Listeria monocytogenes Colonizes Pseudomonas fluorescens Biofilms and Induces Matrix Over-Production.

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 (OD595nm)] 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.

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

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.

Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52.

The polysaccharides associated with free (planktonic) and surface-attached (biofilm) cells from cultures of Pseudomonas fluorescens strain B52 were compared. Variations in the attached matrix due to surface material (glass or stainless steel) were also analyzed. Two digestion methods were used to optimize the recoveries of sugars, uronic acids and acidic substituents. The yield of analyzable material after digestion reached 90% for the material associated to the biofilms, though only 20-30% for that bound to planktonic cells. The polysaccharide(s) in the biofilm had glucuronic and guluronic acids as main components, besides rhamnose, glucose and glucosamine. The proportion of glucuronic to guluronic acid was higher in the polysaccharide(s) found in biofilms formed on stainless steel than in those on glass.

Cooling Raw Milk: Change in the Spoilage Potential of Contaminating Pseudomonas.

A group of 80 Pseudomonas spp. strains isolated from raw milk shortly after milking was compared to another group of 81 obtained from the same sample after incubating it at 7°C for 3 days. Comparison of both collections of strains included growth rates at 7°C and 21°C and production of extracellular proteinase, lipase, and siderophores. The strains selected after cold incubation showed an average to-fold higher growth rate at 7°C, 1,000-fold more proteolytic activity, and 280-fold more lipolytic activity than those found before the incubation. At 21°C, however, they grew half as quickly as the strains isolated before the incubation. In all but one of the 161 Pseudomonas strains tested, there was some production of siderophores, and yields were only moderately increased in the strains obtained after incubation of the milk at 7°C. These changes in spoilage-related properties took place while global Pseudomonas counts increased only 13-fold. Distribution frequencies of the variables tested, their correlation coefficients, and regression models are shown.

Compositional Changes in Cold Raw Milk Supporting Growth of Pseudomonas fluorescens NCDO 2085 before Production of Extracellular Proteinase.

Changes in milk native content of several carbon and nitrogen sources were studied, along with growth at 7°C of a Pseudomonas fluorescens strain. The aim was to characterize the particular compositional environment in milk in which psychrotrophic bacteria produce their extracellular proteinases. Glucose and lactate were depleted from milk, pyruvate and gluconate were significantly diminished, but citrate was mostly unused when proteinase was first detected by the Hide Powder Azure assay, the psychrotrophic count being around 1010 CFU/ml. At that stage, levels of ammonia, amino acids and short peptides had just started to rise and only about 20% of the original urea had been consumed. A procedure to anticipate, in cold stored raw milk batches, the time for production of extracellular proteinase, on the basis of sensitive lactate and ammonia determination, is suggested.