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2.
Nat Immunol ; 21(3): 343-353, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066951

RESUMO

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species.


Assuntos
Colo/imunologia , Colo/microbiologia , Microbioma Gastrointestinal/imunologia , Adulto , Linfócitos B/imunologia , Colo/citologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária , Especificidade de Órgãos , RNA-Seq , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Transcriptoma
3.
Cell ; 180(2): 278-295.e23, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31978345

RESUMO

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Espectrometria de Massas/métodos , Enzimas Multifuncionais/genética , Fosforilação , Proteínas/genética , Nucleotídeos de Purina/metabolismo , Purinas/metabolismo
4.
Nature ; 575(7783): 505-511, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723265

RESUMO

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.


Assuntos
Bacteriófagos/fisiologia , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/virologia , Microbioma Gastrointestinal , Hepatite Alcoólica/microbiologia , Hepatite Alcoólica/terapia , Terapia por Fagos , Alcoolismo/complicações , Alcoolismo/microbiologia , Animais , Enterococcus faecalis/isolamento & purificação , Etanol/efeitos adversos , Fígado Gorduroso/complicações , Fígado Gorduroso/microbiologia , Fezes/microbiologia , Feminino , Vida Livre de Germes , Hepatite Alcoólica/complicações , Hepatite Alcoólica/mortalidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Perforina/metabolismo
5.
Nature ; 574(7776): 117-121, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31534227

RESUMO

Immediately after birth, newborn babies experience rapid colonization by microorganisms from their mothers and the surrounding environment1. Diseases in childhood and later in life are potentially mediated by the perturbation of the colonization of the infant gut microbiota2. However, the effects of delivery via caesarean section on the earliest stages of the acquisition and development of the gut microbiota, during the neonatal period (≤1 month), remain controversial3,4. Here we report the disrupted transmission of maternal Bacteroides strains, and high-level colonization by opportunistic pathogens associated with the hospital environment (including Enterococcus, Enterobacter and Klebsiella species), in babies delivered by caesarean section. These effects were also seen, to a lesser extent, in vaginally delivered babies whose mothers underwent antibiotic prophylaxis and in babies who were not breastfed during the neonatal period. We applied longitudinal sampling and whole-genome shotgun metagenomic analysis to 1,679 gut microbiota samples (taken at several time points during the neonatal period, and in infancy) from 596 full-term babies born in UK hospitals; for a subset of these babies, we collected additional matched samples from mothers (175 mothers paired with 178 babies). This analysis demonstrates that the mode of delivery is a significant factor that affects the composition of the gut microbiota throughout the neonatal period, and into infancy. Matched large-scale culturing and whole-genome sequencing of over 800 bacterial strains from these babies identified virulence factors and clinically relevant antimicrobial resistance in opportunistic pathogens that may predispose individuals to opportunistic infections. Our findings highlight the critical role of the local environment in establishing the gut microbiota in very early life, and identify colonization with antimicrobial-resistance-containing opportunistic pathogens as a previously underappreciated risk factor in hospital births.


Assuntos
Cesárea/efeitos adversos , Microbioma Gastrointestinal , Doenças do Recém-Nascido/microbiologia , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Infecções Oportunistas/congênito , Infecções Oportunistas/microbiologia , Feminino , Humanos , Recém-Nascido , Doenças do Recém-Nascido/etiologia , Infecções Oportunistas/etiologia , Gravidez
6.
Sci Rep ; 9(1): 11293, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383872

RESUMO

Clostridium difficile, the causal agent of antibiotic-associated diarrhea, has a complex epidemiology poorly studied in Latin America. We performed a robust genomic and phenotypic profiling of 53 C. difficile clinical isolates established from diarrheal samples from either intrahospital (IH) or community (CO) populations in central Colombia. In vitro tests were conducted to evaluate the cytopathic effect, the minimum inhibitory concentration of ten antimicrobial agents, the sporulation efficiency and the colony forming ability. Eleven different sequence types (STs) were found, the majority present individually in each sample, however in three samples two different STs were isolated. Interestingly, CO patients were infected with STs associated with hypervirulent strains (ST-1 in Clade-2). Three coexistence events (two STs simultaneously detected in the same sample) were observed always involving ST-8 from Clade-1. A total of 2,502 genes were present in 99% of the isolates with 95% of identity or more, it represents a core genome of 28.6% of the 8,735 total genes identified in the set of genomes. A high cytopathic effect was observed for the isolates positive for the two main toxins but negative for binary toxin (TcdA+/TcdB+/CDT- toxin production type), found only in Clade-1. Molecular markers conferring resistance to fluoroquinolones (cdeA and gyrA) and to sulfonamides (folP) were the most frequent in the analyzed genomes. In addition, 15 other markers were found mostly in Clade-2 isolates. These results highlight the regional differences that C. difficile isolates display, being in this case the CO isolates the ones having a greater number of accessory genes and virulence-associated factors.

7.
Nat Genet ; 51(9): 1315-1320, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406348

RESUMO

Bacterial speciation is a fundamental evolutionary process characterized by diverging genotypic and phenotypic properties. However, the selective forces that affect genetic adaptations and how they relate to the biological changes that underpin the formation of a new bacterial species remain poorly understood. Here, we show that the spore-forming, healthcare-associated enteropathogen Clostridium difficile is actively undergoing speciation. Through large-scale genomic analysis of 906 strains, we demonstrate that the ongoing speciation process is linked to positive selection on core genes in the newly forming species that are involved in sporulation and the metabolism of simple dietary sugars. Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism. Thus, we report the formation of an emerging C. difficile species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission.


Assuntos
Aclimatação/genética , Infecções por Clostridium/transmissão , Clostridium difficile/genética , Especiação Genética , Esporos Bacterianos/crescimento & desenvolvimento , Açúcares/metabolismo , Virulência/genética , Antibacterianos/farmacologia , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Clostridium difficile/isolamento & purificação , Genoma Bacteriano , Genômica , Humanos , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo
8.
Virulence ; 10(1): 657-676, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31304854

RESUMO

Some well-known Clostridiales species such as Clostridium difficile and C. perfringens are agents of high impact diseases worldwide. Nevertheless, other foreseen Clostridiales species have recently emerged such as Clostridium tertium and C. paraputrificum. Three fecal isolates were identified as Clostridium tertium (Gcol.A2 and Gcol.A43) and C. paraputrificum (Gcol.A11) during public health screening for C. difficile infections in Colombia. C. paraputrificum genomes were highly diverse and contained large numbers of accessory genes. Genetic diversity and accessory gene percentage were lower among the C. tertium genomes than in the C. paraputrificum genomes. C. difficile tcdA and tcdB toxins encoding homologous sequences and other potential virulence factors were also identified. EndoA interferase, a toxic component of the type II toxin-antitoxin system, was found among the C. tertium genomes. toxA was the only toxin encoding gene detected in Gcol.A43, the Colombian isolate with an experimentally-determined high cytotoxic effect. Gcol.A2 and Gcol.A43 had higher sporulation efficiencies than Gcol.A11 (84.5%, 83.8% and 57.0%, respectively), as supported by the greater number of proteins associated with sporulation pathways in the C. tertium genomes compared with the C. paraputrificum genomes (33.3 and 28.4 on average, respectively). This work allowed complete genome description of two clostridiales species revealing high levels of intra-taxa diversity, accessory genomes containing virulence-factors encoding genes (especially in C. paraputrificum), with proteins involved in sporulation processes more highly represented in C. tertium. These finding suggest the need to advance in the study of those species with potential importance at public health level.


Assuntos
Clostridium difficile/genética , Clostridium tertium/genética , Genômica , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Infecções por Clostridium/microbiologia , Colômbia , Variação Genética , Genoma Bacteriano , Humanos
9.
PLoS Biol ; 17(5): e3000231, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048876

RESUMO

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.


Assuntos
Mucosa Gástrica/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Microscopia Confocal/métodos , Animais , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Contagem de Colônia Microbiana , Feminino , Mucosa Gástrica/efeitos dos fármacos , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Especificidade da Espécie , Linfócitos T/efeitos dos fármacos
10.
Nat Biotechnol ; 37(2): 186-192, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30718869

RESUMO

Understanding gut microbiome functions requires cultivated bacteria for experimental validation and reference bacterial genome sequences to interpret metagenome datasets and guide functional analyses. We present the Human Gastrointestinal Bacteria Culture Collection (HBC), a comprehensive set of 737 whole-genome-sequenced bacterial isolates, representing 273 species (105 novel species) from 31 families found in the human gastrointestinal microbiota. The HBC increases the number of bacterial genomes derived from human gastrointestinal microbiota by 37%. The resulting global Human Gastrointestinal Bacteria Genome Collection (HGG) classifies 83% of genera by abundance across 13,490 shotgun-sequenced metagenomic samples, improves taxonomic classification by 61% compared to the Human Microbiome Project (HMP) genome collection and achieves subspecies-level classification for almost 50% of sequences. The improved resource of gastrointestinal bacterial reference sequences circumvents dependence on de novo assembly of metagenomes and enables accurate and cost-effective shotgun metagenomic analyses of human gastrointestinal microbiota.


Assuntos
Genoma Bacteriano , Metagenoma , Metagenômica , Bactérias/classificação , Biologia Computacional/métodos , Mapeamento de Sequências Contíguas , Microbioma Gastrointestinal , Genoma Humano , Humanos , Filogenia , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie
11.
Nature ; 568(7753): 499-504, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30745586

RESUMO

The composition of the human gut microbiota is linked to health and disease, but knowledge of individual microbial species is needed to decipher their biological roles. Despite extensive culturing and sequencing efforts, the complete bacterial repertoire of the human gut microbiota remains undefined. Here we identify 1,952 uncultured candidate bacterial species by reconstructing 92,143 metagenome-assembled genomes from 11,850 human gut microbiomes. These uncultured genomes substantially expand the known species repertoire of the collective human gut microbiota, with a 281% increase in phylogenetic diversity. Although the newly identified species are less prevalent in well-studied populations compared to reference isolate genomes, they improve classification of understudied African and South American samples by more than 200%. These candidate species encode hundreds of newly identified biosynthetic gene clusters and possess a distinctive functional capacity that might explain their elusive nature. Our work expands the known diversity of uncultured gut bacteria, which provides unprecedented resolution for taxonomic and functional characterization of the intestinal microbiota.


Assuntos
Bactérias/classificação , Bactérias/genética , Microbioma Gastrointestinal/genética , Genoma Bacteriano/genética , Genômica , Metagenoma/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Humanos , Família Multigênica , Filogenia , Especificidade da Espécie
12.
Sci Signal ; 12(562)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30600262

RESUMO

The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein-coupled receptor GPR35 interacted with the α chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca2+ to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast, a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis, inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover, whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus, GPR35 acts as a central signaling and metabolic pacesetter, which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology.


Assuntos
Proliferação de Células , Glicólise , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Carcinogênese , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas-G/genética , ATPase Trocadora de Sódio-Potássio/genética , Células THP-1 , Quinases da Família src/genética , Quinases da Família src/metabolismo
13.
Proc Natl Acad Sci U S A ; 115(40): 10118-10123, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30217896

RESUMO

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.


Assuntos
Células Epiteliais/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Fagossomos/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/microbiologia , Células-Tronco Pluripotentes Induzidas/patologia , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-21/genética , Subunidade alfa de Receptor de Interleucina-21/imunologia , Interleucinas/genética , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Fagossomos/genética , Fagossomos/microbiologia , Fagossomos/patologia , Infecções por Salmonella/genética , Infecções por Salmonella/patologia , Salmonella typhimurium/genética
14.
Sci Rep ; 8(1): 8181, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29802257

RESUMO

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans. Comparative proteomic analysis of consecutively collected susceptible and resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar. Together these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.


Assuntos
Achromobacter denitrificans/efeitos dos fármacos , Achromobacter denitrificans/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Meropeném/farmacologia , Proteômica , beta-Lactamases/metabolismo , Achromobacter denitrificans/genética , Sequência de Aminoácidos , Humanos , beta-Lactamases/química , beta-Lactamases/genética
15.
Nat Commun ; 9(1): 1557, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29674608

RESUMO

Metagenomic analyses have indicated that the female bladder harbors an indigenous microbiota. However, there are few cultured reference strains with sequenced genomes available for functional and experimental analyses. Here we isolate and genome-sequence 149 bacterial strains from catheterized urine of 77 women. This culture collection spans 78 species, representing approximately two thirds of the bacterial diversity within the sampled bladders, including Proteobacteria, Actinobacteria, and Firmicutes. Detailed genomic and functional comparison of the bladder microbiota to the gastrointestinal and vaginal microbiotas demonstrates similar vaginal and bladder microbiota, with functional capacities that are distinct from those observed in the gastrointestinal microbiota. Whole-genome phylogenetic analysis of bacterial strains isolated from the vagina and bladder in the same women identifies highly similar Escherichia coli, Streptococcus anginosus, Lactobacillus iners, and Lactobacillus crispatus, suggesting an interlinked female urogenital microbiota that is not only limited to pathogens but is also characteristic of health-associated commensals.


Assuntos
Bactérias/isolamento & purificação , Microbiota , Bexiga Urinária/microbiologia , Vagina/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Estudos de Coortes , Feminino , Genoma Bacteriano , Humanos , Pessoa de Meia-Idade , Filogenia , Adulto Jovem
16.
Curr Opin Microbiol ; 42: 47-52, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29112885

RESUMO

Advances in high-throughput sequencing technologies and the development of sophisticated bioinformatics analysis methods, algorithms, and pipelines to handle the large amounts of data generated have driven the field of human microbiome research forward. This specialist knowledge has been crucial to thoroughly mine the human gut microbiota, particularly in the absence of methods for the routine cultivation of most enteric microorganisms. In recent years, however, significant efforts have been made to address the 'great plate count anomaly' and to overcome the barriers to cultivation of the fastidious and mostly strictly anaerobic bacteria that reside in the human gut. As a result, many new species have been discovered, characterised, genome sequenced, and deposited in culture collections. These continually expanding resources enable experimental investigation of the human gut microbiota, validation of hypotheses made with sequence-based analyses, and phenotypic characterisation of its constituent microbes. Herein we propose a variant of Koch's postulates, aimed at providing a framework to establish causation in microbiome studies, with a particular focus on demonstrating the health-promoting role of the commensal gut microbiota.


Assuntos
Microbiota , Simbiose , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Humanos , Metagenômica/métodos , Pesquisa
17.
Nat Commun ; 8(1): 1367, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-29118316

RESUMO

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have "jumped" into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen.


Assuntos
Infecções por Campylobacter/transmissão , Campylobacter fetus/genética , Campylobacter fetus/patogenicidade , Microbioma Gastrointestinal , Animais , Infecções por Campylobacter/veterinária , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/transmissão , Fezes/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Filogenia
18.
Sci Rep ; 7(1): 5648, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28717159

RESUMO

Bifidobacteria are common gut commensals with purported health-promoting effects. This has encouraged scientific research into bifidobacteria, though recalcitrance to genetic manipulation and scarcity of molecular tools has hampered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation. To overcome this problem and facilitate functional genomic analyses in bifidobacteria, we created a large Tn5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Transposon Directed Insertion Sequencing (TraDIS) approach. Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions. These essential genes encode functions involved in the so-called bifid-shunt, most enzymes related to nucleotide biosynthesis and a range of housekeeping functions. Comparison to the Bifidobacterium and B. breve core genomes highlights a high degree of conservation of essential genes at the species and genus level, while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria. This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria.


Assuntos
Proteínas de Bactérias/genética , Bifidobacterium breve/crescimento & desenvolvimento , Mutagênese Insercional , Bifidobacterium breve/genética , Elementos de DNA Transponíveis , Evolução Molecular , Genes Essenciais , Genoma de Planta , Anotação de Sequência Molecular , Filogenia , Homologia de Sequência do Ácido Nucleico , Simbiose
19.
Nat Rev Microbiol ; 15(9): 531-543, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28603278

RESUMO

Transmission of commensal intestinal bacteria between humans could promote health by establishing, maintaining and replenishing microbial diversity in the microbiota of an individual. Unlike pathogens, the routes of transmission for commensal bacteria remain unappreciated and poorly understood, despite the likely commonalities between both. Consequently, broad infection control measures that are designed to prevent pathogen transmission and infection, such as oversanitation and the overuse of antibiotics, may inadvertently affect human health by altering normal commensal transmission. In this Review, we discuss the mechanisms and factors that influence host-to-host transmission of the intestinal microbiota and examine how a better understanding of these processes will identify new approaches to nurture and restore transmission routes that are used by beneficial bacteria.


Assuntos
Translocação Bacteriana/fisiologia , Microbioma Gastrointestinal/fisiologia , Dinâmica Populacional , Humanos
20.
BMC Microbiol ; 16(1): 274, 2016 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-27842515

RESUMO

BACKGROUND: Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. RESULTS: Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset. CONCLUSION: The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.


Assuntos
DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/genética , Sequência de Bases , Biodiversidade , Biologia Computacional/métodos , DNA Ribossômico/genética , Feminino , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos/microbiologia , Metagenoma , Microbiota/genética , Filogenia , Análise de Sequência de DNA , Staphylococcus aureus/genética , Vagina/microbiologia
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