Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats.

In addition to their use in cheese technology, dairy propionibacteria have been identified as potential probiotics. However, to have a probiotic effect, propionibacteria have to survive and to remain metabolically active in the digestive tract. The aim of the present study was to investigate the survival and metabolic activity of Propionibacterium freudenreichii within the gastrointestinal tract of human microbiota-associated rats, and its influence on intestinal microbiota composition and metabolism. Twenty-five dairy Propionibacterium strains were screened for their tolerance towards digestive stresses and their ability to produce propionate in a medium mimicking the content of the human colon. Three strains were selected and a daily dose of 2 x 10(10) colony-forming units was fed to groups of human microbiota-associated rats for 20 d before microbiological, biochemical and molecular investigations being carried out. These strains all reached 8-log values per g faeces, showing their ability to survive in the gastrointestinal tract. Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. The probiotic efficacy of propionibacteria was yet species- and strain-dependent. Indeed, two of the strains, namely TL133 and TL1348, altered the faecal microbiota composition, TL133 also increasing the caecal concentration of acetate, propionate and butyrate, while the third strain, TL3, did not have similar effects. Such alterations may have an impact on gut health and will thus be taken into consideration for further in vivo investigations on probiotic potentialities of P. freudenreichii.

Changes in protein synthesis during thermal adaptation of Propionibacterium freudenreichii subsp. shermanii.

Dairy propionibacteria are present in Graviera Kritis, a traditional Gruyère-type cheese made without added propionic starter. Ten isolated strains were identified by a combination of SDS-PAGE, species-specific PCR and according to their ability to ferment lactose. They were all found to belong to the Propionibacterium freudenreichii subsp. shermanii species. Because of the stressing Gruyère technology, which includes cooking at 52 to 53 degrees C their thermotolerance was investigated at 55 degrees C. Thermotolerant and thermosensitive strains were clearly discriminated. Interestingly, the reference strain CIP 103027 belongs to the sensitive subset. One sensitive strain, ACA-DC 1305 and one tolerant, ACA-DC 1451, were selected for further study and compared to CIP 103027. For the sensitive strains ACA-DC 1305 and CIP 103027, heat pre-treatment at 42 degrees C conferred thermoprotection of cells at the lethal temperature of 55 degrees C, while there was less effect on the tolerant ACA-DC 1451. No cross-protection of salt-adapted cells against heat stress was observed for none of the strains. Differential proteomic analysis revealed distinct but overlapping cell responses to heat stress between sensitive and tolerant strains. Thermal adaptation upregulated typical HSPs involved in protein repair or turnover in the sensitive one. In the tolerant one, a distinct subset of proteins was overexpressed, whatever the temperature used, in addition to HSPs. This included enzymes involved in propionic fermentation, amino acid metabolism, oxidative stress remediation and nucleotide phosphorylation. These results bring new insights into thermoprotection in propionibacteria and the occurrence of divergent phenotypes within a same subspecies.

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii.

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.

Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii.

We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.