Effect of fermented milk from Lactococcus lactis ssp. cremoris strain JFR1 on Salmonella invasion of intestinal epithelial cells.

The process of fermentation contributes to the organoleptic properties, preservation, and nutritional benefits of food. Fermented food may interfere with pathogen infections through a variety of mechanisms, including competitive exclusion or improving intestinal barrier integrity. In this study, the effect of milk fermented with Lactococcus lactis ssp. cremoris JFR1 on Salmonella invasion of intestinal epithelial cell cultures was investigated. Epithelial cells (HT29-MTX, Caco-2, and cocultures of the 2) were treated for 1 h with Lactococcus lactis ssp. cremoris JFR1 fermented milk before infection with Salmonella enterica ssp. enterica Typhimurium. Treatment with fermented milk resulted in increased transepithelial electrical resistance, which remained constant for the duration of infection (up to 3 h), illustrating a protective effect. After gentamicin treatment to remove adhered bacterial cells, enumeration revealed a reduction in numbers of intracellular Salmonella. Quantitative reverse-transcription PCR data indicated a downregulation of Salmonella virulence genes hilA, invA, and sopD after treatment with fermented milk. Fermented milk treatment of epithelial cells also exhibited an immunomodulatory effect reducing the production of proinflammatory IL-8. In contrast, chemically acidified milk (glucono delta-lactone) failed to show the same effect on monolayer integrity, Salmonella Typhimurium invasion, and gene expression as well as immune modulation. Furthermore, an oppA knockout mutant of Salmonella Typhimurium infecting treated epithelial cells did not show suppressed virulence gene expression. Collectively, these results suggest that milk fermented with Lactococcus lactis ssp. cremoris JFR1 is effective in vitro in the reduction of Salmonella invasion into intestinal epithelial cells. A functional OppA permease in Salmonella is required to obtain the antivirulence effect of fermented milk.

Symposium review: Interaction of starter cultures and nonstarter lactic acid bacteria in the cheese environment.

The microbiota of ripening cheese is dominated by lactic acid bacteria, which are either added as starters and adjunct cultures or originate from the production and processing environments (nonstarter or NSLAB). After curd formation and pressing, starters reach high numbers, but their viability then decreases due to lactose depletion, salt addition, and low pH and temperature. Starter autolysis releases cellular contents, including nutrients and enzymes, into the cheese matrix. During ripening, NSLAB may attain cell densities up to 8 log cfu per g after 3 to 9 mo. Depending on the species and strain, their metabolic activity may contribute to defects or inconsistency in cheese quality and to the development of typical cheese flavor. The availability of gene and genome sequences has enabled targeted detection of specific cheese microbes and their gene expression over the ripening period. Integrated systems biology is needed to combine the multiple perspectives of post-genomics technologies to elucidate the metabolic interactions among microorganisms. Future research should delve into the variation in cell physiology within the microbial populations, because spatial distribution within the cheese matrix will lead to microenvironments that could affect localized interactions of starters and NSLAB. Microbial community modeling can contribute to improving the efficiency and reduce the cost of food processes such as cheese ripening.

Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.

Linkage disequilibrium (LD) mapping provides a powerful method for fine-structure localization of rare disease genes, but has not yet been widely applied to common disease. We sought to design a systematic approach for LD mapping and apply it to the localization of a gene (IBD5) conferring susceptibility to Crohn disease. The key issues are: (i) to detect a significant LD signal (ii) to rigorously bound the critical region and (iii) to identify the causal genetic variant within this region. We previously mapped the IBD5 locus to a large region spanning 18 cM of chromosome 5q31 (P<10(-4)). Using dense genetic maps of microsatellite markers and single-nucleotide polymorphisms (SNPs) across the entire region, we found strong evidence of LD. We bound the region to a common haplotype spanning 250 kb that shows strong association with the disease (P< 2 x 10(-7)) and contains the cytokine gene cluster. This finding provides overwhelming evidence that a specific common haplotype of the cytokine region in 5q31 confers susceptibility to Crohn disease. However, genetic evidence alone is not sufficient to identify the causal mutation within this region, as strong LD across the region results in multiple SNPs having equivalent genetic evidence-each consistent with the expected properties of the IBD5 locus. These results have important implications for Crohn disease in particular and LD mapping in general.

Exploring suppression subtractive hybridization (SSH) for discriminating Lactococcus lactis ssp. cremoris SK11 and ATCC 19257 in mixed culture based on the expression of strain-specific genes.

AIM: An approach based on quantitative reverse transcriptase PCR (RT-qPCR) was developed for monitoring two strains of lactococci in co-culture in milk by measuring the expression of specific genes identified by suppression subtractive hybridization (SSH). METHODS AND RESULTS: SSH was used to identify strain-specific genes of Lactococcus lactis ssp. cremoris SK11 and ATCC 19257. RT-qPCR was then employed to validate gene specificity and compare the expression of selected specific genes (glycosyltransferase and amidase genes for L. lactis ssp. cremoris ATCC 19257 and a hypothetical protein for SK11) identified by SSH. The time profile of changes in gene expression relative to ldh transcription differed between pure and mixed cultures as well as between media. At the stationary phase, gene expression of mixed cultures in GM17 attained the highest proportion of ldh transcription while mixed cultures in milk peaked at the postexponential phase. Strain ratios expressed as RNA proportion appear to favour SK11 in GM17 medium, while ATCC 19257 dominated in milk co-cultures. CONCLUSIONS: This approach was useful to determine the contribution of strain SK11 in relation to strain ATCC 19257 during co-culture in milk compared to rich medium. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to track the metabolic contribution of each lactococcal strain during fermentation of milk or cheese ripening will extend our understanding of the impact of process parameters on the production performance of strains.

Comparative antimicrobial activity of two new mutacins.

OBJECTIVES: To compare the in vitro activity of mutacins D-123.1 and F-59.1 against different bacteria including antibiotic-resistant strains, in order to evaluate their application potential. DESIGN AND METHODS: The antibacterial activity spectrum of purified F-59.1 and the MIC and MBC of F-59.1 and D-123.1 against target bacteria were determined. RESULTS: Most bacteria were inhibited by the purified mutacins. Mutacin F-59.1 shows a relatively wide activity spectrum. Mutacin D-123.1 has low Minimum Inhibitory Concentrations [MICs] (0.25-4 microg/ml) against human pathogens while F-59.1 has higher MICs (3.2-12.8 microg/ml) mainly against food-borne pathogens. CONCLUSION: The effectiveness of mutacins D-123.1 and F-59.1 against human and food-borne pathogens is demonstrated. Mutacin D-123.1 shows potential as a new antibiotic while F-59.1 shows promising application in food products.

A comprehensive perspective of food nanomaterials.

Nanotechnology is a rapidly developing toolbox that provides solutions to numerous challenges in the food industry and meet public demands for healthier and safer food products. The diversity of nanostructures and their vast, tunable functionality drives their inclusion in food products and packaging materials to improve their nutritional quality through bioactive fortification and probiotics encapsulation, enhance their safety due to their antimicrobial and sensing capabilities and confer novel sensorial properties. In this food nanotechnology state-of-the-art communication, matrix materials with particular focus on food-grade components, existing and novel production techniques, and current and potential applications in the fields of food quality, safety and preservation, nutrient bioaccessibility and digestibility will be detailed. Additionally, a thorough analysis of potential strategies to assess the safety of these novel nanostructures is presented.

Use of antisense RNA to modulate glycosyltransferase gene expression and exopolysaccharide molecular mass in Lactobacillus rhamnosus.

Discovery of gene function requires inactivation in order to demonstrate the effect of the absence of gene expression on cell phenotype. As gene inactivation can be lethal, such mutations are often unattainable. Antisense RNA provides a method of reducing transcript and protein levels without totally inactivating the targeted gene, thus providing information on the gene's possible function. This study demonstrates the use of antisense RNA to modulate polysaccharide size in Lactobacillus rhamnosus, a bacterial species with technological and health applications in fermented milk products. Production of antisense RNA coding for a glycosyltransferase leads to reduced sense RNA transcript. While the total amount of polysaccharide produced was not significantly affected, size exclusion chromatography showed that polysaccharides of different molecular mass were produced in the presence of antisense RNA. Conditional control over gene expression could thus be useful for metabolic engineering strategies, where gene inactivation is not practicable.

Improvement of texture and structure of reduced-fat Cheddar cheese by exopolysaccharide-producing lactococci.

The objective of this study was to evaluate the effect of capsular and ropy exopolysaccharide (EPS)-producing strains of Lactococcus lactis ssp. cremoris on textural and microstructural attributes during ripening of 50%-reduced-fat Cheddar cheese. Cheeses were manufactured with added capsule- or ropy-forming strains individually or in combination. For comparison, reduced-fat cheese with or without lecithin added at 0.2% (wt/vol) to cheese milk and full-fat cheeses were made using EPS-nonproducing starter, and all cheeses were ripened at 7 degrees C for 6 mo. Exopolysaccharide-producing strains increased cheese moisture retention by 3.6 to 4.8% and cheese yield by 0.28 to 1.19 kg/100 kg compared with control cheese, whereas lecithin-containing cheese retained 1.4% higher moisture and had 0.37 kg/100 kg higher yield over the control cheese. Texture profile analyses for 0-d-old cheeses revealed that cheeses with EPS-producing strains had less firm, springy, and cohesive texture but were more brittle than control cheeses. However, these effects became less pronounced after 6 mo of ripening. Using transmission electron microscopy, fresh and aged cheeses with added EPS-producing strains showed a less compact protein matrix through which larger whey pockets were dispersed compared with control cheese. The numerical analysis of transmission electron microscopy images showed that the area in the cheese matrix occupied by protein was smaller in cheeses with added EPS-producing strains than in control cheese. On the other hand, lecithin had little impact on both cheese texture and microstructure; after 6 mo, cheese containing lecithin showed a texture profile very close to that of control reduced-fat cheese. The protein-occupied area in the cheese matrix did not appear to be significantly affected by lecithin addition. Exopolysaccharide-producing strains could contribute to the modification of cheese texture and microstructure and thus modify the functional properties of reduced-fat Cheddar cheese.

Purification and structure of mutacin B-Ny266: a new lantibiotic produced by Streptococcus mutans.

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post-translationally modified amino acids such as lanthionine. Mutacin B-Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed-phase chromatography (Sep-Pak cartridge) and by HPLC on a C18 column. The mean purification factor was 3240 +/- 81 and the mean yield was 1.0 +/- 0.1%. Molecular mass of mutacin B-Ny266 as determined by mass spectroscopy is 2270.29 +/- 0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty-one amino acids were detected in this analysis. Mutacin B-Ny266 belongs to the type A lantibiotics. The proposed sequence is: F-K-A-W-U-F-A-Abu-P-G-A-A-K-O-G-A-F-N-U-Y-A. The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.

A pilot study on upper esophageal sphincter dilatation for the treatment of dysphagia in patients with oculopharyngeal muscular dystrophy.

Upper esophageal sphincter (UES) dilatation was done for the treatment of moderate to severe dysphagia with a Maloney bougie in 14 patients with oculopharyngeal muscular dystrophy (OPMD) or with an achalasia dilator in three patients. The severity of dysphagia prior to UES dilatation was evaluated by a 15-point dysphagia score, a pharyngeal and esophageal manometry and a radionuclide pharyngoesophageal transit study. Using actuarial life table, the improvement rate after dilatation with Maloney bougie was 64.3% (95% CI 39.2-89.4) at 3- and 6-month follow-ups, and was 55.7% (95% CI 28.9-82.5) at 12- and 18-month follow-ups. At 3-month post-dilatation, we observed a significant reduction of the mean dysphagia score from 9.6 to 7.2 (P = 0.05). No significant manometric or radionuclide factors were found to predict effective dilatation. The results of this pilot study showed that UES dilatation with Maloney bougie or achalasia dilator may be an effective treatment of moderate dysphagia in patients with OPMD. However, further studies with larger sample sizes are needed to corroborate these results and to assess long-term outcome.

Tobacco plants expressing T4 endonuclease V show enhanced sensitivity to ultraviolet light and DNA alkylating agents.

DNA repair processes and UV-filtering pigments protect organisms from the cytotoxicity of UV light and endow plants with a high degree of natural UV resistance. In an attempt to further enhance this UV resistance we have constructed transgenic tobacco lines that express a DNA repair enzyme encoded by the bacteriophage T4 denV gene. The denV gene encodes endonuclease V, an enzyme which initiates base excision repair of cyclobutane pyrimidine dimers. Its presence is expected to provide transgenotes with a repair pathway complementary to, but likely distinct from, the repair pathways found in tobacco. The denV gene, flanked by a CaMV 35S promoter and poly(A) addition site, was introduced into tobacco and mature plants regenerated. The transgenotes expressed high levels of a UV-specific endonuclease and no such activity was found in control plants. Curiously, assays which detected several different biological endpoints showed that the denV+ transgenotes were also hypersensitive to UV-C light. This hypersensitivity segregated with the denV gene and was not caused by altered concentrations of UV-filtering pigments. Moreover, the denV+ transgenotes were also hypersensitive to high levels of baseless lesions that would be generated by a transgenically expressed beta-eliminating lyase such as endonuclease V.

Investigation on the induction of 14-3-3 in white spruce.

A cDNA encoding a 14-3-3 protein was isolated from white spruce. The corresponding polypeptide contains several motifs that are conserved in this type of protein and is predicted to be 260 amino acids in length. Multiple banding in Southern blot analysis suggests that the gene encoding this cDNA is, in fact, part of a small family of genes. Wounding and chitosan treatment of spruce plants followed by Northern blot analysis indicates that these stimuli caused the accumulation of 14-3-3 mRNA. In addition, cell suspension cultures treated with methyl jasmonate showed up-regulation of 14-3-3-encoding mRNA. Chitosan and methyl jasmonate are both signalling molecules in the activation of plant defense response genes. Therefore, our results suggest a possible role for this 14-3-3 protein in the pathogen defense response of coniferous trees.

Computerized product information system: The Toxicological Index.

The Toxicological Index, a division of the Commission de la Santé et de la Sécurité du Travail, was established in 1980 by the Quebec Government. Its main objective, as a database producer, is to provide health and safety related toxicological information to anyone who is involved in risk management in the workplace. In order to meet this requirement, a fully computerized information system was created. It has allowed us to process, over the past 5 years, 250,000 information inquiries, to register 100,000 contaminants, to exchange information with 3,300 product manufacturers and to provide our users with 6,500 fully reviewed toxicological information profiles. The procedure used to assess the information as well as the choices made to achieve our objective in terms of quantity and quality of the information provided are described.

Glucose decreases virulence gene expression of Escherichia coli O157:H7.

Escherichia coli O157:H7 is responsible for a human toxico-infection that can lead to severe complications such as hemolytic uremic syndrome. Inside the intestine, E. coli O157:H7 forms typical attaching-effacing lesions and produces Shiga toxins. The genes that are responsible for these lesions are located in a pathogenicity island called the locus of enterocyte effacement (LEE). LEE gene expression is influenced by quorum sensing through the luxS system. In this study, the effect of glucose on the expression of several genes from LEE, on the expression of Shiga toxin genes, and on the expression of luxS was assessed with real-time, reverse transcription PCR. All concentrations of glucose (from 0.1 to 1%) were able to down-regulate genes from the LEE operon. A slight down-regulation of genes implicated in Shiga toxin expression was also observed but was significant for low doses of glucose (0.1 to 0.5%) only. A slight but significant increase in luxS expression was observed with 1% glucose. This confirms that in addition to quorum sensing, the presence or absence of nutrients such as glucose has an impact on the down- or upregulation of LEE-encoded virulence genes by the bacterium. The influence of glucose on the virulence of E. coli O157:H7 has received little attention, and these results suggest that glucose can have an important effect on the virulence of E. coli O157:H7.

Identification and molecular characterization of the chromosomal exopolysaccharide biosynthesis gene cluster from Lactococcus lactis subsp. cremoris SMQ-461.

The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 x 10(6) Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5' end and orfY, along with an IS981-related tnp gene, at the 3' end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.

Comparative antimicrobial activity of two new mutacins / Actividad microbiana comparativa de dos nuevas mutacinas

OBJECTIVES: To compare the in vitro activity of mutacins D-123.1 and F-59.1 against different bacteria including antibiotic-resistant strains, in order to evaluate their application potential. DESIGN AND METHODS: The antibacterial activity spectrum of purified F-59.1 and the MIC and MBC of F-59.1 and D-123.1 against target bacteria were determined. RESULTS: Most bacteria were inhibited by the purified mutacins. Mutacin F-59.1 shows a relatively wide activity spectrum. Mutacin D-123.1 has low Minimum Inhibitory Concentrations [MICs] (0.25-4 µ/ml) against human pathogens while F-59.1 has higher MICs (3.2-12.8 fig/ml) mainly against food-borne pathogens. CONCLUSION: The effectiveness of mutacins D-123.1 and F-59.1 against human and food-borne pathogens is demonstrated. Mutacin D-123.1 shows potential as a new antibiotic while F-59.1 shows promising application in food products. ABBREVIATIONS: MALDI-TOF MS, matrix assisted laser desorption ionisation-time of flight mass spectrometry; MB(I)C, minimum bactericidal (inhibitory) concentrations; OD, optical density; RP-HPLC, reverse-phase high-pressure liquid chromatography; TSBYE, trypticase soy broth yeast extract.

OBJETIVOS: Comparar la actividad in vitro de las mutacinas D-123.1 y F-59.1 frente a diferentes bacterias incluyendo las cepas resistentes a los antibióticos, a fin de evaluar el potencial de su aplicación. DISEÑO Y MÉTODOS: Se determinó el espectro de actividad antibacteriana de F-59.1 purificada y la CIM y la CBM de F-59.1 y D-123.1 frente a determinadas bacterias. RESULTADOS: La mayor parte de las bacterias eran inhibidas por las mutacinas purificadas. La mutacina F-59.1 muestra un espectro de actividad relativamente amplio. La mutacina D-123.1 posee bajas concentraciones de inhibición mínimas [CIM] (0.25-4 fig/ml) contra los patógenos humanos, mientras que el F-59.1 posee concentraciones CIM más altas (3.2-12.8 fig/ml) principalmente frente a los patógenos alimentarios. CONCLUSIÓN: Queda demostrada la efectividad de las mutacinas D-123.1 y F-59.1 frente a los patógenos humanos y alimentarios. La mutacina D-123.1 muestra poseer un potencial como nuevo antibiótico, en tanto que F-59.1 se presenta como una aplicación promisoria en relación con los productos alimentarios. ABREVIATURAS: MALDI-TOF MS, espectrometría de masas con desorción/ionización mediante láser asistida por matriz asociada a un analizador de tiempo de vuelo (del inglés: matrix assisted laser desorption ionisation-time de flight mass spectrometry). CIM: concentración inhibitoria mínima (inglés MIC). CBM: concentración bactericida mínima (inglés MBC). DO: densidad óptica (inglés OD); RP-HPLC: cromatografía líquida de alta resolución en fase revertida; TSBYE:caldo tripticasa soya- extracto de levadura.