, 1993). Also, L. plantarum is able to form biofilms and cause spoilage of food products ( Kubota et al., 2008 and Kubota et al., 2009). In this study, we investigated the formation of single and mixed species biofilms of L. monocytogenes and L. plantarum and the resistance of these single and mixed species biofilms to the disinfectants benzalkonium chloride and peracetic acid. Benzalkonium chloride and peracetic acid are two of the most widely used disinfectants in the food industry ( Ceragioli et al., 2010). Benzalkonium chloride is a member of the quaternary selleck chemicals llc ammonium compounds that target cell membranes, while peracetic acid is an oxidizing agent that decomposes into safe waste products. Furthermore, we were
able to modulate the contribution of both species to the mixed species biofilm with the addition of manganese sulfate and/or glucose to the growth medium to obtain mixed species biofilms containing equal number of
bacteria from both species or mixed species biofilms in which one of the species is dominant. This allowed us to estimate whether a protective effect from one species to the other in the mixed species biofilm is dependent on the number bacteria from each species in the mixed species biofilm. Single and mixed species biofilms were visualized using phase contrast and fluorescence microscopy on cells constitutively expressing the optimized fluorescent proteins EGFP, ECFP, EYFP, or DsRed. The original genes that encode for these proteins contain codons that are optimal for expression in eukaryotic cells, while they are infrequently
used by bacteria. selleck kinase inhibitor Therefore, we modified these genes by replacing the infrequently used codons by codons that are more frequently used by L. monocytogenes and L. plantarum. L. monocytogenes strains EGD-e and LR-991 and derivatives thereof ( Table 1) were stored in Brain Hearth Infusion (BHI) broth (Becton Dickinson, Le Pont de Claix, France) containing 15% sterile glycerol (Fluka, Buchs, Switserland) at -80 °C. L. plantarum WCFS1 and derivatives ( Table 1) were stored in De Man, Rogosa and Sharpe (MRS) broth (Merck, Darmstadt, Germany) containing 15% sterile glycerol at -80 °C. BHI or MRS agar plates were streaked with cells from the -80 °C bacterial stocks using an inoculation needle CYTH4 and plates were incubated at 30 °C for 24 h. Single colonies were inoculated in BHI broth, BHI broth with addition of 0.005% manganese sulfate (Merck, Darmstadt, Germany) (BHI-Mn), based on the concentration of the specific Lactobacillus medium MRS ( de Man et al., 1960), or BHI broth with addition of 0.005% manganese sulfate and 2% glucose (Merck, Darmstadt, Germany) (BHI-Mn-G) and grown for 18 h at 20 °C. Recombinant DNA techniques were performed according to standard protocols (Sambrook et al., 1989). Sequences of genes expressing the fluorescent proteins EGFP, ECFP, EYFP, and DsRed were optimized to replace codons that are infrequently used by L. monocytogenes and L.