Results: Attachment to and biofilm formation on SS coupons The attachment ability of each strain was expressed as the percentage (%) of cells being attached, compared to the total population of cells contained in bacterial suspension in which the SS coupon was immersed (for 3 h)

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Results: Attachment to and biofilm formation on SS coupons Variations at levels of attachment and biofilm formation for each species Relationship between attachment & biofilm forming ability for the 39 bacterial strains

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Future work (…to be done the next 6 months) Select 3 strains from each species & test biofilm formation on SS in monospecies mixed culture  Study multispecies biofilm formation on SS - L. monocytogenes – S. enterica - L. monocytogenes – S. aureus - L. monocytogenes – Pseudomonas sp. - L. monocytogenes – L. sakei - L. monocytogenes – S. enterica - S. aureus – Pseudomonas sp. – L. sakei Test disinfection efficiency of 3 commercial disinfectants (benzalkonium chloride, chlorine, PAA) against mono- and mixed-culture biofilms DUAL SPECIES

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Thank you Acknowledgments BSc Student Elli Braxou