Safety Characterization and Antimicrobial Properties of Kefir-Isolated <i>Lactobacillus kefiri</i>

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

Safety Characterization and Antimicrobial Properties of Kefir-Isolated <i>Lactobacillus kefiri</i>

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

Impact of kefir derived lactobacillus kefiri on the mucosal immune response and gut microbiota

The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-γ, GM-CSF, and IL-1β genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders.Facultad de Ciencias ExactasInstituto de Estudios Inmunológicos y Fisiopatológico

Association of levels of antibodies from patients with inflammatory bowel disease with extracellular proteins of food and probiotic bacteria

Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n=50), Crohn's disease patients (CD, n=37), and ulcerative colitis patients (UC, n=15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P<0.002; n=52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS- 377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. © 2014 Arancha Hevia et al.Borja Sánchez and Arancha Hevia were recipients of a Ramón y Cajal postdoctoral contract and a FPI Grant, respectively, from the Spanish Ministry of Economy and Competitiveness. Research in our group is supported by Grants AGL2010-14952 and RM2010-00012-00-00 from the Spanish Plan Nacional de I+D.Peer Reviewe

Enterococcus durans EP1 a promising anti-inflammatory probiotic able to stimulate sIgA and to increase Faecalibacterium prausnitzii abundance

Enterococcus species, principally Enterococcus faecium are used as probiotics since a long time with preference in animal applications but safety considerations were updated and also new uses as probiotics can be envisaged. Fifteen Enterococcus strains isolated from different foods were identified and analyzed for virulence factors and antibiotic resistance. Three Enterococcus durans strains were selected to study their immunomodulatory properties on PBMC and Caco2 cells. Two strains presented a profile toward a mild inflammatory Th1 response considering TNF-α/IL-10 and IL-1β/IL-10 cytokines ratios. The third strain EP1, presented an anti-inflammatory potential and was selected for in vivo studies. In mice, the strain was well tolerated and did not cause any adverse effects. EP1 administration increased the amount of IgA+ cells in mesenteric lymph node (MLN) after 7 days of administration. In fecal samples, the IgA content increased gradually and significantly from day 7 to day 21 in treated group. Additionally, IL-17, IL-6, IL-1β, IFN-γ, and CXCL1 gene expression significantly decreased on day 21 in Peyer's patches and IL-17 decreased in MLN. Mice treated with the probiotic showed significant lower mRNA levels of pro-inflammatory cytokines and mucins in the ileum at day 7 while their expression was normalized at day 21. Colonic expression of il-1β, il6, and mucins remain diminished at day 21. Ileum and colon explants from treated mice stimulated in vitro with LPS showed a significant reduction in IL-6 and an increase in IL-10 secretion suggesting an in vivo protective effect of the probiotic treatment against a proinflammatory stimulus. Interestingly, analysis of feces microbiota demonstrated that EP1 administration increase the amount of Faecalibacterium prausnitzii, a butyrate-producing bacteria, which is known for its anti-inflammatory effects. In conclusion, we demonstrated that EP1 strain is a strong sIgA inducer and possess mucosal anti-inflammatory properties. This strain also modulates gut microbiota increasing Faecalibacterium prausnitzii, a functionally important bacterium. Thus, E. durans EP1 is not only a good candidate to increases F. prausnitzii in some cases of dysbiosis but can also be interesting in gut inflammatory disorders therapy.Facultad de Ciencias Exacta

Safety Characterization and Antimicrobial Properties of Kefir-Isolated <i>Lactobacillus kefiri</i>

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

Impact of kefir derived lactobacillus kefiri on the mucosal immune response and gut microbiota

The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-γ, GM-CSF, and IL-1β genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders.Facultad de Ciencias ExactasInstituto de Estudios Inmunológicos y Fisiopatológico

Characterization of the Exopolysaccharide Produced by Salipiger mucosus A3T, a Halophilic Species Belonging to the Alphaproteobacteria, Isolated on the Spanish Mediterranean Seaboard

We have studied the exopolysaccharide produced by the type strain of Salipiger mucosus, a species of halophilic, EPS-producing (exopolysaccharide-producing) bacterium belonging to the Alphaproteobacteria. The strain, isolated on the Mediterranean seaboard, produced a polysaccharide, mainly during its exponential growth phase but also to a lesser extent during the stationary phase. Culture parameters influenced bacterial growth and EPS production. Yield was always directly related to the quantity of biomass in the culture. The polymer is a heteropolysaccharide with a molecular mass of 250 kDa and its components are glucose (19.7%, w/w), mannose (34%, w/w), galactose (32.9%, w/w) and fucose (13.4%, w/w). Fucose and fucose-rich oligosaccharides have applications in the fields of medicine and cosmetics. The chemical or enzymatic hydrolysis of fucose-rich polysaccharides offers a new efficient way to process fucose. The exopolysaccharide in question produces a solution of very low viscosity that shows pseudoplastic behavior and emulsifying activity on several hydrophobic substrates. It also has a high capacity for binding cations and incorporating considerable quantities of sulfates, this latter feature being very unusual in bacterial polysaccharides

Microbiota/Host Crosstalk Biomarkers: Regulatory Response of Human Intestinal Dendritic Cells Exposed to Lactobacillus Extracellular Encrypted Peptide

The human gastrointestinal tract is exposed to a huge variety of microorganisms, either commensal or pathogenic; at this site, a balance between immunity and immune tolerance is required. Intestinal dendritic cells (DCs) control the mechanisms of immune response/tolerance in the gut. In this paper we have identified a peptide (STp) secreted by Lactobacillus plantarum, characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains, and lacks cleavage sites for the major intestinal proteases. When studied in vitro, STp expanded the ongoing production of regulatory IL-10 in human intestinal DCs from healthy controls. STp-primed DC induced an immunoregulatory cytokine profile and skin-homing profile on stimulated T-cells. Our data suggest that some of the molecular dialogue between intestinal bacteria and DCs may be mediated by immunomodulatory peptides, encoded in larger extracellular proteins, secreted by commensal bacteria. These peptides may be used for the development of nutraceutical products for patients with IBD. In addition, this kind of peptides seem to be absent in the gut of inflammatory bowel disease patients, suggesting a potential role as biomarker of gut homeostasis

Extracellular proteins from Lactobacillus plantarum BMCM12 prevent adhesion of enteropathogens to mucin

The aim of this study was to study the interference of the extracellular proteins produced by Lactobacillus plantarum BMCM12 with the adhesion of some well-known gut pathogens. The extracellular proteins secreted by L. plantarum BMCM12 in MRS broth were precipitated, resolved by SDS-PAGE, and identified by tandem mass spectrometry. Discordances between the observed and the theoretical molecular masses of several proteins suggested the presence of protein glycosylation, corroborated with specific glycoprotein staining after protein de-glycosylation using trifluoromethanesulfonic acid. Experiments of exclusion, competition, or prevention of the pathogen adhesion to mucin were performed using BMCM12 extracellular proteins, using Escherichia coli LMG2092 and Salmonella enterica subsp. enterica LMG15860. Extracellular proteins from BMCM12 reduced significantly the adhesion of the pathogens when they were added prior to adhesion assays. These proteins play thus important roles in preventing pathogen adhesion to the mucin layer.This study has been supported with grant (RM2010-00012-00-00) from the Spanish MICINN.Peer Reviewe