Chitosan has proven antimicrobial properties against planktonic cell growth. Little is known, however, about its effects on already established biofilms. Oriented for application in food industry disinfection, the effectiveness of both medium molecular weight (MMW) chitosan and its enzymatically hydrolyzed product was tested against mature biofilms of four pathogenic strains, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus and Salmonella enterica, and a food spoilage species, Pseudomonas fluorescens. Unexpectedly, log reductions were in some cases higher for biofilm than for planktonic cells. One hour exposure to MMW chitosan (1% w/v) caused a 6 log viable cell reduction on L. monocytogenes monospecies mature biofilms and reduced significantly (3–5 log reductions) the attached population of the other organisms tested, except S. aureus. Pronase-treated chitosan was more effective than MMW chitosan on all tested microorganisms, also with the exception of S. aureus, offering best results (8 log units) against the attached cells of B. cereus. These treatments open a new possibility to fight against mature biofilms in the food industry.

In food facilities, biofilms or their debris might act as helpers for attracting free floating microorganisms. In this sense, Pseudomonas fluorescens, a dense biofilm producer frequently isolated from food contact surfaces, could be a good candidate for sheltering other microorganisms, such as Listeria monocytogenes. The main objective of this work was to evaluate the ability of L. monocytogenes to colonize pre-established Pseudomonas biofilms. For this, the movement throughout mature Pseudomonas biofilms of a green fluorescent protein (GFP) – tagged strain of L. monocytogenes was tracked for 24 h by confocal laser scanning microscopy (CLSM). Moreover, in order to check the effect of the incorporation of Listeria on the overall matrix production, attached populations of both microorganisms and total biomass (cells + matrix) of the resulting biofilms were measured over time. Planktonic cells of L. monocytogenes efficiently migrated to preformed P. fluorescens biofilms. Moreover, they moved preferentially toward the bottom layers of these structures, suggesting some kind of tropism. When preformed P. fluorescens biofilms were conditioning the surfaces, the L. monocytogenes attached population was on average, 1–2 Log higher than when this organism grew on bare coupons. Furthermore, the arrival of L. monocytogenes to the already established P. fluorescens biofilms led to a matrix over-production. Indeed, biomass values [optical density (OD595 nm)] of the resulting biofilms were double those of the ordinary L. monocytogenes–P. fluorescens mixed biofilms (1.40 vs. 0.6). The fact that L. monocytogenes cells accumulate in the bottom layers of preformed biofilms provides this microorganism an extra protection toward physical–chemical damages. This might partly explain why this microorganism can persist in food industry environments.

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.

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In this study the use of TiO2 nanoparticles in the preparation of active packaging film materials is investigated.

22This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium 23 chloride (BAC) sequential treatment to remove Listeria monocytogenes-Escherichia 24 coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy 25 and plate count assays. 26The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-27 species samples were determined using a first-order factorial design. Empirical equation

Enzyme-based treatments could therefore be used to complement regular cleaning processes. Most studies using enzymes as anti-biofilms strategy are focused on their outcome in mono-species biofilms. Nevertheless, in real environments mixed biofilms are prevalent. In this work, seven types of dual-species biofilms were selected to serve as targets for enzymatic treatments carrying different environmental strains of L. monocytogenes and accompanying bacteria isolated from dairy, meat and seafood processing plants. The effectiveness of nine commercial enzymatic preparations, including pronase, cellulase, pectinase, DNase I, lysozyme, phospholipase, peroxidase, β-glucanase and chitinase, was evaluated. For this, residual attached viable cells of both L. monocytogenes and its partners were enumerated through swabbing and colony plate counting following the action of each enzyme. Moreover, Confocal Laser Scanning Microscopy (CLSM) images were analyzed pre and after enzymatic treatments in order to quantify changes in biofilm thickness, covered area and volume. The viable attached population of L. monocytogenes was almost unaffected by all of the enzymes here tested, being eliminated on average just the 90% of the initially attached population (around 1 Log 10 cfu•cm-2 reduction). Nevertheless, some of the partner species

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