Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BM medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log(10) cfu/well) and that they contained 1-2 log(10) cfu/well more L. monocytogenes than L plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log(10) cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log(10) more L plantarum than L monocytogenes cells (total count, 9 log(10) cfu/well). Corresponding single species biofilms of L. monocytogenes and L plantarum contained up to 9 log(10) cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L plantarum grown in the mixed species biofilm showed less than 2 log(10) cfu/well inactivation after exposure for 15 min to 100 mu g/ml benzalkonium chloride, while single species biofilms of both L monocytogenes strains showed 4.5 log(10) cfu/well inactivation and single species biofilms of L plantarum showed 3.3 log(10) cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms. (C) 2010 Elsevier B.V. All rights reserved.