Search

Inorganic phosphate (Pi) concentration modulates polyphosphate (polyP) levels in diverse bacteria, affecting their physiology and survival. Lactiplantibacillus paraplantarum CRL 1905 is a lactic acid bacterium isolated from quinoa sourdough with biotechnological potential as starter, for initiating fermentation processes in food, and as antimicrobial-producing organism. The aim of this work was to evaluate the influence of the environmental Pi concentration on different physiological and molecular aspects of the CRL 1905 strain. Cells grown in a chemically defined medium containing high Pi (CDM + P) maintained elevated polyP levels up to late stationary phase and showed an enhanced bacterial survival and tolerance to oxidative stress. In Pi sufficiency condition (CDM-P), cells were ~ 25% longer than those grown in CDM + P, presented membrane vesicles and a ~ 3-fold higher capacity to form biofilm. Proteomic analysis indicated that proteins involved in the “carbohydrate transport and metabolism” and “energy production and conversion” categories were up-regulated in high Pi stationary phase cells, implying an active metabolism in this condition. On the other hand, stress-related chaperones and enzymes involved in cell surface modification were up-regulated in the CDM-P medium. Our results provide new insights to understand the CRL 1905 adaptations in response to differential Pi conditions. The adjustment of environmental Pi concentration constitutes a simple strategy to improve the cellular fitness of L. paraplantarum CRL 1905, which would benefit its potential as a microbial cell factory.

Abstract Background Diarrheal infections caused by Salmonella, are one of the major causes of childhood morbidity and mortality in developing countries. Salmonella causes various diseases that range from mild gastroenteritis to enteric fever, depending on the serovar involved, infective dose, species, age and immune status of the host. Probiotics are proposed as an attractive alternative possibility in the prevention against this pathogen infection. Previously we demonstrated that continuous Lactobacillus casei CRL 431 administration to BALB/c mice before and after challenge with Salmonella enterica serovar Typhimurium (S. Typhimurium) decreased the severity of Salmonella infection. The aim of the present work was to deep into the knowledge about how this probiotic bacterium exerts its effect, by assessing its impact on the expression and secretion of pro-inflammatory (TNFα, IFNγ) and anti-inflammatory (IL-10) cytokines in the inductor and effector sites of the gut immune response, and analyzing toll-like receptor (TLR2, TLR4, TLR5 and TLR9) expressions in both healthy and infected mice. Results Probiotic administration to healthy mice increased the expression of TLR2, TLR4 and TLR9 and improved the production and secretion of TNFα, IFNγ and IL-10 in the inductor sites of the gut immune response (Peyer's patches). Post infection, the continuous probiotic administration, before and after Salmonella challenge, protected the host by modulating the inflammatory response, mainly in the immune effector site of the gut, decreasing TNFα and increasing IFNγ, IL-6 and IL-10 production in the lamina propria of the small intestine. Conclusions The oral administration of L. casei CRL 431 induces variations in the cytokine profile and in the TLRs expression previous and also after the challenge with S. Typhimurium. These changes show some of the immune mechanisms implicated in the protective effect of this probiotic strain against S. Typhimurium, providing an alternative way to reduce the severity of the infection.

Abstract Background Malnutrition affects the immune response, causing a decrease of defence mechanisms and making the host more susceptible to infections. Probiotics can reconstitute the intestinal mucosa and stimulate local and systemic immunity. The aim of this work was evaluate the effects of a probiotic fermented milk as a complement of a re-nutrition diet, on the recovery of the intestinal barrier, and mucosal and systemic immune functions in a murine model of non-severe protein-energy-malnutrition. Its potential protection against Salmonella enterica serovar Typhimurium (S. Typhimurium) infection was also analyzed. Methods Mice were undernourished and divided into 3 groups according to the dietary supplement received during re-nutrition (milk, probiotic fermented milk or its bacterial free supernatant) and compared to well-nourished and malnourished mice. They were sacrificed previous to the re-nutrition and 5 days post re-nutrition. The phagocytic activity of macrophages from spleen and peritoneum and the changes in the intestinal histology and microbiota were evaluated. Different immune cell populations and cytokine productions were analyzed in the small intestine tissues. The effect of the re-nutrition supplements on the systemic immunity using OVA antigen and against an infection with S. Typhimurium was also studied. Results Probiotic fermented milk was the most effective re-nutrition diet that improved the intestinal microbiota. Its administration also increased the number of IgA+ cells, macrophages and dendritic cells. The production of different cytokine (IFN-γ, TNF-α, IL-12) by these cells and the phagocytic activity in peritoneum and spleen was also increased. This re-nutrition diet also stimulated the systemic immune response against OVA antigen which was diminished after the malnutrition period and also improved the host response against S. Typhimurium, decreasing the spread of pathogenic bacteria to the liver and the spleen. The importance of the metabolites released during milk fermentation was also demonstrated through the analysis of the bacterial free supernatant obtained from the probiotic fermented milk, but the whole product showed the best effects in the parameters evaluated in this study. Conclusions The administration of probiotic fermented milk as a dietary supplement during the re-nutrition process in a murine immunodeficiency model by malnutrition could be a good adjuvant diet to improve the gut and systemic immune response for the protection against Salmonella infection.

Abstract Background Microbial colonization of the intestine after birth is an important step for the development of the gut immune system. The acquisition of passive immunity through breast-feeding may influence the pattern of bacterial colonization in the newborn. The aim of this work was to evaluate the effect of the administration of a probiotic fermented milk (PFM) containing yogurt starter cultures and the probiotic bacteria strain Lactobacillus casei DN-114001 to mothers during nursing or their offspring, on the intestinal bacterial population and on parameters of the gut immune system. Results Fifteen mice of each group were sacrificed at ages 12, 21, 28 and 45 days. Large intestines were taken for determination of intestinal microbiota, and small intestines for the study of secretory-IgA (S-IgA) in fluid and the study of IgA+ cells, macrophages, dendritic cells and goblet cells on tissue samples. The consumption of the PFM either by the mother during nursing or by the offspring after weaning modified the development of bifidobacteria population in the large intestine of the mice. These modifications were accompanied with a decrease of enterobacteria population. The administration of this PFM to the mothers improved their own immune system and this also affected their offspring. Offspring from mice that received PFM increased S-IgA in intestinal fluids, which mainly originated from their mother's immune system. A decrease in the number of macrophages, dendritic cells and IgA+ cells during the suckling period in offspring fed with PFM was observed; this could be related with the improvement of the immunity of the mothers, which passively protect their babies. At day 45, the mice reach maturity of their own immune system and the effects of the PFM was the stimulation of their mucosal immunity. Conclusion The present work shows the beneficial effect of the administration of a PFM not only to the mothers during the suckling period but also to their offspring after weaning and until adulthood. This effect positively improved the intestinal microbiota that are related with a modulation of the gut immune response, which was demonstrated with the stimulation of the IgA + cells, macrophages and dendritic cells.

Intestinal barrier integrity is essential in order to maintain the homeostasis of mucosal functions and efficient defensive reactions against chemical and microbial challenges. An impairment of the intestinal barrier has been observed in several chronic diseases. The gut microbiota and its impact on intestinal homeostasis is well described and numerous studies suggest the ability of some probiotic strains to protect the intestinal epithelial integrity and host homeostasis. In this work, we aimed to assess the beneficial effects of three Lactobacillus strains (Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC03, and Lactiplantibacillus plantarum CNCM I-4459) and their mechanism of action in low-grade inflammation or neonatal maternal separation models in mice. We compared the impact of these strains to that of the well-known probiotic Lacticaseibacillus rhamnosus GG. Our results demonstrated that the three strains have the potential to restore the barrier functions by (i) increasing mucus production, (ii) restoring normal permeability, and (iii) modulating colonic hypersensitivity. Moreover, gene expression analysis of junctional proteins revealed the implication of Claudin 2 and Cingulin in the mechanisms that underlie the interactions between the strains and the host. Taken together, our data suggest that LR04, CNCM I-4459, and LC03 restore the functions of an impaired intestinal barrier.

The host immune system is an important factor to combat metastasis. In this work, it was evaluated the immunomodulation exerted by milk fermented with the probiotic strain Lactobacillus casei CRL431 (PFM) when was administered to mice in the metastatic stage of breast cancer. Mice received PFM were compared to animals given milk. PFM administration diminished metastasis in the lungs and increased the survival of the animals. Systemically, PFM administration decreased pro-inflammatory cytokines and locally in the lungs (metastatic organs) decreased F4/80+ cells, principally IL-10/F4/80+ cells. The highest percentages for CD4+ and CD4+CD8+ cells were observed in PFM group. Finally, immune cells from lungs of mice given PFM showed lower production of most cytokines evaluated when they were re-stimulated in vitro with tumour cells. The results obtained suggest that modulation of immune cells in the lungs by PFM could be a strategy to fight against tumour cells in the metastatic sites.

Fibre content, phenolic content, antioxidant and anti-inflammatory activities were evaluated for water extracts of fruit by-products (passion fruit, orange, acerola, and mango). The impact of these extracts on microbial growth, folate production, and adhesion ability of Streptococcus thermophilus TH-4 and Lactobacillus rhamnosus LGG was investigated. Mango water extract (MWE) presented the highest phenolic content and antioxidant activity. Orange water extract (OWE) and MWE showed the best anti-inflammatory potential by decreasing the highest nitric oxide levels. When TH-4 and LGG were grown together, folate production was only stimulated by MWE. Passion fruit water extract and OWE increased the TH-4 adhesion whereas acerola water extracts and MWE improved LGG adhesion when strains were used individually. These results showed that fruit by-product water extracts (FWE), especially from mango, presented potential beneficial biological and functional properties. These FWE could be used to develop new functional antioxidant foods and natural pharmaceutical ingredients.

Riboflavin, vitamin B2, is essential for humans and has to be obtained from the diet. Some lactic acid bacteria (LAB) produce this vitamin, and they can be used for in-situ fortification of foods. This could be an alternative to supplementation with chemically synthesized vitamin, to palliate riboflavin deficiencies in specific groups of people. Moreover, if the producing LAB could survive in the gastrointestinal stress (GIT) they could be added as probiotics in this environment. In the present study we tested two riboflavin-overproducing Lactiplantibacillus plantarum strains (M5MA1-B2 and M9MG6-B2), spontaneous mutants of LAB isolated from chicha, a traditional Andean beverage. These two LAB, and also their isogenic strains M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12], expressing the mCherry protein from the pRCR12 plasmid, were evaluated in vitro under simulated GIT conditions. Among other, specifically developed protein fluorescence assays were used. The four LAB showed similar levels of adhesion (>6.0%) to Caco-2 cells, higher than that of the probiotic Lacticaseibacillus rhamnosus GG strain (4.51%). Thus, LAB biofilm formation was assessed in the labeled cells by intracellular mCherry fluorescence and in the unlabeled parental strains by crystal violet staining. Both methods detected the formation of consistent biofilms by the L. plantarum strains. The quantification of mCherry fluorescence was also used to analyze LAB auto-aggregation properties. High levels of auto-aggregation were detected for both M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12]. Survival of LAB included in a commercial cereal-based food matrix (Incaparina) under GIT conditions was also evaluated. The four LAB were resistant in vitro to the stomach and intestinal stresses, and proliferated in this environment, indicating a protective and nutritional effect of the Incaparina on the bacteria. Also, M9MG6-B2 survival in the presence or absence of Incaparina was evaluated in vivo in a BALB/c mouse model. The administration of the M9MG6-B2 strain alone or together with Incaparina had no adverse effect on the health, growth and/or well-being of the rodents. In addition, an increment in the villus length/crypt depth ratio was observed. The overall results obtained indicate that the LAB studied have probiotic characteristics of interest for the development of functional foods.

This study aimed to evaluate the bioavailability and effects on the intestinal mucosa of a bioenriched fermented milk (BFM) prepared with folate-producing lactic acid bacteria, using a depletion/repletion BALB/c mice model. Five Streptococcus thermophilus strains were combined with Lactobacillus plantarum 16cv and the combination that resulted in the best folate production was used for preparation of BFM. The folate content in BFM, prepared under controlled conditions (pH 6.0, 42 °C, 70 rpm, 24 h), was 321.1 ± 14.1 ng/mL. Results for mice fed with BFM were compared to those fed with other diets. The BFM group presented an increase in hemoglobin, hematocrit and red blood cells, and the villi height/crypt depth ratio was similar to that of animals receiving milk supplemented with commercial folic acid. The strains were sensitive to most tested antibiotics and lacked virulence genes, indicating that consumption of BFM may be a promising alternative to increase intake of folate.

The characteristics of innate immunity have recently been investigated in depth in several research articles, and original findings suggest that innate immunity also has a memory capacity, which has been named “trained immunity”. This notion has revolutionized our knowledge of the innate immune response. Thus, stimulation of trained immunity represents a therapeutic alternative that is worth exploring. In this context, probiotics, live microorganisms which when administered in adequate amounts confer a health benefit on the host, represent attractive candidates for the stimulation of trained immunity; however, although numerous studies have documented the beneficial proprieties of these microorganisms, their mechanisms of action are not yet fully understood. In this review, we propose to explore the putative connection between probiotics and stimulation of trained immunity.

There is now strong evidence to support the interest in using lactic acid bacteria (LAB)in particular, strains of lactococci and lactobacilli, as well as bifidobacteria, for the development of new live vectors for human and animal health purposes. LAB are Gram-positive bacteria that have been used for millennia in the production of fermented foods. In addition, numerous studies have shown that genetically modified LAB and bifodobacteria can induce a systemic and mucosal immune response against certain antigens when administered mucosally. They are therefore good candidates for the development of new mucosal delivery strategies and are attractive alternatives to vaccines based on attenuated pathogenic bacteria whose use presents health risks. This article reviews the most recent research and advances in the use of LAB and bifidobacteria as live delivery vectors for human and animal health.

Inflammatory Bowel Diseases (IBD) are disorders of the gastrointestinal tract characterized by recurrent inflammation that requires lifelong treatments. Probiotic microorganisms appear as an alternative for these patients; however, probiotic characteristics are strain dependent and each probiotic needs to be tested to understand the underlining mechanisms involved in their beneficial properties. Genetic modification of lactic acid bacteria (LAB) was also described as a tool for new IBD treatments. The first part of this review shows different genetically modified LAB (GM-LAB) described for IBD treatment since 2000. Then, the two principally studied strategies are discussed (i) GM-LAB producing antioxidant enzymes and (ii) GM-LAB producing the anti-inflammatory cytokine IL-10. Different delivery systems, including protein delivery and DNA delivery, will also be discussed. Studies show the efficacy of GM-LAB (using different expression systems) for the prevention and treatment of IBD, highlighting the importance of the bacterial strain selection (with anti-inflammatory innate properties) as a promising alternative. These microorganisms could be used in the near future for the development of therapeutic products with anti-inflammatory properties that can improve the quality of life of IBD patients.

Inflammatory bowel diseases (IBD) describe a group of complex intestinal disorders characterized by inflammation in the gastrointestinal tract. Current treatments for IBD include the use of antiinflammatory drugs; furthermore, recombinant lactic acid bacteria have been used as a therapeutic vehicle for anti-inflammatory agents in IBD models. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines; however, its oral administration is limited because it is quickly degraded in the gastrointestinal tract and systemic treatments have led to undesirable side effects. In this study, an engineered invasive strain of Lactococcus (L.) lactis producing Fibronectin Binding Protein A (FnBPA+), from Staphylococcus aureus capable of delivering, directly inside eukaryotic cells, an eukaryotic DNA expression vector containing the ORF coding for IL-10 of Mus musculus (pValac: il-10 ) was developed and its functionality was evaluated using in vitro and in vivo assays. Functionality of the plasmid and the invasive strain was demonstrated by transfection and invasiveness assays using cell cultures and in vivo in mice by fluorescence microscopy. TNBS inoculated mice that received this novel strain showed lower damage scores in their large intestines (at both macroscopic and microscopic levels), lower microbial translocation to liver, and increased anti-inflammatory/pro-inflammatory cytokine ratios compared to mice that received L. lactis FnBPA+ without the pValac: il-10 plasmid. The effectiveness was demonstrated of this novel DNA delivery therapeutic strategy in the prevention of inflammation using a murine model of colitis.

Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are important problems in industrialized countries. The complete aetiology of both diseases is still unknown but likely involves genetic, environmental and immunological factors. The aim of this work is to study the anti-inflammatory mechanisms reported for yoghurt in a colon cancer model in order to evaluate its usefulness in the treatment of intestinal inflammation such as Crohn's disease. A trinitrobenzenesulfonic acid (TNBS)-induced colitis model was used. The influence of yoghurt feeding was studied before and after TNBS inoculation. The effect on the intestinal microbiota and on the host immune response was evaluated. IgA-producing cells and cells positive for specific cytokines (IL-12, IL-17, IFNγ and IL-10) were analyzed. Yoghurt administration diminished the severity of inflammation in the TNBS-inoculated mice. This effect occurred mainly through IL-10, which was increased in the intestinal tissues throughout the study, and by the decrease observed in IL-17 and IL-12 levels. In addition to this immunomodulatory capacity, another mechanism by which yoghurt could exert the anti-inflammatory activity observed in our model would be through beneficial changes in the intestinal microbiota (increases in the bifidobacteria and lactobacilli populations). These changes in the intestinal microbiota could also be considered one of the causes of the intestinal inflammation reduction. These results show that yoghurt administration modulated the immune response, inducing down regulation of the inflammatory cytokines produced by the immune cells involved in the inflammatory process. The protective effect

Lactic acid bacteria (LAB) found in numerous fermented products can interact with the gut associated lymphoid tissue increasing antibody (principally secretory IgA) production. IgA secreting cells can repopulate not only the lamina propria but also they can go to other distant sites such as bronchus, urogenital tract and mammary glands a phenomenon known as the IgA cycle. Later studies have shown that both B cells of other isotypes and T cells from Peyer's patches also exhibit gut-seeking properties. The aim of this study was to study the effect of different feeding periods of L. casei CRL 431 on the interaction with the immune cells in Peyer's patches studying the migration of not only the IgA+ cells, but also other immune cells (T lymphocytes) to other mucosal sites such as bronchus and mammary glands. BALB/c mice were fed with L. casei CRL 431 during 2, 5 or 7 days. At the end of the feeding period, the mice were killed and the small intestine, the lung and the mammary glands were removed. IgA+ cells and CD4+ and CD8+ T lymphocytes were counted in tissue slices using direct immunofluorescence. IgA+ cells increased in the intestine samples taken after 7 days of LAB feeding. In mammary gland and lung tissues, IgA+ cells increased after five days of feeding. CD4+ and CD8+ T lymphocytes were not able to migrate to sites distant from the intestine and their number did not increase in the lamina propria of the small intestine. L. casei CRL 431 was able to stimulate the IgA cycle without proliferation of T population. These results allow us to suggest that this LAB could be used as oral adjuvant to protect mucosal surfaces from intestinal and respiratory diseases, and would also be useful as an adjuvant to protect against mammary glands pathologies such as cancer.

Lactic acid bacteria (LAB) are the most commonly used microorganisms in probiotic products and it is known that these LAB enhanced the immune response and increase resistance to neoplasia and infections. In previous studies using an experimental model of BALB/c mice it was demonstrated that a cyclical diet of yoghurt given to animals previously injected with the carcinogen 1,2 dimethylhydrazine (DMH) inhibited the development of colorectal carcinoma. The animals showed an inflammatory response prior to the development of the tumour, which was diminished with the yoghurt feeding. We examined the immunoregulatory and antiinflammatory mechanisms involved in the inhibition of tumour growth by yoghurt and compared with the mechanisms of a non-steroidal antiinflammatory drug (Indomethacin). Five experimental groups (BALB/c mice) were used in this study: 1) DMH group, injected with 1,2 dimethylhydrazine weekly for 10 weeks. 2) DMH-yoghurt group, yoghurt was supplemented 10 days followed by inoculation with DMH. After tumour induction yoghurt was given every 10 days for six months. 3) Only yoghurt given during six months following the same schedule (yoghurt control. 4) DMH-indomethacin group. After tumour induction, animals were treated with indomethacin, injected cyclically. 5) Non-treatment control group fed with a conventional balanced diet. We studied IgA secreting cells and CD4+ and CD8+ T cells in the large intestine of mice fed long term with yoghurt and others treated with indomethacin. TNFα, INFγ cytokines, Bcl2 protein and iNOS enzyme production was also measured We observed an increase in the number of IgA-secreting cells but not in the CD4+ and CD8+ cells in the mice fed long term with yoghurt. Indomethacin treated mice showed high values of all these cellular populations. Mice injected with indomethacin did not show increased levels of the proinflammatory cytokine TNFα and INFγ. These cytokines were increased in DMH and DMH plus yoghurt groups. iNOS enzyme determinations were increased in DMH and DMH plus indomethacin group. These results coincided with the inflammatory response observed in the histological findings. Bcl-2 protein was increased in mice fed long term with yoghurt. We suggest that the immune mechanisms by which yoghurt operates would be different to those induced with the antiinflammatory drug indomethacin. Yoghurt activated the production of cytokines that could exert a regulation of the immune response by apoptosis induced by TNFα. We conclude that yoghurt down modulate the immune response and exert its antitumour activity by its antiinflammatory activity, a mechanism that is different with that of the antiinflammatory indomethacin.