RESUMO
An altered gut microbiota has been linked to obesity in adulthood, although little is known about childhood obesity. The aim of this study was to characterize the composition of the gut microbiota in obese (n = 42) and normal-weight (n = 36) children aged 6 to 16. Using 16S rRNA gene-targeted sequencing, we evaluated taxa with differential abundance according to age- and sex-normalized body mass index (BMI z-score). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Members of the Bacteroidetes were generally better predictors of BMI z-score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. In accordance with these observations, the main metabolites produced by gut bacteria, short chain fatty acids (SCFAs), were higher in obese children, suggesting elevated substrate utilisation. Multiple taxa were correlated with SCFA levels, reinforcing the tight link between the microbiota, SCFAs and obesity. Our results suggest that gut microbiota dysbiosis and elevated fermentation activity may be involved in the etiology of childhood obesity.
Assuntos
Bacteroidetes/crescimento & desenvolvimento , Disbiose/microbiologia , Ácidos Graxos Voláteis/metabolismo , Firmicutes/crescimento & desenvolvimento , Microbioma Gastrointestinal/genética , Obesidade Infantil/microbiologia , Adolescente , Bacteroidetes/classificação , Bacteroidetes/genética , Criança , Dieta , Fezes/microbiologia , Feminino , Fermentação/genética , Firmicutes/classificação , Firmicutes/genética , Humanos , Masculino , Tipagem Molecular , RNA Ribossômico 16S/genéticaRESUMO
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder affecting 1 in 10,000 live female births. Changes in microbiota composition, as observed in other neurological disorders such as autism spectrum disorders, may account for several symptoms typically associated with RTT. We studied the relationship between disease phenotypes and microbiome by analyzing diet, gut microbiota, and short-chain fatty acid (SCFA) production. We enrolled eight RTT patients and 10 age- and sex-matched healthy women, all without dietary restrictions. The microbiota was characterized by 16S rRNA gene sequencing, and SCFAs concentration was determined by gas chromatographic analysis. The RTT microbiota showed a lower α diversity, an enrichment in Bacteroidaceae, Clostridium spp., and Sutterella spp., and a slight depletion in Ruminococcaceae. Fecal SCFA concentrations were similar, but RTT samples showed slightly higher concentrations of butyrate and propionate, and significant higher levels in branched-chain fatty acids. Daily caloric intake was similar in the two groups, but macronutrient analysis showed a higher protein content in RTT diets. Microbial function prediction suggested in RTT subjects an increased number of microbial genes encoding for propionate and butyrate, and amino acid metabolism. A full understanding of these critical features could offer new, specific strategies for managing RTT-associated symptoms, such as dietary intervention or pre/probiotic supplementation.
Assuntos
Microbioma Gastrointestinal , Fenótipo , Síndrome de Rett/diagnóstico , Síndrome de Rett/etiologia , Adolescente , Adulto , Alelos , Bactérias/classificação , Bactérias/metabolismo , Dieta , Disbiose , Metabolismo Energético , Feminino , Humanos , Redes e Vias Metabólicas , Proteína 2 de Ligação a Metil-CpG/genética , Mutação , Fatores de Risco , Índice de Gravidade de Doença , Adulto JovemRESUMO
Over the past decade, the emergence of biofilm-related invasive fungal diseases has been the subject of numerous studies focused on antifungal resistance and its impact on antifungal therapy in severely ill patients. The majority of the studies investigated the molecular mechanisms involved in antifungal resistance and pathogenicity of biofilm production by Candida albicans and Aspergillus fumigatus, the most common etiologic agents of yeast and mold invasive infections. The main mechanism characterizing biofilm-related antifungal resistance is the production of extracellular matrix, a physical barrier preventing the drugs from entering and expressing their activity. However, over-expression of efflux pumps, genetic changes of drug targets, persister cells, biofilm-host immune system interaction, proteins leading to filamentation, all together contribute to the onset of biofilm antifungal resistance. Some of these mechanisms are shared with planktonic cells and are often related to developmental phases of biofilm formation. All physical and genetic factors leading to biofilm-related antifungal resistance have been briefly discussed.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Fúngica , Fungos/efeitos dos fármacos , Micoses/microbiologia , Animais , Fungos/genética , Fungos/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológicoRESUMO
BACKGROUND: The objective of this study was to determine the species, clonal diversity, antibiotic resistance and virulence of enterococci in different environments. Seventy-one samples of farm origin (34 of food vegetables, 27 of soil and ten of irrigation water) and 19 samples of vegetables from five markets, were inoculated in Slanetz-Bartley agar plates supplemented or not with gentamicin (SB-Gen and SB plates, respectively) for enterococci recovery. RESULTS: Enterococci were obtained from 72.2% of tested samples in SB media (food vegetables from farms, 88.2%; soil and irrigation water, 51%; food vegetables from markets, 84.2%), and 65 enterococcal isolates were obtained. Enterococcus faecium was the most prevalent species (52.3%), followed by E. hirae (35.4%), E. faecalis (6.15%), and E. casseliflavus (6.15%). Antibiotic resistance detected among these enterococci was as follows (percentage/detected gene): ciprofloxacin (60%), erythromycin (18.4%/erm(B)), tetracycline (15.4%/tet(M)-tet(L)), kanamycin (15.4%/aph(3')-III), chloramphenicol (7.7%), streptomycin (3%/ant(6)), vancomycin (6.15%/vanC2)), teicoplanin (0%) and ampicillin (0%). High-level gentamicin-resistant (HLR-G) enterococci were detected in SB-Gen plates in 14 of 90 tested samples (15.5%), and 15 isolates were characterized: ten E. faecalis, four E. faecium and one E. hirae. All HLR-G enterococci carried the aac(6')-aph(2â³), erm(B) and tet(M) genes, among other resistance genes. The HLR-G isolates showed high genetic diversity (ten different PFGE profiles), and were ascribed to the sequence types ST2, ST16, ST28 and new ST528 (in E. faecalis), and ST56, new ST885 and new ST886 (in E. faecium). CONCLUSION: Food vegetables in the farm or market settings are frequently contaminated by HLR-G enterococci, and these microorganisms could reach the human intestine through the food chain, if hygienic conditions are not followed. © 2015 Society of Chemical Industry.
Assuntos
Enterococcus/efeitos dos fármacos , Enterococcus/isolamento & purificação , Microbiologia de Alimentos , Microbiologia do Solo , Verduras/microbiologia , Microbiologia da Água , Irrigação Agrícola , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Enterococcus/genética , TunísiaRESUMO
The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.
Assuntos
Perfilação da Expressão Gênica , Histidina Descarboxilase/biossíntese , Histidina Descarboxilase/genética , Morganella morganii/enzimologia , Morganella morganii/genética , Família Multigênica , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Ordem dos Genes , Klebsiella oxytoca/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Proteínas Recombinantes/genética , Análise de Sequência de DNA , SinteniaRESUMO
Through the analysis of the recently available genome shotgun sequence of Enterococcus italicus DSM 15952(T) type strain (Accession PRJNA61487, ID 61487), we found the presence of a gene encoding a bifunctional enzyme, termed γ-GCS-GS or GshF, involved in glutathione production and not influenced by feedback inhibition. The gshF gene exhibited high nucleotide and amino acid sequence similarity to other reported sequences from the Enterococcus genus and was constitutively expressed both in osmotic shock or in common cultural conditions. Several experimental studies concerning the culture medium, physiological stress, cell extract obtainment, and scaling-up showed that in selected conditions E. italicus was able to accumulate up to 250 µM of intracellular glutathione, which represented the main thiol group present into the cells. This is the first report regarding the production of glutathione by E. italicus, a species that could be used as a safe adjunct culture for glutathione-enriched dairy foods.
Assuntos
Reatores Biológicos , Enterococcus/genética , Enterococcus/metabolismo , Genômica , Glutationa/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus/enzimologia , Fermentação , Genoma Bacteriano/genéticaRESUMO
The diversity of a collection of 19 Enterococcus italicus strains isolated from different dairy sources was explored using a molecular polyphasic approach, comprising random amplification of polymorphic DNA (RAPD-PCR), repetitive element PCR (REP-PCR), plasmid profiling and ribotyping. The data obtained showed a high-level of biodiversity, not always correlated to the niche of isolation. Particularly, REP-PCR with primer BOXA1R and plasmid profiling allowed the best discrimination at strain level. Exploiting the genome shotgun sequence of the type strain of the species, available in public database, genes related to insertion sequences present on enterococcal Pathogenic Islands (ISEf1, IS905), determinants related to virulence factors (codifying for hemolysin and cell wall surface proteins), exogenously DNA (conjugal transfer protein, replication plasmid protein, pheromone shutdown protein, phage integrase/recombinase) and penicillin binding proteins system were detected. The presence of most of these genes seemed a common genetic trait in the Enterococcus genus, sur gene (cell wall surface protein) was only detected in strains of E. italicus. To our knowledge, this is the first time that specific primers, with the expection of the species-specific probe targeted to 16S rRNA gene, have been designed for this species.
Assuntos
Queijo/microbiologia , Enterococcus/classificação , Microbiologia de Alimentos , Animais , Bovinos , Galinhas , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Bacteriano/genética , Enterococcus/genética , Enterococcus/isolamento & purificação , Fezes/microbiologia , Genes Bacterianos , Variação Genética , Genótipo , Humanos , Olea/microbiologia , Filogenia , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico , Especificidade da EspécieRESUMO
BACKGROUND: The association between microbes and cancer has been reported repeatedly; however, it is not clear if molecular tumour properties are connected to specific microbial colonisation patterns. This is due mainly to the current technical and analytical strategy limitations to characterise tumour-associated bacteria. METHODS: Here, we propose an approach to detect bacterial signals in human RNA sequencing data and associate them with the clinical and molecular properties of the tumours. The method was tested on public datasets from The Cancer Genome Atlas, and its accuracy was assessed on a new cohort of colorectal cancer patients. RESULTS: Our analysis shows that intratumoural microbiome composition is correlated with survival, anatomic location, microsatellite instability, consensus molecular subtype and immune cell infiltration in colon tumours. In particular, we find Faecalibacterium prausnitzii, Coprococcus comes, Bacteroides spp., Fusobacterium spp. and Clostridium spp. to be strongly associated with tumour properties. CONCLUSIONS: We implemented an approach to concurrently analyse clinical and molecular properties of the tumour as well as the composition of the associated microbiome. Our results may improve patient stratification and pave the path for mechanistic studies on microbiota-tumour crosstalk.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Microbiota , Humanos , Neoplasias Colorretais/genética , Neoplasias do Colo/genética , Bactérias/genética , Análise de Sequência de RNARESUMO
Lactococcus garvieae is a fish pathogen and an emerging zoonotic opportunistic pathogen as well as a component of natural microbiota in dairy products. Here, we present the first report of a genome sequence of L. garvieae TB25, isolated from a dairy source, and that of L. garvieae LG9, isolated from rainbow trout.
Assuntos
Queijo/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Lactococcus/genética , Lactococcus/isolamento & purificação , Oncorhynchus mykiss/microbiologia , Animais , Itália , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
BACKGROUND: High-grade serous ovarian cancer (HGSOC) is a major unmet need in oncology. The remaining uncertainty on its originating tissue has hampered the discovery of molecular oncogenic pathways and the development of effective therapies. METHODS: We used an approach based on the retention in tumors of a DNA methylation trace (OriPrint) that distinguishes the two putative tissues of origin of HGSOC, the fimbrial (FI) and ovarian surface epithelia (OSE), to stratify HGSOC by several clustering methods, both linear and non-linear. The identified tumor subtypes (FI-like and OSE-like HGSOC) were investigated at the RNAseq level to stratify an in-house cohort of macrodissected HGSOC FFPE samples to derive overall and disease-free survival and identify specific transcriptional alterations of the two tumor subtypes, both by classical differential expression and weighted correlation network analysis. We translated our strategy to published datasets and verified the co-occurrence of previously described molecular classification of HGSOC. We performed cytokine analysis coupled to immune phenotyping to verify alterations in the immune compartment associated with HGSOC. We identified genes that are both differentially expressed and methylated in the two tumor subtypes, concentrating on PAX8 as a bona fide marker of FI-like HGSOC. RESULTS: We show that: - OriPrint is a robust DNA methylation tracer that exposes the tissue of origin of HGSOC. - The tissue of origin of HGSOC is the main determinant of DNA methylation variance in HGSOC. - The tissue of origin is a prognostic factor for HGSOC patients. - FI-like and OSE-like HGSOC are endowed with specific transcriptional alterations that impact patients' prognosis. - OSE-like tumors present a more invasive and immunomodulatory phenotype, compatible with its worse prognostic impact. - Among genes that are differentially expressed and regulated in FI-like and OSE-like HGSOC, PAX8 is a bona fide marker of FI-like tumors. CONCLUSIONS: Through an integrated approach, our work demonstrates that both FI and OSE are possible origins for human HGSOC, whose derived subtypes are both molecularly and clinically distinct. These results will help define a new roadmap towards rational, subtype-specific therapeutic inroads and improved patients' care.
Assuntos
Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Epigênese Genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Imunomodulação , Gradação de Tumores , Fenótipo , Prognóstico , Estudos Retrospectivos , TranscriptomaRESUMO
Previous reports suggest that the internal organization of semantic memory is in terms of different "types of knowledge," including "sensory" (information about perceptual features), "action" (motor-based knowledge of object utilization), and "functional" (abstract properties, as function and context of use). Consistent with this view, a specific loss of action knowledge, with preserved functional knowledge, has been recently observed in patients with left frontoparietal lesions. The opposite pattern (impaired functional knowledge with preserved action knowledge) was reported in association with anterior inferotemporal lesions. In the present study, the cerebral representation of action and functional knowledge was investigated using event-related analysis of functional magnetic resonance imaging data. Fifteen subjects were presented with pictures showing pairs of manipulable objects and asked whether the objects within each pair were used with the same manipulation pattern ("action knowledge" condition) or in the same context ("functional knowledge" condition). Direct comparisons showed action knowledge, relative to functional knowledge, to activate a left frontoparietal network, comprising the intraparietal sulcus, the inferior parietal lobule, and the dorsal premotor cortex. The reverse comparison yielded activations in the retrosplenial and the lateral anterior inferotemporal cortex. These results confirm and extend previous neuropsychological data and support the hypothesis of the existence of different types of information processing in the internal organization of semantic memory.
Assuntos
Córtex Cerebral/fisiologia , Cognição/fisiologia , Imageamento por Ressonância Magnética , Memória/fisiologia , Semântica , Adulto , Feminino , Lobo Frontal/fisiologia , Humanos , Masculino , Lobo Parietal/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Estimulação Luminosa , Lobo Temporal/fisiologiaRESUMO
Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.
Assuntos
Laticínios/microbiologia , Enterococcus/efeitos dos fármacos , Enterococcus/isolamento & purificação , Transferência Genética Horizontal , Plasmídeos/análise , Resistência a Tetraciclina , Animais , Southern Blotting , Cromossomos Bacterianos , Conjugação Genética , Enterococcus/genética , Genes Bacterianos , HumanosRESUMO
Dairy and fish isolates of Lactococcus garvieae were tested for their ability to utilize lactose and to grow in milk. Fish isolates were unable to assimilate lactose, but unexpectedly, they possessed the ability to grow in milk. Genetic studies, carried out constructing different vectorette libraries, provided evidence that in fish isolates, no genes involved in lactose utilization were present. For L. garvieae dairy isolates, a single system for the catabolism of lactose was found. It consists of a lactose transport and hydrolysis depending on a phosphoenolpyruvate-dependent phosphotransferase system combined with a phospho-beta-galactosidase. The genes involved were highly similar at the nucleotide sequence level to their counterparts in Lactococcus lactis; however, while in many L. lactis strains these genes are plasmid encoded, in L. garvieae they are chromosomally located. Thus, in the species L. garvieae, the phospho-beta-galactosidase gene, detectable in all strains of dairy origin but lacking in fish isolates, can be considered a reliable genetic marker for distinguishing biotypes in the two diverse ecological niches. Moreover, we obtained information regarding the complete nucleotide sequence of the gal operon in L. garvieae, consisting of a galactose permease and the Leloir pathway enzymes. This is one of the first reports concerning the determination of the nucleotide sequences of genes (other than the 16S rDNA gene) in L. garvieae and should be considered a step in a continuous effort to explore the genome of this species, with the aim of determining the real relationship between the presence of L. garvieae in dairy products and food safety.
Assuntos
DNA Bacteriano/química , Laticínios/microbiologia , Peixes/microbiologia , Lactococcus/enzimologia , Lactose/metabolismo , beta-Galactosidase/genética , Animais , Sequência de Bases , Técnicas de Cocultura , Amplificação de Genes , Marcadores Genéticos , Lactococcus/classificação , Lactococcus/genética , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos , Hibridização de Ácido Nucleico , Óperon , Filogenia , Plasmídeos , beta-Galactosidase/metabolismoRESUMO
Bitto of Valchiavenna, an artisanal Italian cheese produced without the addition of any starter cultures, has been attributed a protected designation origin (PDO) cheese, but the strain composition of the natural microbial population colonizing this traditional dairy product is still unknown. To obtain preliminary information on the non starter lactic acid bacteria involved in its ripening, a total of 136 NSLAB isolates, randomly selected from MRS and M17 agar plates, were collected from three different cheese samples after 120 days of ripening. The new isolates were identified by combining PCR 16S-23S rDNA spacer analyses, partial 16S rRNA gene sequencing, species-specific probes and colony hybridization. Eighty-two isolates, representing 60% of the total strains selected, were homofermentative cocci: 83% of them were enterococci, with Enterococcus durans being the predominant species found. Pediococcus spp. were also isolated, together with strains of Streptococcus thermophilus. Within lactobacilli, 57% of the isolates were identified as Lactobacillus paracasei; Lact. curvatus, Lact. plantarum, Lact. fermentum, were present in a lower amount. The isolates were differentiated at strain-level by Rep-PCR analysis. This is the first effort to microbiological characterization of Valchiavenna's Bitto; the results suggest the possibility of preserving the wild bacterial population in order to protect the typical organoleptic characteristics of this traditional raw milk cheese and to select new strains for the dairy industry.
Assuntos
Queijo/microbiologia , Microbiologia de Alimentos , Genótipo , Lactobacillaceae/genética , Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Lactobacillaceae/classificação , Lactobacillaceae/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
HIV infection causes the progressive depletion of CD4+ T-lymphocytes and profound modifications of T-cell homeostasis, which persist despite virologically-suppressive treatment and have been linked to a worse clinical outcome. Enduring alterations of the gastrointestinal tract may represent the underlying pathogenic mechanisms of these phenomena. Twenty-six HIV-infected subjects were assessed over a 12-month period following the introduction of antiretroviral therapy. 18 uninfected individuals were enrolled as controls. Parameters of peripheral T-cell homeostasis (activation, maturation), gastrointestinal function (microbial translocation, gut inflammation, fecal microbiota composition) and mucosal immunity (CD4+CCR6+CD161+, CD4+CCR9+α4ß7+, stem cell memory CD4+/CD8+ T-cells) were assessed. CD4+CCR6+CD161+ cells were depleted in HIV-infected untreated subjects and maintained significantly lower levels compared to controls, despite the introduction of effective antiviral treatment. The frequency of gut-homing CD4+CCR9+α4ß7+ cells was also impaired in untreated infection and correlated with the HIV RNA load and CD4+HLADR+CD38+; during therapy, we observed a contraction of this pool in the peripheral blood and the loss of its correlation with antigenic exposure/immune activation. A partial correction of the balance between stem cell memory pools and T-cell homeostasis was registered following treatment. In HIV-infected subjects with moderate immune-suppression, antiretroviral therapy has a marginal impact on mucosal immune populations which feature distinctive kinetics in the periphery, possibly reflecting their diverse recruitment from the blood to the mucosa. The persistent defects in mucosal immunity may fuel peripheral T-cell abnormalities through diverse mechanisms, including the production of IL-17/IL-22, cellular permissiveness to infection and regulation of T-lymphocyte maturation.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Tolerância Imunológica , Imunidade nas Mucosas/efeitos dos fármacos , Mucosa/citologia , Adulto , Fármacos Anti-HIV/farmacologia , Interações Medicamentosas , Fezes/microbiologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Infecções por HIV/microbiologia , Humanos , Masculino , Microbiota/efeitos dos fármacos , Pessoa de Meia-Idade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The widely accepted dogma of intrauterine sterility and initial colonization of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta, and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations and to assess fetal colonization before parturition. To this end, we analyzed sterilely collected intestinal tissues, placenta, and amniotic fluid from rodent fetuses and tissues from autoptic human fetuses. Total bacterial DNA was extracted from collected samples and analyzed by Next Generation Sequencing (NGS) techniques using hypervariable 16S ribosomal RNA (rRNA) regions (V3-V4). Colonizing microbes were visualized in situ, using labeled probes targeting 16S ribosomal DNA by fluorescent in situ hybridization. The NGS analysis showed the presence of pioneer microbes in both rat and human intestines as well as in rodent placentas and amniotic fluids. Microbial communities showed fetus- and dam-dependent clustering, confirming the high interindividual variability of commensal microbiota even in the antenatal period. Fluorescent in situ hybridization analysis confirmed the microbes' presence in the lumen of the developing gut. These findings suggest a possible antenatal colonization of the developing mammalian gut.
Assuntos
Líquido Amniótico/microbiologia , Desenvolvimento Embrionário/fisiologia , Intestinos/microbiologia , Microbiota , Placenta/microbiologia , Animais , Feminino , Humanos , Intestinos/embriologia , Gravidez , RNA Ribossômico 16S/metabolismo , RatosRESUMO
Persistent alteration of plasma neuroactive steroid levels associated with major depression has been recently reported in men after the suspension of the treatment for androgenetic alopecia with finasteride, an inhibitor of the enzyme 5alpha-reductase. Observations in male rats confirmed persistent alterations in neuroactive steroid levels also in the brain. In the present study, we have ascertained possible effects on depressive-like behavior, neurogenesis, gliosis, neuroinflammation and gut microbiota in male rats after subchronic treatment for 20 days with finasteride and after one month of its withdrawal. At the end of treatment there was an increase in the number of pH3 immunoreactive cells in the subgranular zone of the dentate gyrus together with an increase in the mRNA levels of TNF-α in the hippocampus. By one month after the end of finasteride treatment, rats showed depressive-like behavior coupled with a decrease in the number of pH3 immunoreactive cells in the subgranular zone of the dentate gyrus, a decrease in granule cell density in the granule cell layer and an increase in the number of GFAP immunoreactive astrocytes in the dentate gyrus. Finally, alteration of gut microbiota (i.e., an increase in Bacteroidetes phylum and in Prevotellaceae family at the end of the treatment and a decrease in Ruminococcaceae family, Oscillospira and Lachnospira genus at the end of the withdrawal period) was detected. In conclusion, finasteride treatment in male rats has long term effects on depressive-like behavior, hippocampal neurogenesis and neuroinflammation and gut microbiota composition.
Assuntos
Depressão/tratamento farmacológico , Finasterida/farmacologia , Síndrome de Abstinência a Substâncias/fisiopatologia , Inibidores de 5-alfa Redutase/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Colestenona 5 alfa-Redutase/metabolismo , Giro Denteado/efeitos dos fármacos , Finasterida/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Neurogênese/efeitos dos fármacos , Neuroimunomodulação/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Esteroides/sangueRESUMO
Low-phenylalanine diet, the mainstay of treatment for phenylketonuria (PKU), has been shown to increase glycemic index and glycemic load, affecting the availability of substrates for microbial fermentation. Indeed, changes in the PKU gut microbiota compared with healthy controls have been previously reported. In this study we compared the gut microbial communities of children with PKU and with mild hyperphenylalaninemia (MHP, unrestricted diet). For each group, we enrolled 21 children (4-18 years old), for a total dataset of 42 subjects. We assessed dietary intake and performed gut microbiota analysis by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Short chain fatty acids (SCFAs) were quantified by gas chromatographic analysis. While alpha-diversity analysis showed no significant differences between PKU and MHP groups, microbial community analysis highlighted a significant separation of the gut microbiota according to both unweighted (p = 0.008) and weighted Unifrac distances (p = 0.033). Major differences were seen within the Firmicutes phylum. Indeed, PKU children were depleted in Faecalibacterium spp. and enriched in Blautia spp. and Clostridium spp (family Lachnospiraceae). We found a divergent response of members of the Firmicutes phylum with respect to daily glycemic index, higher in PKU children. Faecalibacterium prausnitzii, unclassified Ruminococcaceae and, to a lesser extent Roseburia spp. negatively correlated with glycemic index, whereas unclassified Lachnospiraceae were positively associated. Indicator species analysis suggested F. prausnitzii be related to MHP status and Ruminococcus bromii to be associated with PKU. Despite PKU children having a higher vegetable and fiber intake, resembling a vegan diet, their gut microbial profile is different from the microbiota reported in the literature for individuals consuming a high-fiber/low-protein diet. Indeed, beneficial microorganisms, such as F. prausnitzii, considered a biomarker for a healthy status and one of the main butyrate producers, are depleted in PKU gut microbiota. We suggest that both the quality and quantity of carbohydrates ingested participate in determining the observed Firmicutes shifts on the PKU population.
Assuntos
Dietoterapia/métodos , Dieta/métodos , Firmicutes/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Fenilcetonúrias/terapia , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Fezes/química , Fezes/microbiologia , Humanos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Different gastrointestinal disorders, including inflammatory bowel diseases (IBD), have been linked to alterations of the gut microbiota composition, namely dysbiosis. Fecal microbiota transplantation (FMT) is considered an encouraging therapeutic approach for ulcerative colitis patients, mostly as a consequence of normobiosis restoration. We recently showed that therapeutic effects of FMT during acute experimental colitis are linked to functional modulation of the mucosal immune system and of the gut microbiota composition. Here we analysed the effects of therapeutic FMT administration during chronic experimental colitis, a condition more similar to that of IBD patients, on immune-mediated mucosal inflammatory pathways. Mucus and feces from normobiotic donors were orally administered to mice with established chronic Dextran Sodium Sulphate (DSS)-induced colitis. Immunophenotypes and functions of infiltrating colonic immune cells were evaluated by cytofluorimetric analysis. Compositional differences in the intestinal microbiome were analyzed by 16S rRNA sequencing. Therapeutic FMT in mice undergoing chronic intestinal inflammation was capable to decrease colonic inflammation by modulating the expression of pro-inflammatory genes, antimicrobial peptides, and mucins. Innate and adaptive mucosal immune cells manifested a reduced pro-inflammatory profile in FMT-treated mice. Finally, restoration of a normobiotic core ecology contributed to the resolution of inflammation. Thus, FMT is capable of controlling chronic intestinal experimental colitis by inducing a concerted activation of anti-inflammatory immune pathways, mechanistically supporting the positive results of FMT treatment reported in ulcerative colitis patients.
Assuntos
Transplante de Microbiota Fecal , Microbioma Gastrointestinal/imunologia , Inflamação/patologia , Intestinos/imunologia , Intestinos/microbiologia , Linfócitos T/imunologia , Animais , Doença Crônica , Citocinas/metabolismo , Sulfato de Dextrana , Feminino , Inflamação/induzido quimicamente , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Análise de Componente PrincipalRESUMO
Gut microbiota is considered a separate organ with endocrine capabilities, actively contributing to tissue homeostasis. It consists of at least two separate microbial populations, the lumen-associated (LAM) and the mucosa-associated microbiota (MAM). In the present study, we compared LAM and MAM, by collecting stools and sigmoid brush samples of forty adults without large-bowel symptoms, and through a 16S rRNA gene next-generation sequencing (NGS) approach. MAM sample analysis revealed enrichment in aerotolerant Proteobacteria, probably selected by a gradient of oxygen that decreases from tissue to lumen, and in Streptococcus and Clostridium spp., highly fermenting bacteria. On the other hand, LAM microbiota showed an increased abundance in Bacteroides, Prevotella, and Oscillospira, genera able to digest and to degrade biopolymers in the large intestine. Predicted metagenomic analysis showed LAM to be enriched in genes encoding enzymes mostly involved in energy extraction from carbohydrates and lipids, whereas MAM in amino acid and vitamin metabolism. Moreover, LAM and MAM communities seemed to be influenced by different host factors, such as diet and sex. LAM is affected by body mass index (BMI) status. Indeed, BMI negatively correlates with Faecalibacterium prausnitzii and Flavonifractor plautii abundance, putative biomarkers of healthy status. In contrast, MAM microbial population showed a significant grouping according to sex. Female MAM was enriched in Actinobacteria (with an increased trend of the genus Bifidobacterium), and a significant depletion in Veillonellaceae. Interestingly, we found the species Gemmiger formicilis to be associated with male and Bifidobacterium adolescentis, with female MAM samples. In conclusion, our results suggest that gut harbors microbial niches that differ in both composition and host factor susceptibility, and their richness and diversity may be overlooked evaluating only fecal samples.