Inhibition of the early stage of Salmonella enterica serovar Enteritidis biofilm development on stainless steel by cell-free supernatant of a Hafnia alvei culture.

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.

Quorum sensing in food spoilage and natural-based strategies for its inhibition.

Food can harbor a variety of microorganisms including spoilage and pathogenic bacteria. Many bacterial processes, including production of degrading enzymes, virulence factors, and biofilm formation are known to depend on cell density through a process called quorum sensing (QS), in which cells communicate by synthesizing, detecting and reacting to small diffusible signaling molecules - autoinducers (AI). The disruption of QS could decisively contribute to control the expression of many harmful bacterial phenotypes. Several quorum sensing inhibitors (QSI) have been extensively studied, being many of them of natural origin. This review provides an analysis on the role of QS in food spoilage and biofilm formation within the food industry. QSI from natural sources are also reviewed towards their putative future applications to prolong shelf life of food products and decrease foodborne pathogenicity.

Listeria monocytogenes attachment to and detachment from stainless steel surfaces in a simulated dairy processing environment.

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (10(7) CFU/ml or CFU/g) and incubated at two temperatures (5 and 20 degrees C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5 degrees C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5 degrees C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20 degrees C). The opposite was observed when custard incubated at 20 degrees C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days of incubation (at 5 degrees C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5 degrees C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes cells.

Dynamics of biofilm formation by Listeria monocytogenes on stainless steel under mono-species and mixed-culture simulated fish processing conditions and chemical disinfection challenges.

The progressive ability of a six-strains L. monocytogenes cocktail to form biofilm on stainless steel (SS), under fish-processing simulated conditions, was investigated, together with the biocide tolerance of the developed sessile communities. To do this, the pathogenic bacteria were left to form biofilms on SS coupons incubated at 15°C, for up to 240h, in periodically renewable model fish juice substrate, prepared by aquatic extraction of sea bream flesh, under both mono-species and mixed-culture conditions. In the latter case, L. monocytogenes cells were left to produce biofilms together with either a five-strains cocktail of four Pseudomonas species (fragi, savastanoi, putida and fluorescens), or whole fish indigenous microflora. The biofilm populations of L. monocytogenes, Pseudomonas spp., Enterobacteriaceae, H2S producing and aerobic plate count (APC) bacteria, both before and after disinfection, were enumerated by selective agar plating, following their removal from surfaces through bead vortexing. Scanning electron microscopy was also applied to monitor biofilm formation dynamics and anti-biofilm biocidal actions. Results revealed the clear dominance of Pseudomonas spp. bacteria in all the mixed-culture sessile communities throughout the whole incubation period, with the in parallel sole presence of L. monocytogenes cells to further increase (ca. 10-fold) their sessile growth. With respect to L. monocytogenes and under mono-species conditions, its maximum biofilm population (ca. 6logCFU/cm2) was reached at 192h of incubation, whereas when solely Pseudomonas spp. cells were also present, its biofilm formation was either slightly hindered or favored, depending on the incubation day. However, when all the fish indigenous microflora was present, biofilm formation by the pathogen was greatly hampered and never exceeded 3logCFU/cm2, while under the same conditions, APC biofilm counts had already surpassed 7logCFU/cm2 by the end of the first 96h of incubation. All here tested disinfection treatments, composed of two common food industry biocides gradually applied for 15 to 30min, were insufficient against L. monocytogenes mono-species biofilm communities, with the resistance of the latter to significantly increase from the 3rd to 7th day of incubation. However, all these treatments resulted in no detectable L. monocytogenes cells upon their application against the mixed-culture sessile communities also containing the fish indigenous microflora, something probably associated with the low attached population level of these pathogenic cells before disinfection (<102CFU/cm2) under such mixed-culture conditions. Taken together, all these results expand our knowledge on both the population dynamics and resistance of L. monocytogenes biofilm cells under conditions resembling those encountered within the seafood industry and should be considered upon designing and applying effective anti-biofilm strategies.

The adherence of Salmonella Enteritidis PT4 to stainless steel: the importance of the air-liquid interface and nutrient availability.

Biofilm formation on stainless steel by Salmonella enterica serovar Enteritidis PT4 during growth in three different nutritious conditions was studied. The ability of micro-organisms to generate biofilms on the stainless steel surfaces was studied for a total period of 18 days at 20 degrees C, under three different experimental treatments: (i) growth medium (tryptone soy broth) was not refreshed (no further nutrients were provided) during the incubation period, (ii) growth medium was renewed every 2 days and (iii) growth medium was renewed every 2 days and at the same time the planktonic cells from the old medium were transferred to the new fresh medium. It was found that biofilms developed better and a higher number of adherent cells (ca. 10(7) cfu/cm(2)) were recovered when the organism was grown in periodically renewed nutrient medium than when the growth medium was not refreshed. Regardless of the availability of nutrients, biofilm development was better (range 2-3 logs greater) when coupons were not totally covered by the growth medium and part of the surface was exposed to the air-liquid interface, than when coupons were submerged in the medium. The results suggest that existence of air-liquid interface and adequate nutrient conditions provide the best environment for Salmonella Enteritidis PT4 biofilm formation on stainless steel. The possible role of stationary phase planktonic cells in biofilm development by sessile/attached microbial cells is also discussed.