RESUMO
OBJECTIVE: Altered microbiota composition or dysbiosis is suspected to be implicated in the pathogenesis of chronic inflammatory diseases, such as spondyloarthritis (SpA) and rheumatoid arthritis (RA). METHODS: 16S ribosomal RNA gene sequencing was performed on faecal DNA isolated from stool samples in two consecutive cross-sectional cohorts, each comprising three groups of adult volunteers: SpA, RA and healthy controls (HCs). In the second study, HCs comprised a majority of aged-matched siblings of patients with known HLA-B27 status. Alpha and beta diversities were assessed using QIIME, and comparisons were performed using linear discriminant analysis effect size to examine differences between groups. RESULTS: In both cohorts, dysbiosis was evidenced in SpA and RA, as compared with HCs, and was disease specific. A restriction of microbiota biodiversity was detected in both disease groups. The most striking change was a twofold to threefold increased abundance of Ruminococcus gnavus in SpA, as compared with both RA and HCs that was significant in both studies and positively correlated with disease activity in patients having a history of inflammatory bowel disease (IBD). Among HCs, significant difference in microbiota composition were also detected between HLA-B27+ and HLA-B27 negative siblings, suggesting that genetic background may influence gut microbiota composition. CONCLUSION: Our results suggest that distinctive dysbiosis characterise both SpA and RA and evidence a reproducible increase in R. gnavus that appears specific for SpA and a marker of disease activity. This observation is consistent with the known proinflammatory role of this bacteria and its association with IBD. It may provide an explanation for the link that exists between SpA and IBD.
Assuntos
Artrite Reumatoide/microbiologia , Disbiose/microbiologia , Microbioma Gastrointestinal/genética , RNA Ribossômico 16S/genética , Espondiloartropatias/microbiologia , Adulto , Idoso , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Estudos Transversais , Disbiose/imunologia , Fezes/microbiologia , Feminino , Antígeno HLA-B27/genética , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Ruminococcus/genética , Irmãos , Espondiloartropatias/genética , Espondiloartropatias/imunologiaRESUMO
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
OBJECTIVE: Gut microbiome dysbiosis has previously been reported in spondyloarthritis (SpA) patients and could be critically involved in the pathogenesis of this disorder. The objectives of this study were to further characterize the microbiota structure in SpA patients and to investigate the relationship between dysbiosis and disease activity in light of the putative influence of the genetic background. METHODS: Shotgun sequencing was performed on fecal DNA isolated from stool samples from 2 groups of adult volunteers: SpA patients (n = 102) and healthy controls (n = 63). A subset of the healthy controls comprised the age-matched siblings of patients whose HLA-B27 status was known. Changes in gut microbiota composition were assessed based on species diversity, enterotypes, and taxonomic and functional differences. RESULTS: Dysbiosis was confirmed in SpA patients as compared to healthy controls. The restriction of microbiota diversity was detected in patients with the most active disease, and the abundance of several bacterial species was correlated with Bath Ankylosing Spondylitis Disease Activity Index score. Among healthy controls, significant differences in microbiota composition were also detected between the HLA-B27-positive and the HLA-B27-negative siblings of SpA patients. We highlighted a decreased abundance of several species of bacteria in SpA patients, especially those bacteria belonging to the Clostridiales order. Among the few species of bacteria showing increased abundance, Ruminococcus gnavus was one of the top differentiating species. CONCLUSION: These findings reveal that genetic background and level of disease activity are likely to influence the composition of the gut microbiota of patients with SpA. It may be appropriate for further research on chronic arthritis to focus on these key parameters.
Assuntos
Microbioma Gastrointestinal , Microbiota , Espondilartrite , Adulto , Humanos , Microbioma Gastrointestinal/genética , Antígeno HLA-B27/genética , Disbiose/microbiologia , Espondilartrite/genética , Espondilartrite/complicaçõesRESUMO
Low-grade inflammation observed in obesity is a risk factor for cardiovascular disease. Recent studies revealed that this would be linked to gut-derived endotoxemia during fat digestion in high-fat diets, but nothing is known about the effect of lipid composition. The study was designed to test the impact of oil composition of high-fat diets on endotoxin metabolism and inflammation in mice. C57/Bl6 mice were fed for 8 wk with chow or isocaloric isolipidic diets enriched with oils differing in fatty acid composition: milk fat, palm oil, rapeseed oil, or sunflower oil. In vitro, adipocytes (3T3-L1) were stimulated or not with lipopolysaccharide (LPS; endotoxin) and incubated with different fatty acids. In mice, the palm group presented the highest level of IL-6 in plasma (P < 0.01) together with the highest expression in adipose tissue of IL-1ß and of LPS-sensing TLR4 and CD14 (P < 0.05). The higher inflammation in the palm group was correlated with a greater ratio of LPS-binding protein (LBP)/sCD14 in plasma (P < 0.05). The rapeseed group resulted in higher sCD14 than the palm group, which was associated with lower inflammation in both plasma and adipose tissue despite higher plasma endotoxemia. Taken together, our results reveal that the palm oil-based diet resulted in the most active transport of LPS toward tissues via high LBP and low sCD14 and the greatest inflammatory outcomes. In contrast, a rapeseed oil-based diet seemed to result in an endotoxin metabolism driven toward less inflammatory pathways. This shows that dietary fat composition can contribute to modulate the onset of low-grade inflammation through the quality of endotoxin receptors.
Assuntos
Tecido Adiposo Branco/imunologia , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Doenças Metabólicas/etiologia , Doenças Metabólicas/imunologia , Receptores Imunológicos/metabolismo , Células 3T3-L1 , Proteínas de Fase Aguda , Tecido Adiposo Branco/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteínas de Transporte/sangue , Citocinas/sangue , Ácidos Graxos Monoinsaturados , Ácidos Graxos não Esterificados/efeitos adversos , Ácidos Graxos não Esterificados/sangue , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/imunologia , Bactérias Gram-Positivas/isolamento & purificação , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Receptores de Lipopolissacarídeos/sangue , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Glicoproteínas de Membrana/sangue , Doenças Metabólicas/metabolismo , Doenças Metabólicas/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Óleo de Palmeira , Óleos de Plantas/efeitos adversos , Distribuição Aleatória , Óleo de Brassica napus , Óleo de Girassol , Receptor 4 Toll-Like/metabolismoRESUMO
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 paradox of a host specificity of the human faecal microbiota otherwise acknowledged as characterized by global functionalities conserved between humans led us to explore the existence of a phylogenetic core. We investigated the presence of a set of bacterial molecular species that would be altogether dominant and prevalent within the faecal microbiota of healthy humans. A total of 10 456 non-chimeric bacterial 16S rRNA sequences were obtained after cloning of PCR-amplified rDNA from 17 human faecal DNA samples. Using alignment or tetranucleotide frequency-based methods, 3180 operational taxonomic units (OTUs) were detected. The 16S rRNA sequences mainly belonged to the phyla Firmicutes (79.4%), Bacteroidetes (16.9%), Actinobacteria (2.5%), Proteobacteria (1%) and Verrumicrobia (0.1%). Interestingly, while most of OTUs appeared individual-specific, 2.1% were present in more than 50% of the samples and accounted for 35.8% of the total sequences. These 66 dominant and prevalent OTUs included members of the genera Faecalibacterium, Ruminococcus, Eubacterium, Dorea, Bacteroides, Alistipes and Bifidobacterium. Furthermore, 24 OTUs had cultured type strains representatives which should be subjected to genome sequence with a high degree of priority. Strikingly, 52 of these 66 OTUs were detected in at least three out of four recently published human faecal microbiota data sets, obtained with very different experimental procedures. A statistical model confirmed these OTUs prevalence. Despite the species richness and a high individual specificity, a limited number of OTUs is shared among individuals and might represent the phylogenetic core of the human intestinal microbiota. Its role in human health deserves further study.
Assuntos
Bacteroides/genética , Biodiversidade , Bactérias Gram-Positivas/genética , Intestinos/microbiologia , Filogenia , Bacteroides/isolamento & purificação , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eubacterium/genética , Eubacterium/isolamento & purificação , Fezes/microbiologia , Genoma Bacteriano , Bactérias Gram-Positivas/isolamento & purificação , Humanos , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Ruminococcus/genética , Ruminococcus/isolamento & purificação , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
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
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
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
Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.
Assuntos
Produtos Fermentados do Leite/microbiologia , DNA Bacteriano/análise , Lactobacillus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Streptococcus thermophilus/isolamento & purificação , Sequência de Bases , Contagem de Colônia Microbiana , Primers do DNA , Microbiologia de Alimentos , Lactobacillus/classificação , RNA Ribossômico 16S/análise , Sensibilidade e Especificidade , Especificidade da Espécie , Streptococcus thermophilus/classificaçãoRESUMO
BACKGROUND: The clinical benefit of guaiac fecal occult blood tests (FOBT) is now well established for colorectal cancer screening. Growing evidence has demonstrated that epigenetic modifications and fecal microbiota changes, also known as dysbiosis, are associated with CRC pathogenesis and might be used as surrogate markers of CRC. PATIENTS AND METHODS: We performed a cross-sectional study that included all consecutive subjects that were referred (from 2003 to 2007) for screening colonoscopies. Prior to colonoscopy, effluents (fresh stools, sera-S and urine-U) were harvested and FOBTs performed. Methylation levels were measured in stools, S and U for 3 genes (Wif1, ALX-4, and Vimentin) selected from a panel of 63 genes; Kras mutations and seven dominant and subdominant bacterial populations in stools were quantified. Calibration was assessed with the Hosmer-Lemeshow chi-square, and discrimination was determined by calculating the C-statistic (Area Under Curve) and Net Reclassification Improvement index. RESULTS: There were 247 individuals (mean age 60.8±12.4 years, 52% of males) in the study group, and 90 (36%) of these individuals were patients with advanced polyps or invasive adenocarcinomas. A multivariate model adjusted for age and FOBT led to a C-statistic of 0.83 [0.77-0.88]. After supplementary sequential (one-by-one) adjustment, Wif-1 methylation (S or U) and fecal microbiota dysbiosis led to increases of the C-statistic to 0.90 [0.84-0.94] (pâ=â0.02) and 0.81 [0.74-0.86] (pâ=â0.49), respectively. When adjusted jointly for FOBT and Wif-1 methylation or fecal microbiota dysbiosis, the increase of the C-statistic was even more significant (0.91 and 0.85, p<0.001 and pâ=â0.10, respectively). CONCLUSION: The detection of methylated Wif-1 in either S or U has a higher performance accuracy compared to guaiac FOBT for advanced colorectal neoplasia screening. Conversely, fecal microbiota dysbiosis detection was not more accurate. Blood and urine testing could be used in those individuals reluctant to undergo stool testing.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/diagnóstico , Metilação de DNA , Sangue Oculto , Proteínas Repressoras/genética , Idoso , Neoplasias Colorretais/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
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
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
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 composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10 d supplementation step) and after (10 d follow-up step) the ingestion of a fermented milk containing Lactobacillus casei DN-114 001. Fluorescent in situ hybridisation with group-specific DNA probes, real-time PCR using L. paracasei group-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota. L. casei DNA was detected in the faeces of all of the subjects by TTGE after 10 d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containing L. casei DN-114 001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.
Assuntos
Produtos Fermentados do Leite , Fezes/microbiologia , Lacticaseibacillus casei , Probióticos/administração & dosagem , Administração Oral , Adulto , DNA Bacteriano/análise , Fezes/química , Feminino , Humanos , Lacticaseibacillus casei/enzimologia , Lacticaseibacillus casei/isolamento & purificação , MasculinoRESUMO
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 microM) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and (1)H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction: Bacteroides thetaiotaomicron (n = 2), Clostridium clostridiiforme (n = 3), Clostridium perfringens (n = 1), and Escherichia coli (n = 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.
Assuntos
Bacteroides/metabolismo , Carcinógenos/metabolismo , Clostridium/metabolismo , Escherichia coli/metabolismo , Intestinos/microbiologia , Quinolinas/metabolismo , Adulto , Animais , Ceco/microbiologia , Meios de Cultivo Condicionados , Fezes/microbiologia , Alimentos , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The mRNA expression levels of acyl-CoA oxidase (AOX), a key enzyme in very-long-chain fatty acid peroxisomal oxidation, and of peroxisome proliferator-activated receptor-delta (PPAR-delta), a nuclear receptor possibly involved in the gene regulation of brain lipid metabolism, were determined in human Y79 retinoblastoma cells by using real-time quantitative polymerase chain reaction. Cells were dosed with alpha-linolenic acid (18:3n-3), the essential metabolic precursor of the n-3 polyunsaturated fatty acid series that normally gives rise through terminal peroxisomal oxidation to the synthesis of membrane docosahexaenoic acid (22:6n-3, or DHA). The AOX and PPAR-delta relative expression levels increased 2.3 and 3.4 times, respectively, upon dosing of cells with 7 microM 18:3n-3, whereas AOX cDNA abundance decreased by 50% upon dosing with 70 microM 18:3n-3. Concurrently, the DHA content increased by 23% in the membrane ethanolamine-phosphoglycerides from cells dosed with 7 microM 18:3n-3, whereas it decreased by 38% upon dosing with 70 microM 18:3n-3. The DHA's upstream precursors (20:5n-3 and 22:5n-3) both accumulated in cells dosed with 7 or 70 microM 18:3n-3. The 18:3n-3-induced changes in membrane phospholipid fatty acid composition support the hypothesis that the terminal peroxisomal step of n-3 conversion is rate limiting in the Y79 line. The concurrent 7 microM 18:3n-3-induced increase of mRNAs encoding for AOX and for PPAR-delta suggests that 18:3n-3 (or its metabolites) at low concentration could trigger its proper conversion to DHA, possibly through activation of PPAR-delta-mediated transcription of AOX. Decreased membrane DHA content and mRNA expression level of AOX in 70-microM 18:3n-3-dosed cells corroborated the relationship between AOX expression and DHA synthesis and suggested that simultaneous down-regulating events occurred at high concentrations of 18:3n-3.