Carnobacterium maltaromaticum: identification, isolation tools, ecology and technological aspects in dairy products.

Carnobacterium species constitute a genus of Lactic Acid Bacteria (LAB) present in different ecological niches. The aim of this article is to summarize the knowledge about Carnobacterium maltaromaticum species at different microbiological levels such as taxonomy, isolation and identification, ecology, technological aspects and safety in dairy products. Works published during the last decade concerning C. maltaromaticum have shown that this non-starter LAB (NSLAB) could present major interests in dairy product technology. Four reasons can be mentioned: i) it can grow in milk during the ripening period with no competition with starter LAB, ii) this species synthesizes different flavouring compounds e.g., 3-methylbutanal, iii) it can inhibit the growth of foodborne pathogens as Listeria monocytogenes due to its ability to produce bacteriocins, iv) it has never been reported to be involved in human diseases as no cases of human infection have been directly linked to the consumption of dairy products containing this species.

Interactions between two carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 on target bacteria and Caco-2 cells.

Two purified class IIa carnobacteriocins Cbn BM1 and Cbn B2, from Carnobacterium maltaromaticum CP5, were evaluated for antimicrobial activity against pathogenic, spoilage and lactic acid bacteria. Then, the presence of a synergistic mode of action of these two carnobacteriocins on Listeria sp., Enterococcus sp. and Carnobacterium sp. was investigated. A synergistic mode of action between Cbn BM1 and Cbn B2 on sensitive target bacteria was demonstrated using the FIC index method. Combinations of carnobacteriocins enhanced their antibacterial activities and MICs were significantly reduced, between 2- and 15-fold, by the addition of the second bacteriocin. To improve the safety of the bacteriocins as biopreservative agents, the cytotoxicity of the combination of theses two bacteriocins was determined on Caco-2 cell line. However, these two peptides used alone or in combination, at concentration 100-fold higher than those required for antimicrobial activity, were not cytotoxic. This suggests that the two carnobacteriocins produced by C. maltaromaticum CP5 could be potential natural agents for food preservation.

Functional differences in Leuconostoc sensitive and resistant strains to mesenterocin 52A, a class IIa bacteriocin.

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR52. The interaction of Mes 52A with bacterial membranes of sensitive, resistant and insensitive Leuconostoc strains has been investigated. The degree of insertion of Mes 52A on the phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane, and the consequence for K(+) efflux and proton motive force was analyzed. Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH in the membrane of the sensitive strain L. mesenteroides ssp. mesenteroides LMA 7, indicating that Mes 52A inserts into the cytoplasmic membrane of this sensitive strain. This insertion leads to K(+) efflux, without perturbation of DeltapH and a weak modification of DeltaPsi, and is consistent with pore formation. With the high-level resistant strain L. mesenteroides ssp. mesenteroides LMA 7AR, or with the insensitive strain Leuconostoc citreum CIP 103405, no modification of TMA-DPH or DPH anisotropy occurred, even in the presence of high Mes 52A levels. The membrane potential was not modified and no K(+) efflux was detected. There is a clear correlation between the physico-chemical characteristics of the membrane, the degree of Mes 52A penetration, the mechanism of action and the resistance or insensitivity characteristic of the target strains.

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism.

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K(+) efflux was measured, whereas the Delta Psi component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K(+) efflux, without perturbation of Delta pH and a weak modification of Delta Psi, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.

A selective enumeration medium for Carnobacterium maltaromaticum.

A selective medium was proposed for isolating the species Carnobacterium maltaromaticum from cheeses. This medium, named CM, was elaborated using combinations of three antibiotics (gentamicin, nalidixic acid, vancomycin) and alkaline pH value (8.8). An experimental design (Doehlert matrix) was drawn up to optimize the experimental conditions of the preparation of the medium. Based on the TS-YE agar medium, it contained 3.5 mg L(-1) of vancomycin, 5.0 mg L(-1) of gentamicin, and 20 mg L(-1) of nalidixic acid. The incubation time was 36 to 48 h at 25 degrees C. The selectivity of this medium was tested against bacterial strains present in the dairy industry and controlled by the PCR method. Thanks to this medium, it was easy to detect C. maltaromaticum and to follow this species in the cheese-making process.

Quantitative polymerase chain reaction used for the rapid detection of Carnobacterium species from French soft cheeses.

An identification method by PCR, specific to the Carnobacterium genus, was optimised by testing it on 28 bacterial strains. Primers from the literature were tested to differentiate Carnobacterium strains present among various bacterial species. The DNA of Carnobacterium species (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. viridans), specifically amplified by the Cb1-Cb2R primer couple at a hybridization temperature of 69 degrees C, gave only one band of 340 bp. The validation of this technique was carried out on a French soft cheese made with pasteurised milk inoculated with C. maltaromaticum LMA 28. Using classical PCR, detection was not possible for decimal dilutions of the cheese above 1 g L(-1). With Sybr Green I real time PCR, the specificity of the reaction was confirmed by the T(m) value. The standard curve constructed using the main threshold cycle and various concentrations of C. maltaromaticum LMA 28 (ranging from 10(0) to 10(8) cfu mL(-1)) showed good linearity and a sensitivity limit of > or = 10(4) cfu g(-1) of cheese. This technique was applied on commercially available cheeses made from raw cow's milk. The Sybr Green I real time PCR method constitutes an effective and easy-to-perform method to quantify Carnobacterium sp. in cheese.

Behavior of Escherichia coli cells and Bacillus cereus spores on poplar wood crates by impedance measurements.

Dry poplar wood crates and glass surfaces (bottoms of Pyrex flasks) were contaminated with aqueous suspensions of Escherichia coli Collection de l'Institut Pasteur (CIP) 54.8 cells or Bacillus cereus CIP 78.3 spores. After different periods of storage at 25 degrees C, the number of cells still present on both surfaces was determined by impedance microbiology. The physical and chemical properties of the wood greatly and rapidly decreased the number of cells. After 4 h of contact time, B. cereus residual metabolic activity corresponded to less than 10% of the initial inoculum level. With a low inoculum level of 2.4 x 10(2) E. coli cells, sterility of the wood was obtained after a contact time of 24 h. With a higher inoculum level of 10(6) E. coli cells, only a few viable bacteria, corresponding to the metabolic activity of four cells, could be recovered after a prolonged contact time of 145 h. Conversely, under the same conditions of storage, when bacteria were deposited on inert and nonporous materials such as glass surfaces, growth occurred. After 24 h of contact time at 25 degrees C, bacterial populations were about 10(9) cells for E. coli and 10(7) cells for B. cereus. Under our experimental conditions after a prolonged contact time, wood exhibited growth-inhibiting properties and cells were no longer metabolically active. These results indicate that the potential for cross-contamination of foods stored directly in contact with previously contaminated poplar wood crates is low under our experimental conditions.

Cell envelope analysis of insensitive, susceptible or resistant strains of Leuconostoc and Weissella genus to Leuconostoc mesenteroides FR 52 bacteriocins.

Mesenterocins 52A and 52B belong to class II of lactic acid bacteria bacteriocins. To study susceptibility, insensitivity and resistance to these mesenterocins, four wild-type bacterial strains and four resistant strains, all from Leuconostoc or Weissella genus, were compared. Several cell envelope features were investigated: susceptibilities to antibiotics and to lysozyme, cell morphology and membrane phospholipids contents. The strain insensitive to the two mesenterocins appeared to be resistant to lysozyme and exhibited the highest resistance to antibiotics. Resistant strains displayed cell morphology modifications, several increases in antibiotic resistance and modifications in lysozyme susceptibility. Moreover, mesenterocin 52A-resistant strains displayed modifications in their membrane phospholipids, leading to a more cationic membrane. Insensitivity and resistance of Leuconostoc or Weissella strains seem to be due to various minor modifications of the membrane and/or of the cell wall.

Synergistic mode of action of mesenterocins 52A and 52B produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52.

Few studies have been published on the effects of two bacteriocins combinations and particularly on combinations of two bacteriocins with different structures produced by the same strain. In this work, the actions of mesenterocin 52A (class IIa) and mesenterocin 52B (class II), produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52, were studied on strains susceptible to only one bacteriocin or to both. In broth, combination of mesenterocins enhanced the adaptation time of the strain susceptible to the both mesenterocins (48 h vs 17 h with only one bacteriocin). In agar medium, mesenterocins displayed, as expected, a synergistic effect on this strain (FIC(index) < 1), but also on the two strains susceptible to only one mesenterocin. This original result was probably due to membrane composition modifications induced by the mesenterocin that enhanced bacteriocin action. Thus, this hurdle technique seems to be interesting in food preservation in terms of minimizing bacteriocin concentrations.

Variations in the membrane fatty acid composition of resistant or susceptible Leuconostoc or Weissella strains in the presence or absence of Mesenterocin 52A and Mesenterocin 52B produced by Leuconostoc mesenteroides subsp. mesenteroides FR52.

Mesenterocins 52A (Mes52A) and 52B (Mes52B) are antimicrobial peptides produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52. Mes52A is a class IIa bacteriocin of lactic acid bacteria with a broad spectrum of activity. Mes52B is an atypical class II bacteriocin with a narrow spectrum of activity. Four Leuconostoc and Weissella wild-type strains were selected for their susceptibility or insensitivity to these mesenterocins. Four strains resistant to Mes52A or Mes52B were generated from the three susceptible wild-type strains by increasing bacteriocin concentrations in culture media. These resistant strains were at least 30 times more resistant than the wild-type strains. No cross-resistance to Mes52A and Mes52B was observed in these strains. No significant differences in membrane fatty acid composition were observed among the three susceptible wild-type strains and the four resistant strains cultured in MRS broth. Thus, the mesenterocin resistance is unlikely to be due to changes in membrane fatty acid composition. When cultured with Mes52A or Mes52B, the membranes of insensitive and resistant strains contained more saturated fatty acids (1 to 10% more) and less unsaturated fatty acids (3 to 6% less), resulting in a more rigid membrane. Thus, the presence of mesenterocin in the culture media of insensitive or resistant strains induced a significant increase in saturated fatty acid contents and a decrease in unsaturated fatty acid contents. Weissella paramesenteroides DSM 20288BR, resistant to Mes52B, responded atypically, probably due to the production of an inhibitor.

Characterization of anti-Listeria monocytogenes bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis strains isolated from Raïb, a Moroccan traditional fermented milk.

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis [4], Enterococcus faecium [4], and E. faecalis [1]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30 degrees C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.