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Changes in spatial organization, as observed by confocal laser scanning microscopy (CLSM), viable cell content, biovolume, and substratum surface coverage of the biofilms formed on glass by  Pseudomonas fluorescens  resulting from co-culture with  Listeria monocytogenes , were examined. Two strains of  L. monocytogenes , two culture temperatures and two biofilm developmental stages were investigated. Both  L. monocytogenes  strains, a persistently sampled isolate (collected repeatedly along 3 years from a meat factory) and Scott A, induced shrinkage in matrix volume, both at 20°C and 4°C, in mature or old biofilms, without loss of  P. fluorescens  cell count per surface unit. The nearly homogeneous pattern of surface coverage shown by mono-species  P. fluorescens  biofilms, turned into more irregular layouts in co-culture with  L. monocytogenes . The upper layer of both mono and dual-species biofilms turned to predominantly consist of matrix, with plenty of viable cells underneath, in old biofilms cultured at 20°C, but not in those grown at 4°C. Between 15 and 56% of the substratum area was covered by biofilm, the extent depending on temperature, time and  L. monocytogenes  strain. Real biofilms in food-related surfaces may thus be very heterogeneous regarding their superficial components, i.e., those more accessible to disinfectants. It is therefore a hygienic challenge to choose an adequate agent to disrupt them.

Listeria monocytogenes Impact on Mature or Old Pseudomonas fluorescens Biofilms During Growth at 4 and 20°C