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1.
Scand J Gastroenterol ; 54(8): 1033-1041, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31361979

RESUMO

Objectives: Proton pump inhibitors (PPI), a class of drugs commonly used, are known to be associated with changes in the intestinal microbiota. Published studies were done in heterogeneous cohorts which could hamper conclusions drawn as effects of diseases were not taken into consideration. We aimed to elucidate differences in the intestinal microbiota being associated to the use of PPI in a cohort study of patients with chronic hepatitis C. Material and Methods: The 16S rDNA gene was analyzed in stool samples of patients with and without PPI use. Patients with concomitant medication influencing the microbiota were excluded. Results were compared with the clinical course of hepatitis C patients with decompensated liver cirrhosis. Results: No differences in alpha diversity could be observed, while the microbial community structure differed significantly, especially in patients with liver cirrhosis. The relative abundance of Streptococcus spp., Enterobacter spp. and Haemophilus spp. was significantly increased in patients with PPI use irrespectively of the stage of liver disease. Finally, in patients with decompensated liver cirrhosis due to chronic HCV infection only in these using PPI bacterial phylotypes were isolated. Conclusions: PPI use was associated with significant alterations in the microbial community in patients with chronic hepatitis C, which were even pronounced in patients with liver cirrhosis. In patients with decompensated liver cirrhosis due to chronic HCV infection, the use of PPI may promote infections either directly or indirectly through changes in the microbial community structure. Future studies should further investigate long-term impact on the microbiota and the clinical outcome.

2.
Extremophiles ; 23(1): 35-48, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30284641

RESUMO

Genotypic and morphological diversity of cyanobacteria in the Rupite hot spring (Bulgaria) was investigated by means of optical microscopy, cultivation, single-cell PCR, and 16S rRNA gene amplicon sequencing. Altogether, 34 sites were investigated along the 71-39 °C temperature gradient. Analysis of samples from eight representative sites shown that Illumina, optical microscopy, and Roche 454 identified 72, 45 and 19% respective occurrences of all cumulatively present taxa. Optical microscopy failed to detect species of minor occurrence; whereas, amplicon sequencing technologies suffered from failed primer annealing and the presence of species with extensive extracellular polysaccharides production. Amplicon sequencing of the 16S rRNA gene V5-V6 region performed by Illumina identified the cyanobacteria most reliably to the generic level. Nevertheless, only the combined use of optical microscopy, cultivation and sequencing methods allowed for reliable estimate of the cyanobacterial diversity. Here, we show that Rupite hot-spring system hosts one of the richest cyanobacterial flora reported from a single site above 50 °C. Chlorogloeopsis sp. was the most abundant at the highest temperature (68 °C), followed by Leptolyngbya boryana, Thermoleptolyngbya albertanoae, Synechococcus bigranulatus, Oculatella sp., and Desertifilum sp. thriving above 60 °C, while Leptolyngbya geysericola, Geitlerinema splendidum, and Cyanobacterium aponinum were found above 50 °C.


Assuntos
Cianobactérias/genética , Fontes Termais/microbiologia , Microbiota , Cianobactérias/classificação , Cianobactérias/citologia , Cianobactérias/isolamento & purificação , RNA Ribossômico 16S/genética
3.
ISME J ; 12(11): 2640-2654, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29980795

RESUMO

The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir Rímov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers' removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.


Assuntos
Bactérias/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala/normas , Análise de Sequência de DNA/normas , Bactérias/genética , Cadeia Alimentar , Água Doce/microbiologia , Microbiota , Padrões de Referência
4.
Liver Int ; 38(1): 50-58, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28561276

RESUMO

BACKGROUND & AIMS: The importance of the intestinal microbiota for the onset and clinical course of many diseases, including liver diseases like non-alcoholic steatohepatitis and cirrhosis, is increasingly recognized. However, the role of intestinal microbiota in chronic hepatitis C virus (HCV) infection remains unclear. METHODS: In a cross-sectional approach, the intestinal microbiota of 95 patients chronically infected with HCV (n=57 without cirrhosis [NO-CIR]; n=38 with cirrhosis [CIR]) and 50 healthy controls (HC) without documented liver diseases was analysed. RESULTS: Alpha diversity, measured by number of phylotypes (S) and Shannon diversity index (H'), decreased significantly from HC to NO-CIR to CIR. S and H' correlated negatively with liver elastography. Analysis of similarities revealed highly statistically significant differences in the microbial communities between HC, NO-CIR and CIR (R=.090; P<1.0×10-6 ). Stratifying for HCV genotypes even increased the differences. In addition, we observed distinct patterns in the relative abundance of genera being either positive or negative correlated with diseases status. CONCLUSIONS: This study shows that not only the stage of liver disease but also HCV infection is associated with a reduced alpha diversity and different microbial community patterns. These differences might be caused by direct interactions between HCV and the microbiota or indirect interactions facilitated by the immune system.


Assuntos
Bactérias/classificação , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Hepacivirus/patogenicidade , Hepatite C Crônica/microbiologia , Cirrose Hepática/microbiologia , Adulto , Idoso , Estudos de Casos e Controles , Estudos Transversais , Feminino , Genótipo , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/virologia , Interações Hospedeiro-Patógeno , Humanos , Cirrose Hepática/diagnóstico , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
Environ Microbiol ; 18(7): 2259-71, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27207744

RESUMO

The human nasal passage, from the anterior nares through the nasal vestibule to the nasal cavities, is an important habitat for opportunistic pathogens and commensals alike. This work sampled four different anatomical regions within the human nasal passage across a large cohort of individuals (n = 79) comprising individuals suffering from chronic nasal inflammation clinically known as chronic rhinosinusitis (CRS) and individuals not suffering from inflammation (CRS-free). While individuals had their own unique bacterial fingerprint that was consistent across the anatomical regions, these bacterial fingerprints formed into distinct delineated groups comprising core bacterial members, which were consistent across all four swabbed anatomical regions irrespective of health status. The most significant observed pattern was the difference between the global bacterial profiles of swabbed and tissue biopsy samples from the same individuals, being also consistent across different anatomical regions. Importantly, no statistically significant differences could be observed concerning the global bacterial communities, any of the bacterial species or the range of diversity indices used to compare between CRS and CRS-free individuals, and between two CRS phenotypes (without nasal polyps and with nasal polyps). Thus, the role of bacteria in the pathogenesis of sinusitis remains uncertain.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Microbiota , Cavidade Nasal/microbiologia , Rinite/microbiologia , Sinusite/microbiologia , Adulto , Idoso , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Doença Crônica , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cavidade Nasal/imunologia , Rinite/imunologia , Sinusite/imunologia , Adulto Jovem
6.
Environ Microbiol Rep ; 7(6): 929-35, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26306992

RESUMO

The cotton rat nose is commonly used as a model for Staphylococcus aureus colonization, as it is both physiologically and anatomically comparable to the human nares and can be easily colonized by this organism. However, while the colonization of the human anterior nares has been extensively studied, the microbial community structure of cotton rat noses has not been reported so far. We describe here the microbial community structure of the cotton rat (Sigmodon hispidus) nose through next-generation sequencing of 16S rRNA gene amplicons covering the V1-V2 region and the analysis of nearly full length 16S rRNA genes of the major phylotypes. Roughly half of the microbial community was composed of two undescribed species of the genus Campylobacter, with phylotypes belonging to the genera Catonella, Acholeplasma, Streptobacillus and Capnocytophaga constituting the predominant community members. Thus, the nasal community of the cotton rat is uniquely composed of several novel bacterial species and may not reflect the complex interactions that occur in human anterior nares. Mammalian airway microbiota may, however, be a rich source of hitherto unknown microbes.


Assuntos
Biodiversidade , Microbiota , Nariz/microbiologia , Sigmodontinae/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Metagenoma , Metagenômica , Filogenia , RNA Ribossômico 16S/genética
7.
Gut Microbes ; 3(3): 234-49, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22572831

RESUMO

The structure of the human gut microbial community is determined by host genetics and environmental factors, where alterations in its structure have been associated with the onset of different diseases. Establishing a defined human gut microbial community within inbred rodent models provides a means to study microbial-related pathologies, however, an in-depth comparison of the established human gut microbiota in the different models is lacking. We compared the efficiency of establishing the bacterial component of a defined human microbial community within germ-free (GF) rats, GF mice, and antibiotic-treated specific pathogen-free mice. Remarkable differences were observed between the different rodent models. While the majority of abundant human-donor bacterial phylotypes were established in the GF rats, only a subset was present in the GF mice. Despite the fact that members of the phylum Bacteriodetes were well established in all rodent models, mice enriched for phylotypes related to species of Bacteroides. In contrary to the efficiency of Clostridiales to populate the GF rat in relative proportions to that of the human-donor, members of Clostridia cluster IV only poorly colonize the mouse gut. Thus, the genetic background of the different recipient rodent systems (that is, rats and mice) strongly influences the nature of the populating human gut microbiota, determining each model's biological suitability.


Assuntos
Bactérias/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Biota , Vida Livre de Germes , Humanos , Camundongos , Ratos
8.
Appl Environ Microbiol ; 73(8): 2682-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17322323

RESUMO

Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology 149:1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the bphA gene cluster of Burkholderia xenovorans LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from Pseudomonas sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at meta and para carbons. It attacked ortho-, meta-, and para-chlorinated rings with comparable efficiencies. It gave significantly higher yields in ortho,meta-dioxygenation of recalcitrant congeners containing a doubly ortho-chlorinated ring. While the parental enzyme yielded mainly unproductive meta, para dioxygenation of 2,5,4'-CB, the hybrid predominantly converted this congener into an ortho,meta-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.


Assuntos
Burkholderia/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Bifenilos Policlorados/metabolismo , Engenharia de Proteínas , Pseudomonas/genética , Biotransformação , Burkholderia/enzimologia , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Oxirredução , Pseudomonas/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato
9.
Appl Environ Microbiol ; 72(3): 2191-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16517671

RESUMO

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.


Assuntos
Compostos de Bifenilo/metabolismo , Burkholderia/enzimologia , Dioxigenases/química , Dioxigenases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Burkholderia/genética , Burkholderia/crescimento & desenvolvimento , Dioxigenases/genética , Evolução Molecular Direcionada , Dados de Sequência Molecular , Mutagênese , Especificidade por Substrato
10.
J Bacteriol ; 185(23): 6976-80, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617661

RESUMO

Three regions of the biphenyl dioxygenase (BDO) of Burkholderia sp. strain LB400 have previously been shown to significantly influence the interaction between enzyme and substrates at the active site. For a further discrimination within these regions, we investigated the effects of 23 individual amino acid exchanges. The regiospecificity of substrate dioxygenation was used as a sensitive means to monitor changes in the steric-electronic structure of the active site. Replacements of residues that, according to a model of the BDO three-dimensional structure, directly interact with substrates in most, but not all, cases (Met231, Phe378, and Phe384) very strongly altered this parameter (by factors of >7). On the other hand, a number of amino acids (Ile243, Ile326, Phe332, Pro334, and Trp392) which have no contacts with substrates also strongly changed the site preference of dioxygenation (by factors of between 2.6 and 3.5). This demonstrates that residues which had not been predicted to be influential can play a pivotal role in BDO specificity.


Assuntos
Burkholderia/enzimologia , Oxigenases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Oxigenases/genética , Oxigenases/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Microbiology ; 149(Pt 6): 1475-1481, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12777487

RESUMO

Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds. They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants. This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities. The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments. Previously, it has been shown that neither the N- nor the C-terminal portions but only the core region of the large or alpha-subunit of a class II ARHDO significantly influence substrate and product spectra. On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit. These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds. In 10 cases, a gene fragment of expected length was amplified. DNA sequencing confirmed similarity to ARHDO alpha-subunit gene cores. The heterologously well-expressible bphA gene cluster of Burkholderia sp. strain LB400 was modified to facilitate the in-frame insertion of amplified segments. It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs. The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400. These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids. Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.


Assuntos
Bactérias Aeróbias/enzimologia , Bactérias Aeróbias/genética , Genes Bacterianos , Técnicas Genéticas , Proteínas com Ferro-Enxofre , Oxigenases/genética , Sequência de Aminoácidos , Sequência de Bases , Burkholderia/enzimologia , Burkholderia/genética , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , DNA Bacteriano/genética , Dados de Sequência Molecular , Família Multigênica , Oxigenases/isolamento & purificação , Oxigenases/metabolismo , Subunidades Proteicas , Pseudomonas/enzimologia , Pseudomonas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Homologia de Sequência de Aminoácidos , Sphingomonas/enzimologia , Sphingomonas/genética
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