The response of the immune system to probiotics remains controversial. Some strains modulate the cytokine production of dendritic cells (DCs) in vitro and induce a regulatory response, while others induce conversely a pro-inflammatory response. These strain-dependent effects are thought to be linked to specific interactions between bacteria and pattern recognition receptors. We investigated the effects of a well characterized probiotic strain, Lactobacillus rhamnosus Lcr35, on human monocyte-derived immature DCs, using a wide range of bacterial concentrations (multiplicity of infection, MOI, from 0.01 to 100). DNA microarray and qRT-PCR analysis showed that the probiotic induced a large-scale change in gene expression (nearly 1,700 modulated genes, with 3-fold changes), but only with high doses (MOI, 100). The upregulated genes were mainly involved in immune response and identified a molecular signature of inflammation according to the model of Torri. Flow cytometry analysis also revealed a dose-dependent maturation of the DC membrane phenotype, until DCs reached a semi-mature state, with an upregulation of the membrane expression of CD86, CD83, HLA-DR and TLR4, associated with a down-regulation of DC-SIGN, MR and CD14. Measurement of the DC-secreted cytokines showed that Lcr35 induced a strong dose-dependent increase of the pro-Th1/Th17 cytokine levels (TNFα, IL-1β, IL-12p70, IL-12p40 and IL-23), but only a low increase in IL-10 concentration. The probiotic L. rhamnosus Lcr35 therefore induce a dose-dependent immunomodulation of human DCs leading, at high doses, to the semi-maturation of the cells and to a strong pro-inflammatory effect. These results contribute to a fuller understanding of the mechanism of action of this probiotic, and thus of its potential clinical indications in the treatment of either infectious or IgE-dependent allergic diseases.

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.

Objectives. The ability of a probiotic Lactobacillus rhamnosus strain (Lcr35) to adhere to cervical and vaginal cells and to affect the viability of two main vaginosis-associated pathogens, Prevotella bivia, Gardnerella vaginalis, as well as Candida albicans was investigated. Methods. Adhesion ability was determined in vitro with immortalized epithelial cells from the endocervix, ectocervix, and vagina. Coculture experiments were performed to count viable pathogens cells in the presence of Lcr35. Results. Lcr35 was able to specifically and rapidly adhere to the three cell lines. In coculture assays, a decrease in pathogen cell division rate was observed as from 4 hours of incubation and bactericidal activity after a longer period of incubation, mostly with P. bivia. Conclusion. The ability of Lcr35 to adhere to cervicovaginal cells and its antagonist activities against vaginosis-associated pathogens suggest that this probiotic strain is a promising candidate for use in therapy.

In vivo assays showed that a ⌬eefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the ⌬eefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.Klebsiella pneumoniae is an opportunistic pathogen responsible for many nosocomial infections, and most clinical strains exhibit high levels of resistance to a wide variety of structurally unrelated antibiotics. Epidemiological studies have shown that, whatever the infection site, the first stage in nosocomial infections due to K. pneumoniae consists of the colonization of the patient's gastrointestinal (GI) tract (6,16). This pathogen therefore has to sense and respond to numerous different environments in order to survive and, consequently, to persist in the GI tract of the host. The first major barrier encountered following oral consumption is stomach acidity. The bacteria then enter the small intestine, where they encounter stresses associated with volatile fatty acids, variations in pH and osmolarity, and competition with endogenous flora. Using signature-tagged mutagenesis (STM) in a murine model, we previously identified 13 genes encoding factors required for the in vivo colonization of the GI tract by K. pneumoniae LM21 (14). All of these mutants were selected for their inability to colonize the murine intestinal tract under competitive conditions. One of the identified genes potentially encoded a protein involved in a cryptic efflux pump, EefABC, previously described in Enterobacter aerogenes (18). This pump is thought to belong to the resistance-nodulation-division (RND) family and to be composed of an inner membrane protein, EefB, a periplasmic intermediate, EefA, and an outer membrane protein, EefC (18). No specific function has been attributed to this structure so far, whose expression is repressed by the H-NS repressor in the heterologous host Escherichia coli (18). In addition to the export of antimicrobials, several recent publications have highlighted the natural physiological role of efflux pumps in exporting noxious substances out of the bacterial cell, thereby allowing the bacteria to survive in hostile environments and facilitating intestinal colonization (11,13,24,30). In this report, we characterized the eef-like operon of K. pneumoniae and investigated the role of the potentially enc...

All accessible mucous membranes of the human body are colonized by an abundant and diversified microbial flora called microbiota. Recent studies have shown that these microorganisms, long regarded as purely commensal, have essential beneficial effects on human health. Thus, numerous human ailments are linked to dysbiosis; that is, imbalances in the microflora composition. The administration of probiotic microorganisms could, in some situations, provide substantial relief from such disorders. These live microorganisms, which, according to the definition, confer a health benefit to the host when administered in adequate amounts, are often derived from human flora and belong mostly to lactic acid bacteria, in particular to the genus Lactobacillus. The constant improvement of knowledge of the role of human microbiota and the growing popularity of probiotics are now opening the door to new prophylactic and therapeutic strategies in human health.