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1.
iScience ; 27(2): 108778, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38292428

RESUMO

Bacteriophages are a major component of the gut microbiome and are believed to play a role in establishment and stabilization of microbial communities by influencing taxonomic and functional diversity. We show that the activity of lytic and temperate phages can also significantly affect bacterial community structure in a model of extended colonic retention. Intact fresh human feces were incubated anaerobically at 37°C without homogenization and subjected to metagenomic sequencing. We observed subject-specific blooms and collapses of selected bacteriophage and bacterial populations within some individuals. Most notable were striking collapses of Prevotella populations accompanied by increases in specific bacteriophages. In a number of cases, we even observed a shift from one bacterial "enterotype" to another within 48 h. These results confirm that intact feces represents a highly dynamic ecological system and suggests that colonic retention time could have a profound effect on microbiome composition, including a significant impact by bacteriophages.

2.
Nature ; 617(7960): 409-416, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37138077

RESUMO

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1-4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the 'crass fold', that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.


Assuntos
Vírus de DNA , Intestinos , Proteínas Virais , Vírion , Humanos , Capsídeo/química , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Vírus de DNA/química , Vírus de DNA/classificação , Vírus de DNA/isolamento & purificação , Vírus de DNA/metabolismo , Vírus de DNA/ultraestrutura , Vírion/química , Vírion/metabolismo , Vírion/ultraestrutura , Montagem de Vírus , Intestinos/microbiologia , Intestinos/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Desdobramento de Proteína , Dobramento de Proteína
3.
Gut Microbes ; 15(1): 2194794, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36994608

RESUMO

Ruminococcus gnavus is a prevalent gut microbe reported to occur in higher abundance among individuals with inflammatory bowel disease (IBD). This study reports the isolation and characterization of six bacteriophages (phages) isolated from human fecal material and environmental samples that infect this species. Isolated phages have a siphovirus morphology, with genomes ranging between 36.5 and 37.8 kbp. Genome analysis indicates that the phages have a temperate lifestyle, which was confirmed by their ability to form lysogens on their host bacterial species. In contrast to the finding that phages lyse their host in liquid medium, results from a mouse trial indicate these phages can co-exist with the host bacterium in the gut without causing a significant reduction of R. gnavus. The bacterial counts in the feces of phage-treated mice did not significantly differ in the presence of phage. Furthermore, analysis of publicly available gut virome sequence data indicates a high abundance of these phages among individuals suffering from IBD. This work provides the first insight into how phages interact with R. gnavus in the human gut microbiome.


Assuntos
Bacteriófagos , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Bacteriófagos/genética , Mucinas , Ruminococcus/genética , Microbioma Gastrointestinal/genética , Bactérias
4.
Commun Biol ; 6(1): 221, 2023 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-36841913

RESUMO

Viruses are increasingly recognised as important components of the human microbiome, fulfilling numerous ecological roles including bacterial predation, immune stimulation, genetic diversification, horizontal gene transfer, microbial interactions, and augmentation of metabolic functions. However, our current view of the human gut virome is tainted by previous sequencing requirements that necessitated the amplification of starting nucleic acids. In this study, we performed an original longitudinal analysis of 40 healthy control, 19 Crohn's disease, and 20 ulcerative colitis viromes over three time points without an amplification bias, which revealed and highlighted the interpersonal individuality of the human gut virome. In contrast to a 16 S rRNA gene analysis of matched samples, we show that α- and ß-diversity metrics of unamplified viromes are not as efficient at discerning controls from patients with inflammatory bowel disease. Additionally, we explored the intrinsic properties of unamplified gut viromes and show there is considerable interpersonal variability in viral taxa, infrequent longitudinal persistence of intrapersonal viruses, and vast fluctuations in the abundance of temporal viruses. Together, these properties of unamplified faecal viromes confound the ability to discern disease associations but significantly advance toward an unbiased and accurate representation of the human gut virome.


Assuntos
Colite Ulcerativa , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Vírus , Humanos , Viroma/genética , Microbioma Gastrointestinal/genética , Vírus/genética , Colite Ulcerativa/genética , Colite Ulcerativa/microbiologia , Doenças Inflamatórias Intestinais/genética
5.
Nat Microbiol ; 7(8): 1301-1311, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35918425

RESUMO

The mammalian virome has been linked to health and disease but our understanding of how it is structured along the longitudinal axis of the mammalian gastrointestinal tract (GIT) and other organs is limited. Here, we report a metagenomic analysis of the prokaryotic and eukaryotic virome occupying luminal and mucosa-associated habitats along the GIT, as well as parenchymal organs (liver, lung and spleen), in two representative mammalian species, the domestic pig and rhesus macaque (six animals per species). Luminal samples from the large intestine of both mammals harboured the highest loads and diversity of bacteriophages (class Caudoviricetes, family Microviridae and others). Mucosal samples contained much lower viral loads but a higher proportion of eukaryotic viruses (families Astroviridae, Caliciviridae, Parvoviridae). Parenchymal organs contained bacteriophages of gut origin, in addition to some eukaryotic viruses. Overall, GIT virome composition was specific to anatomical region and host species. Upper GIT and mucosa-specific viruses were greatly under-represented in distal colon samples (a proxy for faeces). Nonetheless, certain viral and phage species were ubiquitous in all samples from the oral cavity to the distal colon. The dataset and its accompanying methodology may provide an important resource for future work investigating the biogeography of the mammalian gut virome.


Assuntos
Bacteriófagos , Vírus , Animais , Bacteriófagos/genética , Fezes , Macaca mulatta , Mamíferos , Metagenoma , Metagenômica , Vírus/genética
6.
BMC Biol ; 19(1): 163, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34407825

RESUMO

BACKGROUND: The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of lysogeny, these viruses demonstrate long-term persistence in the human gut microbiome, dominating the virome in some individuals. RESULTS: Here we show that rapid phase variation of alternate capsular polysaccharides in Bacteroides intestinalis cultures plays an important role in a dynamic equilibrium between phage sensitivity and resistance, allowing phage and bacteria to multiply in parallel. The data also suggests the role of a concomitant phage persistence mechanism associated with delayed lysis of infected cells, similar to carrier state infection. From an ecological and evolutionary standpoint, this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other "benign" forms of phage infection when the host is stably present at high abundance. CONCLUSION: Long-term persistence of bacteriophage and host could result from mutually beneficial mechanisms driving bacterial strain-level diversity and phage survival in complex environments.


Assuntos
Bacteriófagos , Bacteroides , Bactérias , Bacteroides/virologia , Humanos , Variação de Fase , Filogenia
7.
Front Cell Infect Microbiol ; 11: 616918, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33791236

RESUMO

Recent years have been marked by the growing interest towards virulent and temperate bacteriophage populations inhabiting the human lower gastrointestinal tract - the gut phageome. A number of studies demonstrated high levels of specificity and temporal stability of individual gut phageomes, as well as their specific alterations in disease cohorts, in parallel with changes in the bacteriome. It has been speculated that phages might have an active role in shaping the taxonomic composition and functional properties of the human gut bacteriome. An overwhelming majority of gut bacteriophages, however, remain uncultured, unclassified, and their specific hosts and infection strategies are still unknown. They are often referred to as "the viral dark matter". A possible breakthrough in understanding of the phageome can only become possible when a significant proportion of the "the viral dark matter" is identified and linked to bacterial hosts. Here, we describe a method that enables rapid discovery and host-linking of novel bacteriophages in the gut via a combination of serial enrichment cultures and shotgun metagenomics of viral DNA. Using this approach dozens of novel and previously known bacteriophages were detected, including the ones infecting difficult-to-culture anaerobic bacteria. The majority of phages failed to produce lysis and propagate on host cultures in traditional assays. The newly identified phages include representatives of Siphoviridae, Myoviridae, Podoviridae, and crAss-like viruses, infecting diverse bacterial taxa of Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia and Proteobacteria phyla. The proposed new method has a potential for high-throughput screening applications for mass discovery of new phages in different environments.


Assuntos
Bacteriófagos , Metagenômica , Bacteriófagos/genética , DNA Viral/genética , Trato Gastrointestinal , Humanos , Viroma
8.
Microbiome ; 9(1): 89, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33845877

RESUMO

BACKGROUND: The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual's core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible. RESULTS: Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens. ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media. CONCLUSIONS: We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens, at very high levels in liquid culture without impacting on bacterial numbers. Video abstract.


Assuntos
Bacteriófagos , Microbioma Gastrointestinal , Bacteriófagos/genética , Bacteroides , Humanos , Filogenia
9.
Cell Host Microbe ; 26(4): 527-541.e5, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31600503

RESUMO

The human gut contains a vast array of viruses, mostly bacteriophages. The majority remain uncharacterized, and their roles in shaping the gut microbiome and in impacting on human health remain poorly understood. We performed longitudinal metagenomic analysis of fecal viruses in healthy adults that reveal high temporal stability, individual specificity, and correlation with the bacterial microbiome. Using a database-independent approach that uses most of the sequencing data, we uncovered the existence of a stable, numerically predominant individual-specific persistent personal virome. Clustering of viral genomes and de novo taxonomic annotation identified several groups of crAss-like and Microviridae bacteriophages as the most stable colonizers of the human gut. CRISPR-based host prediction highlighted connections between these stable viral communities and highly predominant gut bacterial taxa such as Bacteroides, Prevotella, and Faecalibacterium. This study provides insights into the structure of the human gut virome and serves as an important baseline for hypothesis-driven research.


Assuntos
Bacteroides/virologia , Faecalibacterium/virologia , Microbioma Gastrointestinal/genética , Microviridae/genética , Prevotella/virologia , Bacteroides/isolamento & purificação , Faecalibacterium/isolamento & purificação , Humanos , Estudos Longitudinais , Metagenoma/genética , Microviridae/classificação , Microviridae/isolamento & purificação , Prevotella/isolamento & purificação , Carga Viral
10.
Nat Commun ; 9(1): 4781, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30429469

RESUMO

CrAssphages are an extensive and ubiquitous family of tailed bacteriophages, predicted to infect bacteria of the order Bacteroidales. Despite being found in ~50% of individuals and representing up to 90% of human gut viromes, members of this viral family have never been isolated in culture and remain understudied. Here, we report the isolation of a CrAssphage (ΦCrAss001) from human faecal material. This bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming previous in silico predictions of the likely host. DNA sequencing demonstrates that the bacteriophage genome is circular, 102 kb in size, and has unusual structural traits. In addition, electron microscopy confirms that ΦcrAss001 has a podovirus-like morphology. Despite the absence of obvious lysogeny genes, ΦcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium, and is able to maintain itself in continuous host culture during several weeks.


Assuntos
Bacteriófagos/genética , Bacteroides/virologia , Microbioma Gastrointestinal , Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , DNA Viral , Fezes/microbiologia , Humanos , Microscopia Eletrônica , Podoviridae/genética , Podoviridae/ultraestrutura , Replicação Viral/fisiologia
11.
Int J Syst Evol Microbiol ; 65(12): 4580-4588, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26377180

RESUMO

Culture-based study of the faecal microbiome in two adult female subjects revealed the presence of two obligately anaerobic, non-spore-forming, rod-shaped, non-motile, Gram-negative bacterial strains that represent novel species. The first strain, designated 627T, was a fastidious, slow-growing, indole-positive bacterium with a non-fermentative type of metabolism.The strain was characterized by the production of acetic and succinic acids as metabolic end products, the prevalence of iso-C15 : 0 fatty acid and the presence of menaquinones MK-10 and MK-11. The DNA G+C content was found to be 56.6 mol%. The second strain, designated 177T, was capable of fermenting a rich collection of carbohydrate substrates, producing acetic acid as a terminal product. The strain was indole-negative and resistant to bile. The major cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0 (in a 1 : 1 ratio) and the predominant menaquinone was MK-11.The DNA G+C content was 37.8 mol%. A phylogenomic analysis of the draft genomes of strains 627T and 177T placed these bacteria in the genera Alistipes(family Rikenellaceae) and Coprobacter (family Porphyromonadaceae), respectively.On the basis of the phenotypic and genotypic properties of strains 627T and 177T, we conclude that these strains from human faeces represent two novel bacterial species, for which the names Alistipes inops sp. nov. (type strain 627T5DSM 28863T5VKM B-2859T) and Coprobacter secundus sp. nov. (type strain 177T=DSM 28864T=VKM B-2857T) are proposed.


Assuntos
Bacteroidetes/classificação , Fezes/microbiologia , Filogenia , Adolescente , Adulto , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/genética , Bactérias Anaeróbias/isolamento & purificação , Técnicas de Tipagem Bacteriana , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/química
12.
Int J Syst Evol Microbiol ; 63(Pt 11): 4181-4188, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23771624

RESUMO

A novel obligately anaerobic, non-spore-forming, rod-shaped, non-motile Gram-reaction-negative bacterium was isolated from infant faeces. The strain, designated NSB1(T), was able to grow on rich media at 30-37 °C, in the presence of up to 2 % (w/v) Oxgall and 2 % (w/v) NaCl. Cells of strain NSB1(T) produced catalase, but not urease and indole. Aesculin was not hydrolysed. The strain was able to utilize d-glucose, lactose, maltose, mannose and raffinose as electron donors. When grown on d-glucose, the main metabolic end products were propionic and acetic acids, with a minor product being succinic acid. The major cellular fatty acids, iso-C15 : 0 and anteiso-C15 : 0, were present at a 1 : 1 molar ratio. The major menaquinone was MK-11. The DNA G+C content was found to be 38.5 mol%. According to 16S rRNA gene sequence analysis strain NSB1(T) is a member of the family Porphyromonadaceae, phylum Bacteroidetes. The closest relatives of the strain were Barnesiella viscericola (88.2 % identity) and Barnesiella intestinihominis (87.4 % identity). On the basis of phenotypic and genotypic properties of strain NSB1(T) we conclude that this strain represent a novel species in a new genus within the family of Porphyromonadaceae for which the name Coprobacter fastidiosus gen. nov., sp. nov. is proposed. The type strain of the species is NSB1(T) ( = DSM 26242(T), = VKM B-2743(T)).


Assuntos
Bacteroidetes/classificação , Fezes/microbiologia , Filogenia , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/genética , Bactérias Anaeróbias/isolamento & purificação , Técnicas de Tipagem Bacteriana , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Lactente , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/química
13.
Plasmid ; 69(2): 146-59, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23201047

RESUMO

The analysis of plasmid content in dominant Bacteroidales order intestinal strains isolated from the same child at a 5 year interval identified a 8.9 kb plasmid in Bacteroides uniformis BUN24 strain isolated at age 6 and indistinguishably sized plasmids in the isolates of B. uniformis, B. vulgatus, B. intesinalis, and Parabacteroides distasonis at age 11. We sequenced a B. uniformis BUN24 plasmid, designated pBUN24, and using molecular surveys of diverse species we established that this 8944bp molecule (G+C content 43.5%) represents a novel family of small cryptic Bacteroidales plasmids. The replication region of pBUN24 was experimentally localized to a 1707-bp fragment that includes a putative repA gene, coding for a protein of Rep_3 superfamily of replication proteins of theta-type plasmids preceded by a putative iteron-containing origin of replication. The other open reading frames (ORFs) identified in pBUN24 sequence include a putative tad-ata-type toxin-antitoxin and mobA-mobB mobilization modules, as well as seven additional cryptic ORFs. The interaction of Tad and Ada components demonstrated by a pull-down assay and the toxicity of Tad in Escherichia coli host suggests the functionality of the plasmid addiction module. Re-sequencing of plasmids in two Bacteroides strains isolated at the age of 11 showed 100% nucleotide identity to pBUN24. This data supports the notion that this plasmid is transmissible to other Bacteroidales strains in the natural ecosystem. The possible roles of toxin-antitoxin system and other proteins encoded by pBUN24 in providing an apparent ecological advantage to the plasmid-harbouring strains of a bacterial symbiont in the human gut deserve further investigation.


Assuntos
Bacteroides/genética , Pareamento de Bases/genética , Microbiota/genética , Plasmídeos/genética , Sequência de Aminoácidos , Antitoxinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Sequência de Bases , Criança , Humanos , Intestinos/microbiologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Origem de Replicação/genética
14.
Microbiol Immunol ; 56(1): 27-39, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22040047

RESUMO

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti-inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor-α (TNF-α) and lipopolysaccharide- (LPS) induced inflammatory responses in the HT-29 cell line. In contrast, neither killed bifidobacterial cells, nor cell-free extracts showed such activities. Further investigations resulted in attribution of this activity to heat-stable, non-lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3 kDa. The anti-inflammatory effects were dose- and time-dependent and associated with inhibition of IκB phosphorylation and nuclear factor-κ light chain enhancer of activated B cells (NF-κB)-dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF-α, but not with CMs alone, resulted in upregulation of transforming growth factor-ß1, IκBζ, and p21(CIP) mRNAs. Our data suggest certain species-specificities of the anti-inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.


Assuntos
Bifidobacterium/imunologia , Bifidobacterium/isolamento & purificação , Inflamação/microbiologia , Intestinos/microbiologia , Apoptose , Bifidobacterium/genética , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta Imunológica , Escherichia coli/genética , Escherichia coli/metabolismo , Fezes/microbiologia , Regulação Bacteriana da Expressão Gênica , Células HT29 , Humanos , Proteínas I-kappa B/metabolismo , Lactente , Inflamação/induzido quimicamente , Interleucina-8/metabolismo , Lipopolissacarídeos/efeitos adversos , Peso Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Fatores de Tempo , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador alfa/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia
15.
Plasmid ; 60(2): 136-48, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18652842

RESUMO

A survey of infant fecal Bifidobacterium isolates for plasmid DNA revealed that a significant portion of the strains, 17.6%, carry small plasmids. The majority of plasmid-harboring strains belonged to the Bifidobacterium longum/infantis group. Most of the plasmids could be assigned into two groups based on their sizes and the restriction profiles. Three plasmids, pB44 (3.6 kb) from B. longum, pB80 (4.9 kb) from Bifidobacterium bifidum, and pB21a (5.2kb) from Bifidobacterium breve were sequenced. While the former two plasmids were found to be highly similar to previously characterized rolling-circle replicating pKJ36 and pKJ56, respectively, the third plasmid, pB21a, does not share significant nucleotide homology with known plasmids. However, it might be placed into the pCIBb1-like group of bifidobacterial rolling-plasmids based on the homology of its Rep protein and the overall molecular organization. Two sets of Escherichia coli-Bifidobacterium shuttle vectors constructed based on pB44 and pB80 replicons were capable of transforming B. bifidum and B. breve strains with efficiency up to 3x10(4)cfu/microg DNA. Additionally, an attempt was made to employ a broad host range conjugation element, RP4, in developing of E. coli-Bifidobacterium gene transfer system.


Assuntos
Bifidobacterium/genética , Fezes/microbiologia , Vetores Genéticos/genética , Plasmídeos/genética , Sequência de Bases , Bifidobacterium/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Feminino , Genes Bacterianos , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Análise de Sequência
16.
Biosci Biotechnol Biochem ; 72(3): 742-8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18323636

RESUMO

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Bacteroides/isolamento & purificação , Bifidobacterium/isolamento & purificação , Fezes/microbiologia , Técnicas Bacteriológicas , Bacteroides/genética , Bifidobacterium/genética , DNA Bacteriano , DNA Ribossômico , Seguimentos , Humanos , Lactente , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição
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