Since most food plants have cold wet growth niches in production and storage areas, susceptibility testing should be performed on biofilms produced at refrigeration temperatures. Moreover, the efficiency of the sanitizers used in food industries should be performed on mixed culture biofilms, since in field conditions these will predominate. The results presented here highlight the importance of the temperature used for biofilm formation, when susceptibility to disinfectants is being assessed, as biofilms produced at lower temperature were less susceptible to sanitizers.

Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella’s cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella’s survival during desiccation.

Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.

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The aim of this work was to determine the ability of six yeast and two bacterial species associated with wine spoilage to form biofilms in mono-or co-culture using the Calgary Biofilm Device (CBD). Moreover, the efficacy of several disinfectants was evaluated against these spoilage microorganisms, both in the planktonic and the biofilm states. Results showed that Dekkera bruxellensis, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Schizosaccharomyces pombe and Acetobacter aceti formed biofilms both in wine and in synthetic medium. Zygosaccharomyces bailii formed biofilm only in wine and Pichia guilliermondii and Lactobacillus hilgardii formed biofilms only in synthetic medium. In wine, D. bruxellensis presented the same biofilm population when grown in pure culture or in mixed culture with acetic acid bacteria. There was a 3-log increase in biofilm formed by A. aceti in mixed culture with L. hilgardii. Alkaline chlorine-based disinfectant was the most effective in decontaminating spoilage yeast and bacteria both in planktonic and biofilm tests. Sodium hydroxide-based detergents and peracetic-based disinfectant were also efficient against suspended cells, but at least 10-fold more concentrated solutions were needed to remove the biofilms. Furthermore, the results showed that, except for the neutral detergent VK10, the tested agents were actually effective when used under the conditions recommended by manufacturers. In any case, biofilms showed greater tolerance to biocides when compared to the same microorganisms in the planktonic state. To our knowledge, this is the first study in which the CBD is used to assess the ability of wine spoilage microorganisms to form biofilms and their susceptibilities to disinfectant agents.

The food-borne pathogen Listeria monocytogenes is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119⌬lmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119⌬lmo2504 showed a significantly (P < 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities. Listeria monocytogenes is a food-borne pathogen able to persist in food processing environments. It is the causative agent of the severe human and animal disease invasive listeriosis, whose features frequently include meningitis or sepsis. Listeriosis has a mortality rate of 23.7% (1) and is especially severe in the elderly and in immunocompromised persons. In the case of pregnant women, it may lead to premature birth, abortion, or stillbirth.The ability of these bacteria to form biofilms is often associated with their ability to survive adverse conditions in food processing environments. The biofilm-forming ability of L. monocytogenes has been evaluated by several methods more or less correlated to the conditions found in the food industry, namely, the type of surface material (2). In addition to microbial cells, the biofilm matrix is composed of exopolysaccharides, lipids, glycolipids, DNA, and proteins (3). The evaluation of biofilm-forming ability may be performed by several methods that target different biofilm components. Methods such as the crystal violet assay (4) are directed toward the viable cells within the biofilm, and methods such as the ruthenium red assay (4) target the biofilm matrix exopolysaccharides. A more reliable comparison of strains is obtained when agreement...

The contamination of ready-to-eat products with Listeria monocytogenes has been related to the presence of biofilms in production lines, as biofilms protect cells from chemical sanitizers. The ability of L. monocytogenes to produce biofilms is often evaluated using in vitro methodologies. This work aims to compare the most frequently used methodologies, including high-throughput screening methods based on microplates (crystal violet and the Calgary Biofilm Device) and methods based on CFU enumeration and microscopy after growth on stainless steel. Thirty isolates with diverse origins and genetic characteristics were evaluated. No (or low) correlations between methods were observed. The only significant correlation was found between the methods using stainless steel. No statistically significant correlation (P > 0.05) was detected among genetic lineage, serovar, and biofilm-forming ability. Because results indicate that biofilm formation is influenced by the surface material, the extrapolation of results from high-throughput methods using microplates to more industrially relevant surfaces should be undertaken with caution.