RESUMO
Gut microbiota richness and stability are important parameters in host-microbe symbiosis. Diet modification, notably using dietary fibres, might be a way to restore a high richness and stability in the gut microbiota. In this work, during a 6-week nutritional trial, 19 healthy adults consumed a basal diet supplemented with 10 or 40 g dietary fibre per day for 5 days, followed by 15-day washout periods. Fecal samples were analysed by a combination of 16S rRNA gene pyrosequencing, intestinal cell genotoxicity assay, metatranscriptomics sequencing approach and short-chain fatty analysis. This short-term change in the dietary fibre level did not have the same impact for all individuals but remained significant within each individual gut microbiota at genus level. Higher microbiota richness was associated with higher microbiota stability upon increased dietary fibre intake. Increasing fibre modulated the expression of numerous microbiota metabolic pathways such as glycan metabolism, with genes encoding carbohydrate-active enzymes active on fibre or host glycans. High microbial richness was also associated with high proportions of Prevotella and Coprococcus species and high levels of caproate and valerate. This study provides new insights on the role of gut microbial richness in healthy adults upon dietary changes and host microbes' interaction.
Assuntos
Dieta/métodos , Fibras na Dieta/administração & dosagem , Ácidos Graxos/análise , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Adulto , Clostridiales/genética , Clostridiales/isolamento & purificação , Suplementos Nutricionais , Feminino , Humanos , Masculino , Prevotella/genética , Prevotella/isolamento & purificação , RNA Ribossômico 16S/genética , Simbiose , Adulto JovemRESUMO
SCOPE: Cow's milk allergy is the most prevalent food allergy in infants whose immune system development is critically stimulated during postnatal gut colonization by commensal bacteria. Allergenic potential of cow's milk ß-lactoglobulin (BLG) and caseins (CAS) was investigated in germ-free (GF) BALB/c mice and in GF mice conventionalized (CVd) at 6 weeks of age. METHODS AND RESULTS: Oral sensitization to cow's milk in the presence of cholera toxin led to higher BLG-specific IgE, IgG1, and IgG2a responses in GF mice than in conventional (CV) mice. No significant difference was observed for CAS-specific IgE responses although IgG1 responses to αS1- and κ-caseins were higher in GF mice than in CV mice. CVd mice, orally inoculated with fecal preparations from CV mice, also displayed biased antibody responses compared to CV mice. Secretion of Th2 cytokines by BLG- and CAS-reactivated splenocytes of CVd mice was similar to that of GF mice whereas cytokine production by reactivated cells from mesenteric lymph nodes of CVd mice was equivalent to that of CV mice. CONCLUSION: Oral sensitization to BLG and CAS was differentially affected by the absence of gut microbiota and delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.
Assuntos
Caseínas/imunologia , Trato Gastrointestinal/microbiologia , Lactoglobulinas/imunologia , Metagenoma , Hipersensibilidade a Leite/imunologia , Animais , Toxina da Cólera/metabolismo , Citocinas/metabolismo , DNA/genética , Fezes/microbiologia , Feminino , Vida Livre de Germes , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Leite/imunologia , RNA Ribossômico 16S/genética , Baço/citologia , Baço/efeitos dos fármacosRESUMO
SCOPE: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ-free (GF) mice exhibit Th2-polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant. METHODS AND RESULTS: ß-lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ- and αS1-caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG-reactivation, IL-4, IL-5, IL-13 and IFN-γ productions by splenocytes were higher in GF mice than in CV mice. Heat-treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat-treatment increased protein immunogenicity and led to the production of anti-BLG and anti-κ-casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL-4, IL-5 and IL-13 in BLG-reactivated splenocytes from CV mice. CONCLUSION: Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.
Assuntos
Alérgenos/efeitos adversos , Caseínas/efeitos adversos , Modelos Animais de Doenças , Lactoglobulinas/efeitos adversos , Hipersensibilidade a Leite/imunologia , Alérgenos/química , Animais , Especificidade de Anticorpos , Células Cultivadas , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Vida Livre de Germes , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Temperatura Alta , Isotipos de Imunoglobulinas/análise , Intestinos/imunologia , Intestinos/microbiologia , Lactoglobulinas/química , Camundongos , Camundongos Endogâmicos BALB C , Hipersensibilidade a Leite/metabolismo , Desnaturação Proteica , Isoformas de Proteínas/efeitos adversos , Baço/imunologia , Baço/metabolismo , Baço/patologiaRESUMO
Health claims for probiotics are evaluated by the Panel on Dietetic Products, Nutrition and Allergies of the European Food Safety Authority. Despite a substantial amount of basic and clinical research on the beneficial effects of probiotics, all of the evaluated claim applications thus far have received a negative opinion. With the restrictions on the use of clinical endpoints, validated biomarkers for gut health and immune health in relation to reduction in disease risk are needed. Clear-cut criteria for design as well as evaluation of future studies are needed. An open dialogue between basic and clinical scientists, regulatory authorities, food and nutrition industry, and consumers could bridge the gap between science and marketing of probiotics.
Assuntos
Rotulagem de Medicamentos , Saúde , Legislação de Medicamentos , Marketing , Avaliação de Resultados em Cuidados de Saúde , Probióticos , Projetos de Pesquisa , Biomarcadores , Comunicação , Indústria Farmacêutica/legislação & jurisprudência , Rotulagem de Medicamentos/legislação & jurisprudência , Europa (Continente) , Trato Gastrointestinal , Órgãos Governamentais , Humanos , Sistema Imunitário , Marketing/legislação & jurisprudência , Avaliação de Resultados em Cuidados de Saúde/legislação & jurisprudência , Projetos de Pesquisa/legislação & jurisprudência , CiênciaRESUMO
UNLABELLED: The composition of the human intestinal microbiota is linked to health status. The aim was to analyze the microbiota of normal and colon cancer patients in order to establish cancer-related dysbiosis. PATIENTS AND METHODS: Stool bacterial DNA was extracted prior to colonoscopy from 179 patients: 60 with colorectal cancer, and 119 with normal colonoscopy. Bacterial genes obtained by pyrosequencing of 12 stool samples (6 Normal and 6 Cancer) were subjected to a validated Principal Component Analysis (PCA) test. The dominant and subdominant bacterial population (C. leptum, C. coccoides, Bacteroides/Prevotella, Lactobacillus/Leuconostoc/Pediococcus groups, Bifidobacterium genus, and E. coli, and Faecalibacterium prausnitzii species) were quantified in all individuals using qPCR and specific IL17 producer cells in the intestinal mucosa were characterized using immunohistochemistry. FINDINGS: Pyrosequencing (Minimal sequence 200 nucleotide reads) revealed 80% of all sequences could be assigned to a total of 819 taxa based on default parameter of Classifier software. The phylogenetic core in Cancer individuals was different from that in Normal individuals according to the PCA analysis, with trends towards differences in the dominant and subdominant families of bacteria. Consequently, All-bacteria [log(10) (bacteria/g of stool)] in Normal, and Cancer individuals were similar [11.88±0.35, and 11.80±0.56, respectively, (Pâ=â0.16)], according to qPCR values whereas among all dominant and subdominant species only those of Bacteroides/Prevotella were higher (All bacteria-specific bacterium; Pâ=â0.009) in Cancer (-1.04±0.55) than in Normal (-1.40±0.83) individuals. IL17 immunoreactive cells were significantly expressed more in the normal mucosa of cancer patients than in those with normal colonoscopy. CONCLUSION: This is the first large series to demonstrate a composition change in the microbiota of colon cancer patients with possible impact on mucosal immune response. These data open new filed for mass screening and pathophysiology investigations.
Assuntos
Neoplasias Colorretais/microbiologia , Intestinos/microbiologia , Metagenoma , Bactérias/isolamento & purificação , Bacteroides , Estudos de Casos e Controles , DNA Bacteriano/análise , Fezes/microbiologia , Humanos , Interleucina-17 , Mucosa Intestinal/patologia , Prevotella , Análise de Sequência de DNARESUMO
OBJECTIVE: Obesity alters gut microbiota ecology and associates with low-grade inflammation in humans. Roux-en-Y gastric bypass (RYGB) surgery is one of the most efficient procedures for the treatment of morbid obesity resulting in drastic weight loss and improvement of metabolic and inflammatory status. We analyzed the impact of RYGB on the modifications of gut microbiota and examined links with adaptations associated with this procedure. RESEARCH DESIGN AND METHODS: Gut microbiota was profiled from fecal samples by real-time quantitative PCR in 13 lean control subjects and in 30 obese individuals (with seven type 2 diabetics) explored before (M0), 3 months (M3), and 6 months (M6) after RYGB. RESULTS: Four major findings are highlighted: 1) Bacteroides/Prevotella group was lower in obese subjects than in control subjects at M0 and increased at M3. It was negatively correlated with corpulence, but the correlation depended highly on caloric intake; 2) Escherichia coli species increased at M3 and inversely correlated with fat mass and leptin levels independently of changes in food intake; 3) lactic acid bacteria including Lactobacillus/Leuconostoc/Pediococcus group and Bifidobacterium genus decreased at M3; and 4) Faecalibacterium prausnitzii species was lower in subjects with diabetes and associated negatively with inflammatory markers at M0 and throughout the follow-up after surgery independently of changes in food intake. CONCLUSIONS: These results suggest that components of the dominant gut microbiota rapidly adapt in a starvation-like situation induced by RYGB while the F. prausnitzii species is directly linked to the reduction in low-grade inflammation state in obesity and diabetes independently of calorie intake.
Assuntos
Cirurgia Bariátrica , Inflamação/microbiologia , Inflamação/fisiopatologia , Redução de Peso/fisiologia , Adaptação Fisiológica , Bacteroides/genética , Bacteroides/isolamento & purificação , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , Glicemia/metabolismo , Clostridium/genética , Clostridium/isolamento & purificação , Primers do DNA , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Feminino , Humanos , Inflamação/etiologia , Lactobacillus/isolamento & purificação , Leuconostoc/isolamento & purificação , Masculino , Obesidade/microbiologia , Pediococcus/isolamento & purificação , Reação em Cadeia da Polimerase , Inanição/microbiologia , Magreza/microbiologiaRESUMO
Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.
Assuntos
Catalase , Colite/microbiologia , Colite/terapia , Lacticaseibacillus casei/enzimologia , Lacticaseibacillus casei/genética , Estresse Oxidativo , Superóxido Dismutase , Animais , Anti-Inflamatórios/uso terapêutico , Catalase/genética , Catalase/metabolismo , Catalase/uso terapêutico , Células Cultivadas , Colite/induzido quimicamente , Colite/enzimologia , Colite/patologia , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Humanos , Inflamação/genética , Inflamação/patologia , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase/uso terapêuticoRESUMO
BACKGROUND: In the last years, the use of probiotics such as lactic acid bacteria (LAB) has been proposed as an attractive alternative for the management of allergic diseases. A partial prevention from sensitization to bovine beta-lactoglobulin (BLG), one of the major cows' milk allergens, could be achieved in mice after intranasal administration with a recombinant LAB strain, Lactococcus lactis, producing BLG (LL-BLG). This study aimed to evaluate the effects of the LL-BLG strain in a therapeutic protocol. METHODS: Three groups of mice were first orally sensitized to cows' milk and then intranasally administered with either the LL-BLG strain, BLG protein alone or saline solution. Serum samples were collected to analyze BLG-specific IgE, IgG1 and IgG2a, and mice were further intranasally challenged with BLG to elicit a specific allergic reaction. RESULTS: Treatment with LL-BLG, but not with BLG alone, contributed to diminish IgG1 production in serum and bronchoalveolar lavage fluids. This was associated with decreased IL-4 production and enhanced IFN-gamma production by BLG-reactivated splenocytes, suggesting a switch from Th2- to Th1-immune response. Furthermore, we observed that administration of LL-BLG or LL locally reduced the allergic reaction induced after intranasal challenge, as evidenced by decreased release of IL-4 in bronchoalveolar lavage fluids. CONCLUSION: These preliminary results demonstrate the efficiency of the intranasal administration of LL-BLG for specific therapy against cows' milk-related allergy.
Assuntos
Dessensibilização Imunológica/métodos , Lactococcus lactis/imunologia , Lactoglobulinas/administração & dosagem , Hipersensibilidade a Leite/prevenção & controle , Administração Intranasal , Animais , Bovinos , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Interleucina-4/biossíntese , Lactoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Hipersensibilidade a Leite/imunologiaRESUMO
The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log10 copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log10 copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log10 copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution.
Assuntos
Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Rios/microbiologia , Suínos/microbiologia , Poluição da Água , Animais , Bovinos , Análise por Conglomerados , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , França , Humanos , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups (Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.
Assuntos
Bacteroides/isolamento & purificação , Bifidobacterium/isolamento & purificação , Clostridium/isolamento & purificação , Fezes/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , Animais Domésticos/microbiologia , Bacteroides/classificação , Bacteroides/genética , Bifidobacterium/classificação , Bifidobacterium/genética , Clostridium/classificação , Clostridium/genética , DNA Bacteriano/genética , Humanos , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-kappaB activity, F. prausnitzii had no effect on IL-1beta-induced NF-kappaB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-gamma production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-kappaB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.
Assuntos
Anti-Inflamatórios/administração & dosagem , Doença de Crohn/terapia , Mucosa Intestinal/microbiologia , Probióticos/uso terapêutico , Ruminococcus/isolamento & purificação , Animais , Células Cultivadas , Colite , Doença de Crohn/microbiologia , Citocinas/biossíntese , Modelos Animais de Doenças , Humanos , Leucócitos/imunologia , Leucócitos/microbiologia , Camundongos , NF-kappa B/metabolismo , Probióticos/administração & dosagem , Probióticos/farmacologia , Resultado do TratamentoRESUMO
The objective of this study was to determine i) if Camembert cheese micro-organisms could be detected in fecal samples after regular consumption by human subjects and ii) the consequence of this consumption on global metabolic activities of the host colonic microbiota. An open human protocol was designed where 12 healthy volunteers were included: a 2-week period of fermented products exclusion followed by a 4-weeks Camembert ingestion period where 2x40 g/day of Camembert cheese was consumed. Stools were collected from the volunteers before consumption, twice during the ingestion period (2nd and 4th week) and once after a wash out period of 2 weeks. During the consumption of Camembert cheese, high levels of Lactococcus lactis and Leuconostoc mesenteroides were measured in fecal samples using real-time quantitative PCR, reaching median values of 8.2 and 7.5 Log(10) genome equivalents/g of stool. For Ln. mesenteroides, persistence was observed 15 days after the end of Camembert consumption. The survival of Geotrichum candidum was also assessed and the fecal concentration reached a median level of 7.1 Log(10) CFU/g in stools. Except a decreasing trend of the nitrate reductase activity, no significant modification was shown in the metabolic activities during this study.
Assuntos
Queijo/microbiologia , Colo/microbiologia , Fezes/microbiologia , Lactobacillus/crescimento & desenvolvimento , Streptococcus thermophilus/crescimento & desenvolvimento , Adulto , Contagem de Colônia Microbiana , Estudos Cross-Over , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Fúngico/química , DNA Fúngico/genética , Feminino , Microbiologia de Alimentos , Geotrichum/crescimento & desenvolvimento , Geotrichum/isolamento & purificação , Geotrichum/metabolismo , Humanos , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Masculino , Nitrato Redutase/metabolismo , Reação em Cadeia da Polimerase/métodos , Streptococcus thermophilus/isolamento & purificação , Streptococcus thermophilus/metabolismo , Fatores de TempoRESUMO
Lactic acid bacteria are used on an industrial scale for the manufacturing of dairy products. It is now intended to develop novel applications of lactic acid bacteria that could be used as living vehicles for the targeting of antigens or therapeutics to the digestive mucosa. The aim of this study was to analyze the adaptations of Lactococcus lactis, a model lactic acid bacteria to the digestive tract and to identify functions required for colonization of the intestine. For this purpose, we combined gnotobiology with proteomics: axenic mice were colonized with a dairy L. lactis strain and the bacterial proteome was examined by 2-DE. As compared to cultures in broth, the proteome profile of bacteria grown in the intestine indicates the activation of metabolic pathways involved in various carbon sources assimilation and suggests the adoption of a mixed acids fermentative metabolism. We identified the product of the ywcC gene as essential for the colonization of the digestive tract and demonstrated that the corresponding gene product (YwcC) possesses a phosphogluconolactonase activity, suggesting an important role of the pentose phosphate pathway for the development of L. lactis in the digestive environment.
Assuntos
Adaptação Fisiológica , Trato Gastrointestinal/microbiologia , Lactococcus lactis/metabolismo , Via de Pentose Fosfato , Proteoma/análise , Sequência de Aminoácidos , Animais , Hidrolases de Éster Carboxílico/metabolismo , Primers do DNA , Eletroforese em Gel Bidimensional , Trato Gastrointestinal/metabolismo , Vida Livre de Germes , Lactococcus lactis/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The aim of this study was to evaluate the survival of Lactobacillus rhamnosus R11 and Lactobacillus acidophilus R52 in the human digestive tract and their effects on the microbiota homeostasis. We designed an open human trial including 14 healthy volunteers. A 3-week exclusion period of fermented products was followed by a 12-day consumption period of 4 capsules daily containing 2 x 10(9)L. rhamnosus R11 and 1 x 10(8)L. acidophilus R52, and a 12-day wash-out period. The 2 strains and dominant bacterial groups of the microbiota were quantified by real-time polymerase chain reaction. At the end of the capsule consumption period, high levels of L. rhamnosus R11 were detected in faecal samples from all volunteers, reaching a mean value of 7.1 log(10) colony-forming unit (CFU) equivalents/g of stool. L. acidophilus R52 was detected in the stools of only 1 volunteer, reaching a maximum level of 6.1 log(10) CFU equivalents/g of stool. Dilution plating enumerations performed in parallel provided less consistent and generally lower levels. No significant effect of capsule consumption was observed on microbiota homeostasis for the dominant faecal populations. Mean values of 8.8, 9.2, 9.9 and 10.6 log(10) CFU equivalents/g of stool were obtained for the Clostridium coccoides, Bifidobacterium sp., Bacteroides sp. and Clostridium leptum groups, respectively.
Assuntos
Trato Gastrointestinal/microbiologia , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , Probióticos/administração & dosagem , Administração Oral , Adolescente , Adulto , Bacteroides/crescimento & desenvolvimento , Bifidobacterium/crescimento & desenvolvimento , Clostridium/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Fezes/microbiologia , Feminino , Humanos , Lacticaseibacillus rhamnosus/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Resultado do TratamentoRESUMO
The present study demonstrates for the first time the transfer of vancomycin resistance (vanA cluster) from enterococci to a Lactobacillusacidophilus commercial strain. Transfers were observed in vitro, but also in vivo in the gut of mice (in the absence of antibiotic pressure) where transconjugants arose at relatively high frequencies and could persist in the digestive environment. Since transfer of vancomycin resistance genes might also take place in the human digestive tract, lactobacilli probiotics should be carefully considered especially in either immunocompromised patients or during antibiotherapy. Acquisition and retransfer of resistance genes should be addressed in the safety evaluation of probiotics.
Assuntos
Conjugação Genética , Enterococcus faecium/genética , Trato Gastrointestinal/microbiologia , Lactobacillus acidophilus/genética , Probióticos/administração & dosagem , Resistência a Vancomicina/genética , Animais , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases/genética , Transferência Genética Horizontal , Humanos , Camundongos , Camundongos Endogâmicos C3HRESUMO
Lactococcus lactis is a model of food-grade lactic acid bacterium, which can durably colonize the digestive tract of germ-free mice. To study in vivo the bacterial adaptation to a novel nutritional resource brought by alimentation, the lactose-catabolizing strain IL2661 of L. lactis was established in monoxeny in mice. Half of the mice then received a lactose-rich diet. The mouse has no efficient intestinal lactase and is well adapted to a follow-up of the metabolic activity of microbial origin. The analysis of lactose and lactate in the feces suggested that L. lactis was able to use lactose in vivo. We developed a proteomic approach to evaluate in deeper details the metabolic response of the bacterium. We observed that L. lactis switched its metabolism to use the novel carbon source and reduced the level of proteins involved in an alternative mode of ATP production. In parallel, we also found that the amount of proteins involved in transcriptional regulation, transport and catabolism decreased in the presence of lactose. The proteome analysis informed us about the resources used by the bacteria in absence of lactose. In competition experiments, we found that the metabolic adaptation gives a strong ecological advantage to the bacteria able to efficiently utilize lactose.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/metabolismo , Lactose/metabolismo , Proteoma , Animais , Proteínas de Bactérias/genética , Vida Livre de Germes , Lactococcus lactis/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C3HRESUMO
Probiotics and prebiotics have long been appreciated for their positive influences on gut health. Research on the mechanisms and effects of these agents shows that their impact reaches beyond the intestine. Effects on the microecology and pathology of the oral cavity, stomach, and vaginal tract have been observed. Likely mediated through immune influences, systemic effects such as reduced severity of colds or other respiratory conditions, impact on allergy incidence and symptoms, and reduced absences from work or daycare have also been noted. These observations, among others, suggest a broader spectrum of influence than commonly considered for these unique substances.
Assuntos
Sistema Digestório/microbiologia , Sistema Imunitário/microbiologia , Probióticos , Sistema Respiratório/microbiologia , Sistema Urogenital/microbiologia , Animais , Galinhas , Suplementos Nutricionais/microbiologia , Sistema Digestório/imunologia , Feminino , Humanos , Hipersensibilidade , Sistema Imunitário/imunologia , Masculino , Ensaios Clínicos Controlados Aleatórios como Assunto , Ratos , Sistema Respiratório/imunologia , Sistema Urogenital/imunologiaRESUMO
Enterococci are natural inhabitants of the human gastrointestinal tract and the main Gram-positive and facultative anaerobic cocci recovered in human faeces. They are also present in a variety of fermented dairy and meat products, and some rare isolates are responsible for severe infections such as endocarditis and meningitis. The aim of the present study was to evaluate the effect of Camembert cheese consumption by healthy human volunteers on the faecal enterococcal population. A highly specific real-time quantitative PCR approach was designed and used to type enterococcal species in human faeces. Two species were found, Enterococcus faecalis and Enterococcus faecium, and only the Enterococcus faecalis population was significantly enhanced after Camembert cheese consumption, whereas Escherichia coli population and the dominant microbiota remained unaffected throughout the trial.
Assuntos
Queijo/microbiologia , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecium/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Contagem de Colônia Microbiana/métodos , DNA Bacteriano/genética , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Fezes/microbiologia , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
Mucosal immunity plays a major role in the prevention of infectious diseases. Genetically engineered lactic acid bacteria (LAB) have been tested in the last 10 years as safe mucosal delivery vectors. We previously showed that intranasal co-administration of recombinant lactococci displaying human papillomavirus type 16 (HPV-16) E7 antigen at its surface (LL-E7) and secreting biologically active interleukine-12 (LL-IL-12) has therapeutic effects on HPV-16-induced tumors in mice. In this work, to optimize the immunization protocol, a comparison between intragastric and intranasal routes of administration was performed and two different LAB strains (Lactococcus lactis and Lactobacillus plantarum) were tested as delivery vector. E7-specific systemic and mucosal responses as well as potent anti-tumor effects were higher after intranasal immunization with LL-E7 and LL-IL-12 strains than intragastric administration. Comparisons of the immune responses induced by intranasal administration of either LL-E7 or Lb. plantarum anchoring E7 antigen (LP-E7) revealed highest systemic responses with recombinant Lactobacillus. Furthermore, although only a modest mucosal immune response was observed with LP-E7, this strain was able to induce a significant regression of HPV-induced tumors in contrast to LL-E7. Taken together, our results demonstrate the advantage of intranasal over intragastric route of immunization to induce an antigen-specific immune response and suggest that intrinsic immunomodulatory properties of Lb. plantarum play an important role in the immunogenicity of the expressed antigen.
Assuntos
Imunidade nas Mucosas/imunologia , Imunização/métodos , Lactobacillus plantarum/genética , Lactococcus lactis/genética , Proteínas Recombinantes de Fusão/imunologia , Administração Intranasal , Animais , Vias de Administração de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Immunoblotting , Interleucina-12/genética , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neoplasias/etiologia , Neoplasias/imunologia , Neoplasias/prevenção & controle , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: Human immune cells generate large amounts of reactive oxygen species (ROS) throughout the respiratory burst that occurs during inflammation. In inflammatory bowel diseases, a sustained and abnormal activation of the immune system results in oxidative stress in the digestive tract and in a loss of intestinal homeostasis. We previously showed that the heterologous production of the Lactobacillus plantarum ATCC14431 manganese-dependant catalase (MnKat) in Lb. casei BL23 successfully enhances its survival when exposed to oxidative stress. In this study, we evaluated the preventive effects of this antioxidative Lb. casei strain in a murine model of dextran sodium sulfate (DSS)-induced moderate colitis. RESULTS: Either Lb. casei BL23 MnKat- or MnKat+ was administered daily to mice treated with DSS for 10 days. In contrast to control mice treated with PBS for which DSS induced bleeding diarrhea and mucosal lesions, mice treated with both Lb. casei strains presented a significant (p < 0.05) reduction of caecal and colonic inflammatory scores. CONCLUSION: No contribution of MnKat to the protective effect from epithelial damage has been observed in the tested conditions. In contrast, these results confirm the high interest of Lb. casei as an anti-inflammatory probiotic strain.