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1.
Microbiome ; 12(1): 50, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38468305

RESUMO

BACKGROUND: Antibiotics notoriously perturb the gut microbiota. We treated healthy volunteers either with cefotaxime or ceftriaxone for 3 days, and collected in each subject 12 faecal samples up to day 90. Using untargeted and targeted phenotypic and genotypic approaches, we studied the changes in the bacterial, phage and fungal components of the microbiota as well as the metabolome and the ß-lactamase activity of the stools. This allowed assessing their degrees of perturbation and resilience. RESULTS: While only two subjects had detectable concentrations of antibiotics in their faeces, suggesting important antibiotic degradation in the gut, the intravenous treatment perturbed very significantly the bacterial and phage microbiota, as well as the composition of the metabolome. In contrast, treatment impact was relatively low on the fungal microbiota. At the end of the surveillance period, we found evidence of resilience across the gut system since most components returned to a state like the initial one, even if the structure of the bacterial microbiota changed and the dynamics of the different components over time were rarely correlated. The observed richness of the antibiotic resistance genes repertoire was significantly reduced up to day 30, while a significant increase in the relative abundance of ß-lactamase encoding genes was observed up to day 10, consistent with a concomitant increase in the ß-lactamase activity of the microbiota. The level of ß-lactamase activity at baseline was positively associated with the resilience of the metabolome content of the stools. CONCLUSIONS: In healthy adults, antibiotics perturb many components of the microbiota, which return close to the baseline state within 30 days. These data suggest an important role of endogenous ß-lactamase-producing anaerobes in protecting the functions of the microbiota by de-activating the antibiotics reaching the colon. Video Abstract.


Assuntos
Microbioma Gastrointestinal , Resiliência Psicológica , Adulto , Humanos , Microbioma Gastrointestinal/genética , beta-Lactamases/genética , beta-Lactamas/farmacologia , Voluntários Saudáveis , Antibacterianos , Bactérias/genética , Fezes/microbiologia
3.
PLoS Pathog ; 19(2): e1011170, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36802406

RESUMO

Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.


Assuntos
Interferons , Mitocôndrias , Camundongos , Animais , Mitocôndrias/metabolismo , Vírus do Sarampo , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , DNA Mitocondrial
4.
Microbiome ; 11(1): 12, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670449

RESUMO

BACKGROUND: The plasmid-mediated resistance gene mcr-1 confers colistin resistance in Escherichia coli and paves the way for the evolution to pan-drug resistance. We investigated the impact of mcr-1 in gut colonization in the absence of antibiotics using isogenic E. coli strains transformed with a plasmid encoding or devoid of mcr-1. RESULTS: In gnotobiotic and conventional mice, mcr-1 significantly enhanced intestinal anchoring of E. coli but impaired their lethal effect. This improvement of intestinal fitness was associated with a downregulation of intestinal inflammatory markers and the preservation of intestinal microbiota composition. The mcr-1 gene mediated a cross-resistance to antimicrobial peptides secreted by the microbiota and intestinal epithelial cells (IECs), enhanced E. coli adhesion to IECs, and decreased the proinflammatory activity of both E. coli and its lipopolysaccharides. CONCLUSION: Overall, mcr-1 changed multiple facets of bacterial behaviour and appeared as a factor enhancing commensal lifestyle and persistence in the gut even in the absence of antibiotics. Video Abstract.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Animais , Camundongos , Escherichia coli/genética , Simbiose , Proteínas de Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Infecções por Escherichia coli/microbiologia , Testes de Sensibilidade Microbiana
5.
mBio ; 13(6): e0288022, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36448778

RESUMO

Antibiotics disturb the intestinal bacterial microbiota, leading to gut dysbiosis and an increased risk for the overgrowth of opportunistic pathogens. It is not fully understood to what extent antibiotics affect the fungal fraction of the intestinal microbiota, the mycobiota. There is no report of the direct role of antibiotics in the overgrowth in healthy humans of the opportunistic pathogenic yeast Candida albicans. Here, we have explored the gut mycobiota of 22 healthy subjects before, during, and up to 6 months after a 3-day regimen of third-generation cephalosporins (3GCs). Using ITS1-targeted metagenomics, we highlighted the strong intra- and interindividual diversity of the healthy gut mycobiota. With a specific quantitative approach, we showed that C. albicans prevalence was much higher than previously reported, with all subjects but one being carriers of C. albicans, although with highly variable burdens. 3GCs significantly altered the mycobiota composition and the fungal load was increased both at short and long term. Both C. albicans relative and absolute abundances were increased but 3GCs did not reduce intersubject variability. Variations in C. albicans burden in response to 3GC treatment could be partly explained by changes in the levels of endogenous fecal ß-lactamase activity, with subjects characterized by a high increase of ß-lactamase activity displaying a lower increase of C. albicans levels. A same antibiotic treatment might thus affect differentially the gut mycobiota and C. albicans carriage, depending on the treated subject, suggesting a need to adjust the current risk factors for C. albicans overgrowth after a ß-lactam treatment. IMPORTANCE Fungal infections are redoubtable healthcare-associated complications in immunocompromised patients. Particularly, the commensal intestinal yeast Candida albicans causes invasive infections in intensive care patients and is, therefore, associated with high mortality. These infections are preceded by an intestinal expansion of C. albicans before its translocation into the bloodstream. Antibiotics are a well-known risk factor for C. albicans overgrowth but the impact of antibiotic-induced dysbiosis on the human gut mycobiota-the fungal microbiota-and the understanding of the mechanisms involved in C. albicans overgrowth in humans are very limited. Our study shows that antibiotics increase the fungal proportion in the gut and disturb the fungal composition, especially C. albicans, in a subject-dependent manner. Indeed, variations across subjects in C. albicans burden in response to ß-lactam treatment could be partly explained by changes in the levels of endogenous fecal ß-lactamase activity. This highlighted a potential new key factor for C. albicans overgrowth. Thus, the significance of our research is in providing a better understanding of the factors behind C. albicans intestinal overgrowth, which might lead to new means to prevent life-threatening secondary infections.


Assuntos
Candida albicans , Disbiose , Humanos , Candida albicans/fisiologia , Monobactamas , Antibacterianos , beta-Lactamases
6.
Nat Immunol ; 22(11): 1428-1439, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34471264

RESUMO

Coordinated local mucosal and systemic immune responses following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection either protect against coronavirus disease 2019 (COVID-19) pathologies or fail, leading to severe clinical outcomes. To understand this process, we performed an integrated analysis of SARS-CoV-2 spike-specific antibodies, cytokines, viral load and bacterial communities in paired nasopharyngeal swabs and plasma samples from a cohort of clinically distinct patients with COVID-19 during acute infection. Plasma viral load was associated with systemic inflammatory cytokines that were elevated in severe COVID-19, and also with spike-specific neutralizing antibodies. By contrast, nasopharyngeal viral load correlated with SARS-CoV-2 humoral responses but inversely with interferon responses, the latter associating with protective microbial communities. Potential pathogenic microorganisms, often implicated in secondary respiratory infections, were associated with mucosal inflammation and elevated in severe COVID-19. Our results demonstrate distinct tissue compartmentalization of SARS-CoV-2 immune responses and highlight a role for the nasopharyngeal microbiome in regulating local and systemic immunity that determines COVID-19 clinical outcomes.


Assuntos
COVID-19/imunologia , Microbiota/imunologia , Nasofaringe/imunologia , SARS-CoV-2/fisiologia , Doença Aguda , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Estudos de Coortes , Feminino , Humanos , Imunidade Humoral , Imunidade nas Mucosas , Interferons/sangue , Masculino , Pessoa de Meia-Idade , Nasofaringe/microbiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Carga Viral , Adulto Jovem
7.
ISME J ; 15(3): 702-719, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33077888

RESUMO

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.


Assuntos
Microbiota , Peixe-Zebra , Animais , Disbiose , Flavobacterium/genética
8.
BMC Bioinformatics ; 21(1): 345, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778056

RESUMO

BACKGROUND: Comparing the composition of microbial communities among groups of interest (e.g., patients vs healthy individuals) is a central aspect in microbiome research. It typically involves sequencing, data processing, statistical analysis and graphical display. Such an analysis is normally obtained by using a set of different applications that require specific expertise for installation, data processing and in some cases, programming skills. RESULTS: Here, we present SHAMAN, an interactive web application we developed in order to facilitate the use of (i) a bioinformatic workflow for metataxonomic analysis, (ii) a reliable statistical modelling and (iii) to provide the largest panel of interactive visualizations among the applications that are currently available. SHAMAN is specifically designed for non-expert users. A strong benefit is to use an integrated version of the different analytic steps underlying a proper metagenomic analysis. The application is freely accessible at http://shaman.pasteur.fr/ , and may also work as a standalone application with a Docker container (aghozlane/shaman), conda and R. The source code is written in R and is available at https://github.com/aghozlane/shaman . Using two different datasets (a mock community sequencing and a published 16S rRNA metagenomic data), we illustrate the strengths of SHAMAN in quickly performing a complete metataxonomic analysis. CONCLUSIONS: With SHAMAN, we aim at providing the scientific community with a platform that simplifies reproducible quantitative analysis of metagenomic data.


Assuntos
Classificação , Internet , Metagenômica/métodos , Software , Estatística como Assunto , Interface Usuário-Computador , Líquidos Corporais/microbiologia , Pré-Escolar , Fezes/microbiologia , Humanos , Metagenoma , Microbiota , RNA Ribossômico 16S/genética , Fluxo de Trabalho
9.
PLoS Genet ; 16(5): e1008794, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463828

RESUMO

Although specific interactions between host and pathogen genotypes have been well documented in invertebrates, the identification of host genes involved in discriminating pathogen genotypes remains a challenge. In the mosquito Aedes aegypti, the main dengue virus (DENV) vector worldwide, statistical associations between host genetic markers and DENV types or strains were previously detected, but the host genes underlying this genetic specificity have not been identified. In particular, it is unknown whether DENV type- or strain-specific resistance relies on allelic variants of the same genes or on distinct gene sets. Here, we investigated the genetic architecture of DENV resistance in a population of Ae. aegypti from Bakoumba, Gabon, which displays a stronger resistance phenotype to DENV type 1 (DENV-1) than to DENV type 3 (DENV-3) infection. Following experimental exposure to either DENV-1 or DENV-3, we sequenced the exomes of large phenotypic pools of mosquitoes that are either resistant or susceptible to each DENV type. Using variation in single-nucleotide polymorphism (SNP) frequencies among the pools, we computed empirical p values based on average gene scores adjusted for the differences in SNP counts, to identify genes associated with infection in a DENV type-specific manner. Among the top 5% most significant genes, 263 genes were significantly associated with resistance to both DENV-1 and DENV-3, 287 genes were only associated with DENV-1 resistance and 290 were only associated with DENV-3 resistance. The shared significant genes were enriched in genes with ATP binding activity and sulfur compound transmembrane transporter activity, whereas the genes uniquely associated with DENV-3 resistance were enriched in genes with zinc ion binding activity. Together, these results indicate that specific resistance to different DENV types relies on largely non-overlapping sets of genes in this Ae. aegypti population and pave the way for further mechanistic studies.


Assuntos
Aedes/genética , Vírus da Dengue/classificação , Resistência à Doença , Sequenciamento do Exoma/métodos , Proteínas de Insetos/genética , Aedes/virologia , Animais , Células Cultivadas , Vírus da Dengue/patogenicidade , Feminino , Gabão , Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Especificidade da Espécie
10.
Front Microbiol ; 11: 306, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32174902

RESUMO

The commensal gut microbiome is contained by the enteric epithelial barrier, but little is known about the degree of specificity of host immune barrier interactions for particular bacterial taxa. Here, we show that depletion of leucine-rich repeat immune factor APL1 in the Asian malaria mosquito Anopheles stephensi is associated with higher midgut abundance of just the family Enterobacteraceae, and not generalized dysbiosis of the microbiome. The effect is explained by the response of a narrow clade containing two main taxa related to Klebsiella and Cedecea. Analysis of field samples indicate that these two taxa are recurrent members of the wild Anopheles microbiome. Triangulation using sequence and functional data incriminated relatives of C. neteri and Cedecea NFIX57 as candidates for the Cedecea component, and K. michiganensis, K. oxytoca, and K.sp. LTGPAF-6F as candidates for the Klebsiella component. APL1 presence is associated with host ability to specifically constrain the abundance of a narrow microbiome clade of the Enterobacteraceae, and the immune factor may promote homeostasis of this clade in the enteric microbiome for host benefit.

11.
Nat Commun ; 11(1): 1344, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165618

RESUMO

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host.


Assuntos
Adenosina/análogos & derivados , Ceco/metabolismo , Microbioma Gastrointestinal , Fígado/metabolismo , Adenosina/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Ceco/microbiologia , Feminino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
12.
Sci Rep ; 9(1): 11331, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383878

RESUMO

The microbiota of the human gut is a complex and rich community where bacteria and their viruses, the bacteriophages, are dominant. There are few studies on the phage community and no clear standard for isolating them, sequencing and analysing their genomes. Since this makes comparisons between studies difficult, we aimed at defining an easy, low-cost, and reproducible methodology. We analysed five different techniques to isolate phages from human adult faeces and developed an approach to analyse their genomes in order to quantify contamination and classify phage contigs in terms of taxonomy and lifestyle. We chose the polyethylene glycol concentration method to isolate phages because of its simplicity, low cost, reproducibility, and of the high number and diversity of phage sequences that we obtained. We also tested the reproducibility of this method with multiple displacement amplification (MDA) and showed that MDA severely decreases the phage genetic diversity of the samples and the reproducibility of the method. Lastly, we studied the influence of sequencing depth on the analysis of phage diversity and observed the beginning of a plateau for phage contigs at 20,000,000 reads. This work contributes to the development of methods for the isolation of phages in faeces and for their comparative analysis.


Assuntos
Bacteriófagos/genética , Intestinos/virologia , Metagenoma/genética , Filogenia , Bacteriófagos/isolamento & purificação , Biologia Computacional , Análise Custo-Benefício , Fezes , Genoma Viral , Humanos , Metagenômica , Microbiota/genética
13.
Nat Microbiol ; 4(4): 603-613, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833729

RESUMO

Methanogenesis is an ancient metabolism of key ecological relevance, with direct impact on the evolution of Earth's climate. Recent results suggest that the diversity of methane metabolisms and their derivations have probably been vastly underestimated. Here, by probing thousands of publicly available metagenomes for homologues of methyl-coenzyme M reductase complex (MCR), we have obtained ten metagenome-assembled genomes (MAGs) belonging to potential methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea. Five of these MAGs represent under-sampled (Verstraetearchaeota, Methanonatronarchaeia, ANME-1 and GoM-Arc1) or previously genomically undescribed (ANME-2c) archaeal lineages. The remaining five MAGs correspond to lineages that are only distantly related to previously known methanogens and span the entire archaeal phylogeny. Comprehensive comparative annotation substantially expands the metabolic diversity and energy conservation systems of MCR-bearing archaea. It also suggests the potential existence of a yet uncharacterized type of methanogenesis linked to short-chain alkane/fatty acid oxidation in a previously undescribed class of archaea ('Candidatus Methanoliparia'). We redefine a common core of marker genes specific to methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea, and propose a possible scenario for the evolutionary and functional transitions that led to the emergence of such metabolic diversity.


Assuntos
Alcanos/química , Archaea/metabolismo , Biodiversidade , Metano/metabolismo , Alcanos/metabolismo , Archaea/classificação , Archaea/genética , Archaea/crescimento & desenvolvimento , DNA Arqueal , Metagenoma , Metano/química , Oxirredução , Filogenia
14.
Nat Microbiol ; 4(1): 112-123, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30478291

RESUMO

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Microbioma Gastrointestinal/genética , Intestinos/microbiologia , Conformação Proteica , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , beta-Lactamases/química , beta-Lactamases/genética
15.
PLoS One ; 13(8): e0201174, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30074988

RESUMO

BACKGROUND: The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. METHODS: Fecal samples from healthy adults were stored at -80°C as such or diluted in RNAlater® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. RESULTS: We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. CONCLUSION: Our results showed that RNAlater® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.


Assuntos
Microbioma Gastrointestinal/genética , Adulto , Biodiversidade , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Fezes/microbiologia , Feminino , Humanos , Masculino , Consórcios Microbianos/genética , Técnicas Microbiológicas , Pessoa de Meia-Idade , Micobioma/genética
16.
Parasit Vectors ; 11(1): 207, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29587819

RESUMO

BACKGROUND: Host-associated microbes, collectively known as the microbiota, play an important role in the biology of multicellular organisms. In mosquito vectors of human pathogens, the gut bacterial microbiota influences vectorial capacity and has become the subject of intense study. In laboratory studies of vector biology, genetic effects are often inferred from differences between geographically and genetically diverse colonies of mosquitoes that are reared in the same insectary. It is unclear, however, to what extent genetic effects can be confounded by uncontrolled differences in the microbiota composition among mosquito colonies. To address this question, we used 16S metagenomics to compare the midgut bacterial microbiome of six laboratory colonies of Aedes aegypti recently derived from wild populations representing the geographical range and genetic diversity of the species. RESULTS: We found that the diversity, abundance, and community structure of the midgut bacterial microbiome was remarkably similar among the six different colonies of Ae. aegypti, regardless of their geographical origin. We also confirmed the relatively low complexity of bacterial communities inhabiting the mosquito midgut. CONCLUSIONS: Our finding that geographically diverse colonies of Ae. aegypti reared in the same insectary harbor a similar gut bacterial microbiome supports the conclusion that the gut microbiota of adult mosquitoes is environmentally determined regardless of the host genotype. Thus, uncontrolled differences in microbiota composition are unlikely to represent a significant confounding factor in genetic studies of vector biology.


Assuntos
Aedes/microbiologia , Bactérias/classificação , Bactérias/genética , Microbioma Gastrointestinal , Animais , Análise por Conglomerados , DNA Ribossômico/química , DNA Ribossômico/genética , Trato Gastrointestinal/microbiologia , Metagenômica , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
17.
J Crohns Colitis ; 12(3): 337-346, 2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29194468

RESUMO

BACKGROUND: Crohn's disease [CD] is known to be associated with gut microbial dysbiosis. Infliximab [IFX] is increasingly used to treat paediatric CD; however, it is not clear how the gut microbiota is modified during IFX treatment. The aim of this study was to characterise the faecal microbiota community composition in paediatric CD patients and to assess its dynamic changes during IFX therapy. METHODS: A 16S rRNA sequencing approach was applied to determine the compositions of microbial communities in faecal samples. The composition and function of the faecal microbiota were compared between CD patients and healthy controls. RESULTS: Characteristics of faecal microbiome composition in paediatric CD patients before IFX treatment were represented by a lower biodiversity, a gain in Enterococcus, and a significant loss in multiple short-chain fatty acid [SCFA]-producing bacteria, including Anaerostipes, Blautia, Coprococcus, Faecalibacterium, Lachnospira, Odoribacter, Roseburia, Ruminococcus, and Sutterella. Additionally, alterations were observed in metabolic functions of the gut microbial community in CD. IFX treatment increased the biodiversity of gut microbiota and shifted its composition as well as its functional capabilities in the paediatric CD patients toward a healthy status. However, multiple SCFA-producing taxa were not significantly expanded. The sustained response of paediatric CD patients to IFX was associated with abundance of SCFA-producing bacteria. CONCLUSIONS: A lower biodiversity with alterations in the composition and function of faecal microbial community, characterising gut microbial dysbiosis, was observed in Chinese paediatric CD patients. IFX diminished the CD-associated gut microbial dysbiosis but was deficient in increasing certain SCFA-producing taxa.


Assuntos
Clostridiales/isolamento & purificação , Doença de Crohn/tratamento farmacológico , Doença de Crohn/microbiologia , Fármacos Gastrointestinais/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Infliximab/farmacologia , Adolescente , Bacteroidetes/isolamento & purificação , Bacteroidetes/metabolismo , Burkholderiales/isolamento & purificação , Burkholderiales/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Clostridiales/metabolismo , Doença de Crohn/complicações , Disbiose/tratamento farmacológico , Disbiose/etiologia , Disbiose/microbiologia , Enterococcus/isolamento & purificação , Faecalibacterium/isolamento & purificação , Faecalibacterium/metabolismo , Ácidos Graxos Voláteis/biossíntese , Fezes/química , Fezes/microbiologia , Feminino , Fármacos Gastrointestinais/uso terapêutico , Humanos , Infliximab/uso terapêutico , Masculino , RNA Ribossômico 16S/análise
18.
J Infect Dis ; 217(4): 628-636, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29186529

RESUMO

Background: Antibiotics are life-saving drugs but severely affect the gut microbiome with short-term consequences including diarrhea and selection of antibiotic-resistant bacteria. Long-term links to allergy and obesity are also suggested. We devised a product, DAV132, and previously showed its ability to deliver a powerful adsorbent, activated charcoal, in the late ileum of human volunteers. Methods: We performed a randomized controlled trial in 28 human volunteers treated with a 5-day clinical regimen of the fluoroquinolone antibiotic moxifloxacin in 2 parallel groups, with or without DAV132 coadministration. Two control goups of 8 volunteers each receiving DAV132 alone, or a nonactive substitute, were added. Results: The coadministration of DAV132 decreased free moxifloxacin fecal concentrations by 99%, while plasmatic levels were unaffected. Shotgun quantitative metagenomics showed that the richness and composition of the intestinal microbiota were largely preserved in subjects co-treated with DAV132 in addition to moxifloxacin. No adverse effect was observed. In addition, DAV132 efficiently adsorbed a wide range of clinically relevant antibiotics ex vivo. Conclusions: DAV132 was highly effective to protect the gut microbiome of moxifloxacin-treated healthy volunteers and may constitute a clinical breakthrough by preventing adverse health consequences of a wide range of antibiotic treatments. Clinical Trials Registration: NCT02176005.


Assuntos
Antibacterianos/administração & dosagem , Carvão Vegetal/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Moxifloxacina/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/análise , Fezes/química , Fezes/microbiologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , Moxifloxacina/análise , Resultado do Tratamento , Adulto Jovem
19.
Sci Adv ; 3(8): e1700585, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28835919

RESUMO

Conditions experienced during larval development of holometabolous insects can affect adult traits, but whether differences in the bacterial communities of larval development sites contribute to variation in the ability of insect vectors to transmit human pathogens is unknown. We addressed this question in the mosquito Aedes aegypti, a major arbovirus vector breeding in both sylvatic and domestic habitats in Sub-Saharan Africa. Targeted metagenomics revealed differing bacterial communities in the water of natural breeding sites in Gabon. Experimental exposure to different native bacterial isolates during larval development resulted in significant differences in pupation rate and adult body size but not life span. Larval exposure to an Enterobacteriaceae isolate resulted in decreased antibacterial activity in adult hemolymph and reduced dengue virus dissemination titer. Together, these data provide the proof of concept that larval exposure to different bacteria can drive variation in adult traits underlying vectorial capacity. Our study establishes a functional link between larval ecology, environmental microbes, and adult phenotypic variation in a holometabolous insect vector.


Assuntos
Bactérias , Variação Biológica da População , Microbiologia Ambiental , Mosquitos Vetores/crescimento & desenvolvimento , Mosquitos Vetores/microbiologia , Característica Quantitativa Herdável , Animais , Antibiose , Cruzamento , Dengue/transmissão , Ecossistema , Meio Ambiente , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno , Humanos , Larva , Estágios do Ciclo de Vida , Metagenoma , Metagenômica , Microbiota , Mosquitos Vetores/virologia
20.
Proc Natl Acad Sci U S A ; 113(20): 5706-11, 2016 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-27140611

RESUMO

Listeria monocytogenes is responsible for gastroenteritis in healthy individuals and for a severe invasive disease in immunocompromised patients. Among the three identified L. monocytogenes evolutionary lineages, lineage I strains are overrepresented in epidemic listeriosis outbreaks, but the mechanisms underlying the higher virulence potential of strains of this lineage remain elusive. Here, we demonstrate that Listeriolysin S (LLS), a virulence factor only present in a subset of lineage I strains, is a bacteriocin highly expressed in the intestine of orally infected mice that alters the host intestinal microbiota and promotes intestinal colonization by L. monocytogenes, as well as deeper organ infection. To our knowledge, these results therefore identify LLS as the first bacteriocin described in L. monocytogenes and associate modulation of host microbiota by L. monocytogenes epidemic strains to increased virulence.


Assuntos
Bacteriocinas/metabolismo , Microbioma Gastrointestinal , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Animais , Epidemias , Feminino , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/patogenicidade , Listeriose/epidemiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Virulência
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