Biodiversity of Listeria monocytogenes sensitivity to bacteriocin-producing Carnobacterium strains and application in sterile cold-smoked salmon.

AIMS: The aim of this study was to demonstrate the inhibitory capacity of Carnobacterium strains against a collection of Listeria monocytogenes strains in cold-smoked salmon (CSS). METHODS AND RESULTS: Three bacteriocin-producing strains, Carnobacterium divergens V41, C. piscicola V1 and C. piscicola SF668, were screened for their antilisterial activity against a collection of 57 L. monocytogenes strains selected from the French smoked salmon industry, using an agar spot test. All the Listeria strains were inhibited but three different groups could be distinguished differing in sensitivity to the three Carnobacterium strains. However, C. divergens V41 always had the highest inhibitory effect. The antilisterial capacity was then tested in sterile CSS blocks co-inoculated with Carnobacterium spp. and mixtures of L. monocytogenes strains. C. divergens V41 was the most efficient strain, maintaining the level of L. monocytogenes at <50 CFU g(-1) during the 4 weeks of vacuum storage at 4 and 8 degrees C, whatever the sensitivity of the set of L. monocytogenes strains. CONCLUSIONS: C. divergens V41 may be a good candidate for biopreservation in CSS. SIGNIFICANCE AND IMPACT OF THE STUDY: A biopreservation strategy for CSS against the risk of L. monocytogenes was investigated using bacteriocin-producing lactic acid bacteria.

Impact of preheating on the behavior of Listeria monocytogenes in a broth that mimics Camembert cheese composition.

The effect of preheating on the survival of L. monocytogenes in Richard's broth, which mimics the composition of Camembert cheese composition, was examined. Experiments were carried out to reproduce contamination of cheese with environmental heat-stressed cells of L. monocytogenes surviving hot-cleaning procedures. Cells in mid-log phase were heated for 30 min at 56 degrees C before being inoculated into Richard's broth. The pHs and temperatures of Richard's broth were chosen to recreate the conditions of curd dripping (pH 5, 25 degrees C), of the beginning of cheese ripening (pH 5, 12 degrees C), and of the beginning (pH 5, 4 degrees C) and the end (pH 7, 4 degrees C) of cheese storage. Immediately after heat treatment, the viability loss was especially high for strain 306715, which exhibited only 0.6% +/- 0.2% survival, compared with 22% +/- 8.7% for strain EGD. The percentages of the surviving heated cells that were injured were 93% +/- 8% for strain 306715 and 98% +/- 3% for strain EGD. The destruction of the surviving L. monocytogenes cells was accelerated when they encountered the pH and temperature conditions of Camembert cheese during manufacturing, ripening, and cold storage (pH 5 at 25, 12, and 4 degrees C, respectively). The multiplication of the surviving heated cells was retarded under favorable growth conditions similar to those of storage by the distributor and the consumer (pH 7 at 4 and 12 degrees C, respectively).

Predictive models of the combined effects of curvaticin 13, NaCl and pH on the behaviour of Listeria monocytogenes ATCC 15313 in broth.

Thirty-three strains of Listeria monocytogenes belonging to different serotypes were tested for their sensitivity to curvaticin 13, an antilisterial bacteriocin produced by Lactobacillus curvatus SB13, using the well diffusion method in Institut Pasteur agar plates at 37 degrees C. No relationship between serotype and sensitivity was observed. The sensitivity of this species was strain-dependent and a large variation in tolerance to curvaticin 13 was observed. The combined effects of curvaticin 13 (0-160 AU ml-1), NaCl (0-6% w/v), pH values (5.0-8.2) and incubation time (0-24 h) were investigated on L. monocytogenes ATCC 15313 in trypcase soy-yeast extract broth at 22 degrees C. For this study, two Doehlert matrices were used in order to investigate the main effects of these factors and their different interactions. The results were analysed using the Response Surface Methodology. Curvaticin 13 had a major inhibitory effect and the response was NaCl concentration-, time- and pH-dependent. This inhibitory activity was the same at pH values between 6.6 and 8.2. Curvaticin 13 was bactericidic at acidic pH values, but the surviving cells resumed growth. For a short incubation time (12 h), the effectiveness of curvaticin 13 was maximal in the absence of NaCl. For longer incubation times (12-48 h), with high NaCl (6%) and curvaticin 13 concentrations (160 AU ml-1), the inhibition of L. monocytogenes was greater than that observed with NaCl or curvaticin 13 alone.

Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach.

The influence of pH (5.0-8.2), NaCl concentrations (0-6% w/v), and incubation time (0-24 h) on the inhibitory activity of nisin (0-100 I.U./ml) against Listeria monocytogenes (10(3) cfu/ml) was studied using the Doehlert experimental design and was confirmed by kinetic experiments. Predicted values were in agreement with experimental values. Experiments were carried out at 22 degrees C in reconstituted TSB-YE1 broth with or without NaCl. Nisin had an immediate pH-dependent bactericidal effect, which increased with decreasing pH values. In modified TSB-YE1 broth without NaCl, the bactericidal efficacy of nisin (50 I.U./ml) was maximum at pH 6.6, with no L. monocytogenes survivors until 120 h at 22 degrees C. Nisin (50 I.U./ml) action decreased in the presence of NaCl, with a minimal inhibitory effect between 2 and 4%. This partially protective effect was cancelled at higher levels of nisin.

Nisin-curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313.

Nisin (25-100 IU/ml) and curvaticin 13 (160 AU/ml), a bacteriocin produced by Lactobacillus curvatus SB13, were shown to have a bactericidal effect against Listeria monocytogenes ATCC 15313 in TSB-YE broth (pH 6.5), but it was only transitory. Regrowth was not due to the loss of bacteriocin activity. Cells surviving nisin or curvaticin 13 were more resistant to the respective bacteriocin than the parental strain. Survivors to curvaticin 13 were resistant to the class IIa bacteriocins (camocin CP5, pediocin AcH) but remained sensitive to nisin. The frequencies of spontaneous nisin resistants decreased with increasing bacteriocin concentration and the presence of salts (NaCl, K2HPO4). The behaviour of nisin (1000 IU/ml) or curvaticin 13 (640 AU/ml) resistant variants (Nis1000, Curv645) was investigated in the presence of nisin (100 IU/ml) or curvaticin 13 (320 AU/ml) at 22 and 37 degrees C, and compared with that of the parental strain. The effectiveness of nisin was the same at both temperatures, whereas curvaticin 13 displayed a faster bactericidal action at 37 degrees C. Nis1000 cells were less sensitive to curvaticin 13 than the parental strain, whereas Curv640 cells were more sensitive to nisin than the parental strain. Simultaneous or sequential additions of nisin (50 IU/ml) and curvaticin 13 (160 AU/ml) were performed at 22 degrees C in broth inoculated with the parental strain. All combinations induced a greater inhibitory effect than the use of a single bacteriocin. Simultaneous addition of bacteriocins at t0 led to the absence of viable cells in the broth after 48 h.

Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations.

The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 degrees C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml-1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1.25 x 10(8) cfu ml-1) after 7 d. Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) cfu ml-1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml-1), monolaurin (ranging from 0 to 300 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) spores ml-1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P < or = 0.001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml-1) and monolaurin (100 micrograms ml-1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6.0, without regrowth, within 7 d at 37 degrees C.

Prevalence of Listeria sp. in droppings from urban rooks (Corvus frugilegus).

Droppings from 112 urban rooks (Corvus frugilegus) were cultured for the presence of Listeria sp. Overall, 46% of rooks sampled harboured one or more Listeria species. Of all birds examined, 33%, 24% and 8%, respectively, were infected with Listeria monocytogenes, Listeria innocua and Listeria seeligeri. Differentiation of L. monocytogenes and L. seeligeri carried out by several phenotypic typing methods proved the diversity of strains and the major role of rooks which widely contribute to spreading this bacteria in our environment. The results also suggest that the ability to recover specific Listeria strains from the same sample is at least partially dependent on the methodology. These findings reinforce the need for strain-specific typing of multiple L. monocytogenes isolates from the same sample.