Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestines using a single reactor model

<p>Abstract</p> <p>Background</p> <p>Probiotic bacteria are thought to play an important role in the digestive system and therefore have to survive the passage from stomach to intestines. Recently, a novel approach to simulate the passage from stomach to intestines in a single bioreactor was developed. The advantage of this automated one reactor system was the ability to test the influence of acid, bile salts and pancreatin.</p> <p><it>Lactobacillus gasseri </it>K7 is a strain isolated from infant faeces with properties making the strain interesting for cheese production. In this study, a single reactor system was used to evaluate the survival of <it>L. gasseri </it>K7 and selected bifidobacteria from our collection through the stomach-intestine passage.</p> <p>Results</p> <p>Initial screening for acid resistance in acidified culture media showed a low tolerance of <it>Bifidobacterium dentium </it>for this condition indicating low survival in the passage. Similar results were achieved with <it>B. longum </it>subsp. <it>infantis </it>whereas <it>B. animalis </it>subsp. <it>lactis </it>had a high survival.</p> <p>These initial results were confirmed in the bioreactor model of the stomach-intestine passage. <it>B. animalis </it>subsp. <it>lactis </it>had the highest survival rate (10%) attaining approximately 5 × 10⁶cfu ml^-1compared to the other tested bifidobacteria strains which were reduced by a factor of up to 10⁶. <it>Lactobacillus gasseri </it>K7 was less resistant than <it>B. animalis </it>subsp. <it>lactis </it>but survived at cell concentrations approximately 1000 times higher than other bifidobacteria.</p> <p>Conclusion</p> <p>In this study, we were able to show that <it>L. gasseri </it>K7 had a high survival rate in the stomach-intestine passage. By comparing the results with a previous study in piglets we could confirm the reliability of our simulation. Of the tested bifidobacteria strains, only <it>B. animalis </it>subsp. <it>lactis </it>showed acceptable survival for a successful passage in the simulation system.</p

Isothermal micro calorimetry – a new method for MIC determinations: results for 12 antibiotics and reference strains of E. coli and S. aureus

Antimicrobial susceptibility testing of microorganisms is performed by either disc diffusion or broth dilution tests. In clinical use, the tests are often still performed manually although automated systems exist. Most systems, however, are based on turbidometric methods which have well-known drawbacks

Potassium Lactate as a Strategy for Sodium Content Reduction without Compromising Salt-Associated Antimicrobial Activity in Salami

Reformulating recipes of ready-to-eat meat products such as salami to reduce salt content can mitigate the negative health impacts of a high salt diet. We evaluated the potential of potassium lactate (KL) as a sodium chloride (NaCl) replacer during salami production. NaCl and KL stress tolerance comparisons showed that four food-derived Listeria innocua isolates were suitable as biologically safe Listeria monocytogenes surrogates. Effects of the high salt (4% NaCl) concentration applied in standard salami recipes and a low salt (2.8% NaCl) plus KL (1.6%) combination on product characteristics and growth of contaminating Listeria and starter culture were compared. Simulated salami-ripening conditions applied in meat simulation broth and beef showed that the low salt plus KL combination retained similar to superior anti-Listeria activity compared to the high salt concentration treatment. Salami challenge tests showed that the low NaCl plus KL combination had comparable anti-Listeria activity as the high NaCl concentration during ripening and storage. No significant differences were detected in starter culture growth profiles and product characteristics between the high NaCl and low NaCl plus KL combination treated salami. In conclusion, KL replacement enabled a 30% NaCl reduction without compromising the product quality and antimicrobial benefits of high NaCl concentration inclusion

Lactic Acid Bacteria as Markers for the Authentication of Swiss Cheeses

The manufacture of traditional Swiss-type cheeses adheres to strict rules, so as to guarantee quality and purity of the end product. This raises production costs and means consumers pay more. It also opens the door to cut-rate forgeries claiming to be made to the stringent standards and causing considerable economic losses to the entire dairy sector. In order to combat product counterfeiting, Agroscope has developed proof-of-origin cultures that allow the identification of copycats. Carefully selected lactic acid bacteria, having uniquely located insertion sequence elements, are proliferated by fermentation and subsequently dried by lyophilization. The proof-of-origin culture is added during the cheese production process and sustains maturation. These so-called 'biological markers' can be traced using polymerase chain reaction (PCR) methods, which allow authentication even if the cheese is cut into pieces or grated. They do not lead to any alteration of the cheese's taste or texture, and are compatible with the strict 'protected designation of origin' (PDO) specifications. The proof-of-origin cultures are used for the protection of several traditional Swiss-cheese varieties, such as Emmental PDO, Tête de Moine PDO, and Appenzeller®. A market survey of Emmental PDO showed that the system is effective in revealing fraud and has the power to enforce corrective measures

Deciphering the global roles of Cold shock proteins in Listeria monocytogenes nutrient metabolism and stress tolerance

Listeria monocytogenes (Lm) accounts for serious public health and food safety problems owing to its stress resilience and pathogenicity. Based on their regulatory involvement in global gene expression events, cold-shock domain family proteins (Csps) are crucial in expression of various stress fitness and virulence phenotypes in bacteria. Lm possesses three Csps (CspA, CspB, and CspD) whose regulatory roles in the context of the genetic diversity of this bacterium are not yet fully understood. We examined the impacts of Csps deficiency on Lm nutrient metabolism and stress tolerance using a set of csp deletion mutants generated in different genetic backgrounds. Phenotype microarrays (PM) analysis showed that the absence of Csps in ∆cspABD reduces carbon (C-) source utilization capacity and increases Lm sensitivity to osmotic, pH, various chemical, and antimicrobial stress conditions. Single and double csp deletion mutants in different Lm genetic backgrounds were used to further dissect the roles of individual Csps in these phenotypes. Selected PM-based observations were further corroborated through targeted phenotypic assays, confirming that Csps are crucial in Lm for optimal utilization of various C-sources including rhamnose and glucose as well as tolerance against NaCl, β-phenyethylamine (PEA), and food relevant detergent stress conditions. Strain and genetic lineage background-based differences, division of labour, epistasis, and functional redundancies among the Csps were uncovered with respect to their roles in various processes including C-source utilization, cold, and PEA stress resistance. Finally, targeted transcriptome analysis was performed, revealing the activation of csp gene expression under defined stress conditions and the impact of Csps on expression regulation of selected rhamnose utilization genes. Overall, our study shows that Csps play important roles in nutrient utilization and stress responses in Lm strains, contributing to traits that are central to the public health and food safety impacts of this pathogen

Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum

<p>Abstract</p> <p>Background</p> <p>Bifidobacteria are found at varying prevalence in human microbiota and seem to play an important role in the human gastrointestinal tract (GIT). Bifidobacteria are highly adapted to the human GIT which is reflected in the genome sequence of a <it>Bifidobacterim longum </it>isolate. The competitiveness against other bacteria is not fully understood yet but may be related to the production of antimicrobial compounds such as bacteriocins. In a previous study, 34 <it>Bifidobacterium </it>isolates have been isolated from baby faeces among which six showed proteinaceous antilisterial activity against <it>Listeria monocytogenes</it>. In this study, one of these isolates, RBL67, was further identified and characterized.</p> <p>Results</p> <p><it>Bifidobacterium </it>isolate RBL67 was classified and characterized using a polyphasic approach. RBL67 was classified as <it>Bifidobacterium thermophilum </it>based on phenotypic and DNA-DNA hybridization characteristics, although 16S rDNA analyses and partial <it>gro</it>EL sequences showed higher homology with <it>B. thermacidophilum </it>subsp. <it>porcinum </it>and <it>B. thermacidophilum </it>subsp. <it>thermacidophilum</it>, respectively. RBL67 was moderately oxygen-tolerant and was able to grow at pH 4 and at a temperature of 47°C.</p> <p>Conclusion</p> <p>In order to assign RBL67 to a species, a polyphasic approach was used. This resulted in the classification of RBL67 as a <it>Bifidobacterium thermophilum </it>strain. To our knowledge, this is the first report about <it>B. thermophilum </it>isolated from baby faeces since the <it>B. thermophilum </it>strains were related to ruminants and swine faeces before. <it>B. thermophilum </it>was previously only isolated from animal sources and was therefore suggested to be used as differential species between animal and human contamination. Our findings may disapprove this suggestion and further studies are now conducted to determine whether <it>B. thermophilum </it>is distributed broader in human faeces. Furthermore, the postulated differentiation between human and animal strains by growth above 45°C is no longer valid since <it>B. thermophilum </it>is able to grow at 47°C. In our study, 16S rDNA and partial <it>gro</it>EL sequence analysis were not able to clearly assign RBL67 to a species and were contradictory. Our study suggests that partial <it>gro</it>EL sequences may not be reliable as a single tool for species differentiation.</p

Detection of the pediocin gene pedA in strains from human faeces by real-time PCR and characterization of Pediococcus acidilactici UVA1

<p>Abstract</p> <p>Background</p> <p>Bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus <it>Pediococcus </it>are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen <it>Listeria monocytogenes</it>. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a <it>Bifidobacterium thermophilum </it>strain, and characterized its proteinaceous compound with strong antilisterial activity.</p> <p>Results</p> <p>The newly isolated strain UVA1 was identified as a <it>Pediococcus acidilactici </it>by carbohydrate fermentation profile, growth at 50°C and 16S rDNA sequencing. The partially purified bacteriocin was heat resistant up to 100°C, active over a wide range of pH (2 to 9) and susceptible to proteolytic enzymes. The molecular weight, estimated by SDS-PAGE, was similar to that of pediocin AcH/PA-1 (4.5 kDa). <it>P. acidilactici </it>UVA1 harboured a 9.5-kb plasmid that could be cured easily, which resulted in the loss of the antimicrobial activity. Southern hybridization using the DIG-labelled <it>pedA</it>-probe established that the bacteriocin gene was plasmid-borne as for all pediocin described so far. Nucleotide sequence of the whole operon (3.5 kb) showed almost 100 % similarity to the pediocin AcH/PA-1 operon. The mRNA transcript for <it>pedA </it>could be detected in <it>P. acidilactici </it>UVA1 but not in the cured derivative, confirming the expression of the <it>pedA</it>-gene in UVA1. Using a new real-time PCR assay, eleven out of seventeen human faecal samples tested were found to contain <it>pedA</it>-DNA.</p> <p>Conclusion</p> <p>We identified and characterised the first pediocin produced by a human intestinal <it>Pediococcus acidilactici </it>isolate and successfully developed a new real-time PCR assay to show the large distribution of <it>pedA</it>-containing strains in baby faecal samples.</p

Extensive diversity and rapid turnover of phage defense repertoires in cheese-associated bacterial communities.

BACKGROUND Phages are key drivers of genomic diversity in bacterial populations as they impose strong selective pressure on the evolution of bacterial defense mechanisms across closely related strains. The pan-immunity model suggests that such diversity is maintained because the effective immune system of a bacterial species is the one distributed across all strains present in the community. However, only few studies have analyzed the distribution of bacterial defense systems at the community-level, mostly focusing on CRISPR and comparing samples from complex environments. Here, we studied 2778 bacterial genomes and 188 metagenomes from cheese-associated communities, which are dominated by a few bacterial taxa and occur in relatively stable environments. RESULTS We corroborate previous laboratory findings that in cheese-associated communities nearly identical strains contain diverse and highly variable arsenals of innate and adaptive (i.e., CRISPR-Cas) immunity systems suggesting rapid turnover. CRISPR spacer abundance correlated with the abundance of matching target sequences across the metagenomes providing evidence that the identified defense repertoires are functional and under selection. While these characteristics align with the pan-immunity model, the detected CRISPR spacers only covered a subset of the phages previously identified in cheese, providing evidence that CRISPR does not enable complete immunity against all phages, and that the innate immune mechanisms may have complementary roles. CONCLUSIONS Our findings show that the evolution of bacterial defense mechanisms is a highly dynamic process and highlight that experimentally tractable, low complexity communities such as those found in cheese, can help to understand ecological and molecular processes underlying phage-defense system relationships. These findings can have implications for the design of robust synthetic communities used in biotechnology and the food industry. Video Abstract

Blood lactose after dairy product intake in healthy men.

The absence of a dedicated transport for disaccharides in the intestine implicates that the metabolic use of dietary lactose relies on its prior hydrolysis at the intestinal brush border. Consequently, lactose in blood or urine has mostly been associated with specific cases in which the gastrointestinal barrier is damaged. On the other hand, lactose appears in the blood of lactating women and has been detected in the blood and urine of healthy men, indicating that the presence of lactose in the circulation of healthy subjects is not incompatible with normal physiology. In this cross-over study we have characterised the postprandial kinetics of lactose, and its major constituent, galactose, in the serum of fourteen healthy men who consumed a unique dose of 800 g milk or yogurt. Genetic testing for lactase persistence and microbiota profiling of the subjects were also performed. Data revealed that lactose does appear in serum after dairy intake, although with delayed kinetics compared with galactose. Median serum concentrations of approximately 0·02 mmol/l lactose and approximately 0·2 mmol/l galactose were observed after the ingestion of milk and yogurt respectively. The serum concentrations of lactose were inversely correlated with the concentrations of galactose, and the variability observed between the subjects' responses could not be explained by the presence of the lactase persistence allele. Finally, lactose levels have been associated with the abundance of the Veillonella genus in faecal microbiota. The measurement of systemic lactose following dietary intake could provide information about lactose metabolism and nutrient transport processes under normal or pathological conditions

Mikrobielle Schutzkulturen

In der Abteilung Food Science & Management (FSM) der Berner Fachhochschule laufen derzeit mehrere Projekte zur Erforschung antagonistischer Mikroorganismen zur Bekämpfung der Vor- und Nacherntefäule bei Frischprodukten wie Obst und Gemüse