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1.
J Gastroenterol Hepatol ; 30(2): 268-78, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25087692

RESUMO

BACKGROUND AND AIM: Crohn's disease pathogenesis involves alterations in the gut microbiota. We characterized the mucosa-associated microbiota at the time of surgical resection and 6 months later to identify bacterial profiles associated with recurrence and remission. METHODS: Tissue samples were collected from surgical resection specimens in 12 Crohn's disease patients, and at 6 months postoperative colonoscopy from the neoterminal ileum and anastomosis. Endoscopic recurrence was assessed using the Rutgeerts score. Microbiota was characterized using microarray and 454 pyrosequencing. Longitudinal comparisons were made within patients, and cross-sectional comparisons made with colonoscopic biopsies from the terminal ileum and cecum of 10 healthy subjects. RESULTS: Microbiota of healthy subjects had high diversity and was dominated by the Firmicutes, Bacteroidetes, and Proteobacteria phyla. Biodiversity was lower in Crohn's disease patients at the time of surgery, increased after surgery, but still differed from healthy subjects. Crohn's disease patients with recurrent disease retained a microbiota favoring proteolytic-fueled fermentation and lactic acid-producing bacteria, including Enterococcus and Veillonella spp., while those maintaining remission demonstrated predominant saccharolytic Bacteroides, Prevotella, and Parabacteroides spp., and saccharolytic, butyrate-producing Firmicutes. CONCLUSION: In Crohn's disease, the mucosa-associated microbiota diversity is reduced at the time of surgery, but also differs between patients with different clinical outcomes at 6 months. These findings may provide prognostic information at the time of surgery, allowing identification of patients at increased risk of recurrence, and provide basis for a more targeted approach for therapeutic interventions after surgery.


Assuntos
Doença de Crohn/microbiologia , Doença de Crohn/cirurgia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Projetos Piloto , Adalimumab/administração & dosagem , Adulto , Idoso , Biópsia , Ceco/microbiologia , Ceco/cirurgia , Colonoscopia , Doença de Crohn/tratamento farmacológico , Estudos Transversais , Quimioterapia Combinada , Feminino , Previsões , Humanos , Íleo/microbiologia , Íleo/cirurgia , Estudos Longitudinais , Masculino , Metiltransferases/administração & dosagem , Metronidazol/administração & dosagem , Pessoa de Meia-Idade , Cuidados Pós-Operatórios , Prognóstico , Recidiva , Risco , Fatores de Tempo , Adulto Jovem
2.
Anaerobe ; 29: 100-7, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24225531

RESUMO

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.


Assuntos
Eremophila (Planta)/metabolismo , Metano/biossíntese , Consórcios Microbianos/genética , Oxirredutases/genética , RNA Ribossômico 16S/genética , Animais , Avena/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Biomarcadores/metabolismo , Reatores Biológicos , Euryarchaeota/genética , Euryarchaeota/crescimento & desenvolvimento , Euryarchaeota/metabolismo , Fermentação , Fibrobacter/genética , Fibrobacter/crescimento & desenvolvimento , Fibrobacter/metabolismo , Concentração de Íons de Hidrogênio , Pressão , Reação em Cadeia da Polimerase em Tempo Real , Rúmen/microbiologia , Ruminantes , Ruminococcus/genética , Ruminococcus/crescimento & desenvolvimento , Ruminococcus/metabolismo , Temperatura
3.
Anim Biosci ; 37(2): 396-403, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38186254

RESUMO

OBJECTIVE: Monofluoroacetate (MFA) is a potent toxin that blocks ATP production via the Krebs cycle and causes acute toxicity in ruminants consuming MFA-containing plants. The rumen bacterium, Cloacibacillus porcorum strain MFA1 belongs to the phylum Synergistota and can produce fluoride and acetate from MFA as the end-products of dehalorespiration. The aim of this study was to identify the genomic basis for the metabolism of MFA by this bacterium. METHODS: A draft genome sequence for C. porcorum strain MFA1 was assembled and quantitative transcriptomic analysis was performed thus highlighting a candidate operon encoding four proteins that are responsible for the carbon-fluorine bond cleavage. Comparative genome analysis of this operon was undertaken with three other species of closely related Synergistota bacteria. RESULTS: Two of the genes in this operon are related to the substrate-binding components of the glycine reductase protein B (GrdB) complex. Glycine shares a similar structure to MFA suggesting a role for these proteins in binding MFA. The remaining two genes in the operon, an antiporter family protein and an oxidoreductase belonging to the radical S-adenosyl methionine superfamily, are hypothesised to transport and activate the GrdB-like protein respectively. Similar operons were identified in a small number of other Synergistota bacteria including type strains of Cloacibacillus porcorum, C. evryensis, and Pyramidobacter piscolens, suggesting lateral transfer of the operon as these genera belong to separate families. We confirmed that all three species can degrade MFA, however, substrate degradation in P. piscolens was notably reduced compared to Cloacibacillus isolates possibly reflecting the loss of the oxidoreductase and antiporter in the P. piscolens operon. CONCLUSION: Identification of this unusual anaerobic fluoroacetate metabolism extends the known substrates for dehalorespiration and indicates the potential for substrate plasticity in amino acid-reducing enzymes to include xenobiotics.

4.
Exerc Immunol Rev ; 19: 102-19, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977723

RESUMO

BACKGROUND: Butyrate delivery to the large bowel may positively modulate commensal microbiota and enhance immunity. OBJECTIVE: To determine the effects of increasing large bowel butyrate concentration through ingestion of butyrylated high amylose maize starch (HAMSB) on faecal biochemistry and microbiota, and markers of immunity in healthy active individuals. DESIGN: Male and female volunteers were assigned randomly to consume either two doses of 20 g HAMSB (n = 23; age 37.9 +/- 7.8 y; mean +/- SD) or a low amylose maize starch (LAMS) (n = 18; age 36.9 = 9.5 y) twice daily for 28 days. Samples were collected on days 0, 10 and 28 for assessment of faecal bacterial groups, faecal biochemistry, serum cytokines and salivary antimicrobial proteins. RESULTS: HAMSB led to relative increases in faecal free (45%; 12-86%; mean; 90% confidence interval; P = 0.02), bound (950%; 563-1564%; P < 0.01) and total butyrate (260%; 174-373%; P < 0.01) and faecal propionate (41%; 12-77%; P = 0.02) from day 0 to day 28 compared to LAMS. HAMSB was also associated with a relative 1.6-fold (1.2- to 2.0-fold; P < 0.01) and 2.5-fold (1.4- to 4.4-fold; P = 0.01) increase in plasma IL-10 and TNF-alpha but did not alter other indices of immunity. There were relative greater increases in faecal P. distasonis (81-fold (28- to 237-fold; P < 0.01) and F. prausnitzii (5.1-fold (2.1- to 12-fold; P < 0.01) in the HAMSB group. CONCLUSIONS: HAMSB supplementation in healthy active individuals promotes the growth of bacteria that may improve bowel health and has only limited effects on plasma cytokines.


Assuntos
Butiratos/farmacologia , Colo/efeitos dos fármacos , Colo/microbiologia , Citocinas/biossíntese , Amido/farmacologia , Adulto , Butiratos/imunologia , Colo/imunologia , Fibras na Dieta/administração & dosagem , Suplementos Nutricionais , Método Duplo-Cego , Fezes/química , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Saliva/química , Saliva/imunologia , Amido/imunologia
5.
BMC Pregnancy Childbirth ; 13: 50, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23442391

RESUMO

BACKGROUND: Obesity is increasing in the child-bearing population as are the rates of gestational diabetes. Gestational diabetes is associated with higher rates of Cesarean Section for the mother and increased risks of macrosomia, higher body fat mass, respiratory distress and hypoglycemia for the infant. Prevention of gestational diabetes through life style intervention has proven to be difficult. A Finnish study showed that ingestion of specific probiotics altered the composition of the gut microbiome and thereby metabolism from early gestation and decreased rates of gestational diabetes in normal weight women. In SPRING (the Study of Probiotics IN the prevention of Gestational diabetes), the effectiveness of probiotics ingestion for the prevention of gestational diabetes will be assessed in overweight and obese women. METHODS/DESIGN: SPRING is a multi-center, prospective, double-blind randomized controlled trial run at two tertiary maternity hospitals in Brisbane, Australia. Five hundred and forty (540) women with a BMI > 25.0 kg/m(2) will be recruited over 2 years and receive either probiotics or placebo capsules from 16 weeks gestation until delivery. The probiotics capsules contain > 1x10(9) cfu each of Lactobacillus rhamnosus GG and Bifidobacterium lactis BB-12 per capsule. The primary outcome is diagnosis of gestational diabetes at 28 weeks gestation. Secondary outcomes include rates of other pregnancy complications, gestational weight gain, mode of delivery, change in gut microbiome, preterm birth, macrosomia, and infant body composition. The trial has 80% power at a 5% 2-sided significance level to detect a >50% change in the rates of gestational diabetes in this high-risk group of pregnant women. DISCUSSION: SPRING will show if probiotics can be used as an easily implementable method of preventing gestational diabetes in the high-risk group of overweight and obese pregnant women.


Assuntos
Diabetes Gestacional/prevenção & controle , Obesidade/terapia , Sobrepeso/terapia , Complicações na Gravidez/terapia , Fenômenos Fisiológicos da Nutrição Pré-Natal/fisiologia , Probióticos/uso terapêutico , Adulto , Austrália , Diabetes Gestacional/diagnóstico , Inquéritos sobre Dietas , Comportamento Alimentar , Feminino , Teste de Tolerância a Glucose , Humanos , Obesidade/complicações , Sobrepeso/complicações , Gravidez , Estudos Prospectivos , Análise de Regressão , Centros de Atenção Terciária
6.
Aliment Pharmacol Ther ; 58(4): 417-428, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37386938

RESUMO

BACKGROUND: Carbohydrate fermentation plays a pivotal role in maintaining colonic health with excessive proximal and deficient distal fermentation being detrimental. AIMS: To utilise telemetric gas- and pH-sensing capsule technologies for defining patterns of regional fermentation following dietary manipulations, alongside conventional techniques of measuring fermentation. METHODS: In a double-blind crossover trial, 20 patients with irritable bowel syndrome were fed low FODMAP diets that included no extra fibre (total fibre content 24 g/day), or additional poorly fermented fibre, alone (33 g/day) or with fermentable fibre (45 g/day) for 2 weeks. Plasma and faecal biochemistry, luminal profiles defined by tandem gas- and pH-sensing capsules, and faecal microbiota were assessed. RESULTS: Plasma short-chain fatty acid (SCFA) concentrations (µmol/L) were median (IQR) 121 (100-222) with fibre combination compared with 66 (44-120) with poorly fermented fibre alone (p = 0.028) and 74 (55-125) control (p = 0.069), but no differences in faecal content were observed. Luminal hydrogen concentrations (%), but not pH, were higher in distal colon (mean 4.9 [95% CI: 2.2-7.5]) with fibre combination compared with 1.8 (0.8-2.8) with poorly fermented fibre alone (p = 0.003) and 1.9 (0.7-3.1) control (p = 0.003). Relative abundances of saccharolytic fermentative bacteria were generally higher in association with supplementation with the fibre combination. CONCLUSIONS: A modest increase in fermentable plus poorly fermented fibres had minor effects on faecal measures of fermentation, despite increases in plasma SCFA and abundance of fermentative bacteria, but the gas-sensing capsule, not pH-sensing capsule, detected the anticipated propagation of fermentation distally in the colon. The gas-sensing capsule technology provides unique insights into localisation of colonic fermentation. TRIAL REGISTRATION: ACTRN12619000691145.


Assuntos
Dieta FODMAP , Hidrogênio , Humanos , Hidrogênio/análise , Fermentação , Colo/metabolismo , Fibras na Dieta/metabolismo , Ácidos Graxos Voláteis , Fezes/microbiologia , Dieta
8.
ScientificWorldJournal ; 2012: 178254, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22919294

RESUMO

The objective of this paper was to report the isolation of two fluoroacetate degrading bacteria from the rumen of goats. The animals were adult goats, males, crossbred, with rumen fistula, fed with hay, and native pasture. The rumen fluid was obtained through the rumen fistula and immediately was inoculated 100 µL in mineral medium added with 20 mmol L(-1) sodium fluoroacetate (SF), incubated at 39°C in an orbital shaker. Pseudomonas fluorescens (strain DSM 8341) was used as positive control for fluoroacetate dehalogenase activity. Two isolates were identified by 16S rRNA gene sequencing as Pigmentiphaga kullae (ECPB08) and Ancylobacter dichloromethanicus (ECPB09). These bacteria degraded sodium fluoroacetate, releasing 20 mmol L(-1) of fluoride ion after 32 hours of incubation in Brunner medium containing 20 mmol L(-1) of SF. There are no previous reports of fluoroacetate dehalogenase activity for P. kullae and A. dichloromethanicus. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil. In this way, P. kullae and A. dichloromethanicus may be used to colonize the rumen of susceptible animals to avoid intoxication by fluoroacetate containing plants.


Assuntos
Bactérias/metabolismo , Fluoracetatos/metabolismo , Rúmen/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Brasil , Meios de Cultura , Filogenia , RNA Ribossômico 16S/genética , Navios
9.
ScientificWorldJournal ; 2012: 149893, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22619595

RESUMO

The aim of this work was to isolate and identify bacteria able to degrade sodium fluoroacetate from soil and plant samples collected in areas where the fluoroacetate-containing plants Mascagnia rigida and Palicourea aenofusca are found. The samples were cultivated in mineral medium added with 20 mmol L(-1) sodium fluoroacetate. Seven isolates were identified by 16S rRNA gene sequencing as Paenibacillus sp. (ECPB01), Burkholderia sp. (ECPB02), Cupriavidus sp. (ECPB03), Staphylococcus sp. (ECPB04), Ancylobacter sp. (ECPB05), Ralstonia sp. (ECPB06), and Stenotrophomonas sp. (ECPB07). All seven isolates degraded sodium-fluoroacetate-containing in the medium, reaching defluorination rate of fluoride ion of 20 mmol L(-1). Six of them are reported for the first time as able to degrade sodium fluoroacetate (SF). In the future, some of these microorganisms can be used to establish in the rumen an engineered bacterial population able to degrade sodium fluoroacetate and protect ruminants from the poisoning by this compound.


Assuntos
Bactérias/metabolismo , Flúor/metabolismo , Fluoracetatos/metabolismo , Plantas/microbiologia , Microbiologia do Solo , Animais , Bactérias/classificação , Bactérias/genética , Biodegradação Ambiental , Brasil , Bovinos , Fluoracetatos/intoxicação , Filogenia , Intoxicação/mortalidade , Intoxicação/prevenção & controle , Intoxicação/veterinária , RNA Ribossômico 16S/genética
10.
Appl Environ Microbiol ; 77(17): 6310-2, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21764953

RESUMO

The use and validation of a strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product is described. The strategy allows a modular approach, in that the same bar code can be used with two or more target-specific primer sets, even simultaneously.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Análise de Sequência de DNA/métodos , Bactérias/classificação , Bactérias/genética , Primers do DNA/genética
11.
Appl Environ Microbiol ; 77(24): 8795-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21984239

RESUMO

Several subsampling-based normalization strategies were applied to different high-throughput sequencing data sets originating from human and murine gut environments. Their effects on the data sets' characteristics and normalization efficiencies, as measured by several ß-diversity metrics, were compared. For both data sets, subsampling to the median rather than the minimum number appeared to improve the analysis.


Assuntos
Trato Gastrointestinal/microbiologia , Metagenoma , Metagenômica/métodos , Metagenômica/normas , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Camundongos , Estatística como Assunto
12.
Am J Gastroenterol ; 105(11): 2420-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20648002

RESUMO

OBJECTIVES: Mucosa-associated bacteria are increased in inflammatory bowel disease (IBD), which suggests the possibility of an increased source of digestible endogenous mucus substrate. We hypothesized that mucolytic bacteria are increased in IBD, providing increased substrate to sustain nonmucolytic mucosa-associated bacteria. METHODS: Mucolytic bacteria were characterized by the ability to degrade human secretory mucin (MUC2) in pure and mixed anaerobic cultures. Real-time PCR was used to enumerate mucosa-associated mucolytic bacteria in 46 IBD and 20 control patients. Bacterial mucolytic activity was tested in vitro using purified human MUC2. RESULTS: We confirm increased total mucosa-associated bacteria 16S rRNA gene in macroscopically and histologically normal intestinal epithelium of both Crohn's disease (CD) (mean 1.9-fold) and ulcerative colitis (UC) (mean 1.3-fold). We found a disproportionate increase in some mucolytic bacteria. Mean Ruminococcus gnavus were increased >4-fold and Ruminococcus torques ∼100-fold in macroscopically and histologically normal intestinal epithelium of both CD and UC. The most abundantly detected mucolytic bacterium in controls, Akkermansia muciniphila, was reduced many fold in CD and in UC. Coculture of A. muciniphila with MUC2 as the sole carbon source led to reduction in its abundance while it augmented growth of other bacteria. CONCLUSIONS: Mucolytic bacteria are present in healthy humans, where they are an integral part of the mucosa-associated bacterial consortium. The disproportionate increase in R. gnavus and R. torques could explain increased total mucosa-associated bacteria in IBD.


Assuntos
Colo/microbiologia , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/microbiologia , Mucinas/genética , Ruminococcus/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/patologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Mucinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ruminococcus/metabolismo
13.
FEMS Microbiol Ecol ; 96(7)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32353874

RESUMO

Among the natural halogenic compounds, the plant toxin fluoroacetate (FA) causes livestock fatalities in southern hemisphere countries. Here, we report on the isolation of a rumen bacterium, strain C12-8 that degrades FA under anaerobic conditions. 16S rRNA gene sequence analysis showed this bacterium belonged to the Pyramidobacter genus within the Synergistetes phylum and was 98% similar to Pyramidobacter piscolens W5455 isolated from the human oral cavity. Transmission electron microscopy showed the cell envelope to be unusual, with only one membrane and no obvious external wall. Growth and FA degradation were enhanced by peptide-rich protein hydrolysates but not carbohydrates. End products of metabolism were mainly acetate, propionate/isovalerate and isobutyrate. Strain C12-8 preferentially used peptide-bound amino acids rather than free amino acids. Glycine, serine, threonine, leucine, histidine and isoleucine were utilized as free and peptide-bound amino acids, but there was minimal utilization of alanine, proline, methionine, aspartic acid, lysine and arginine in either form. A survey of several cattle properties in northern Australia showed that strain C12-8 and other FA degrading bacteria affiliated with Cloacibacillus porcorum strain MFA1 were endemic to cattle in the northern beef herd and may help to reduce toxicity.


Assuntos
Fluoracetatos , Rúmen , Animais , Arginina , Austrália , Bactérias , Composição de Bases , Bovinos , DNA Bacteriano/genética , Humanos , Leucina , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Inquéritos e Questionários
14.
J Anim Sci Biotechnol ; 11: 80, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32832076

RESUMO

BACKGROUND: This study investigated changes in rumen protozoal and methanogenic communities, along with the correlations among microbial taxa and methane (CH4) production of six Belmont Red Composite beef steers fed tea seed saponins (TSS). Animals were fed in three consecutive feeding periods, a high-grain basal diet for 14 d (BD period) then a period of progressive addition of TSS to the basal diet up to 30 g/d for 20 d (TSS period), followed by the basal diet for 13 d without TSS (BDP post-control period). RESULTS: The study found that TSS supplementation decreased the amount of the protozoal genus Entodinium and increased Polyplastron and Eudiplodinium genera. During BDP period, the protozoa community of steers did not return to the protozoal profiles observed in BD period, with higher proportions of Metadinium and Eudiplodinium and lower Isotricha. The addition of TSS was found to change the structure of methanogen community at the sub-genus level by decreasing the abundance of methanogens in the SGMT clade and increasing the abundance of methanogens in the RO clade. The correlation analysis indicated that the abundance of SGMT clade methanogens were positively correlated with Isotricha, and Isotricha genus and SGMT clade methanogens were positively correlated with CH4 production. While RO clade were positively correlated with the proportion of Metadinium genus, which was negatively correlated with CH4 emission. CONCLUSIONS: These results suggest that different genera of rumen protozoa ciliates appear to be selectively inhibited by TSS, and the change in methanogen community at the subgenus level may be due to the mutualistic relationships between methanogens and rumen ciliates.

15.
Biochem Biophys Res Commun ; 385(4): 605-11, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19486892

RESUMO

Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C(12)), palmitate (C(16)) and stearate (C(18)). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism.


Assuntos
Lipase/metabolismo , Rúmen/microbiologia , Veillonellaceae/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Ácidos Graxos/metabolismo , Feminino , Biblioteca Gênica , Genômica , Lipase/classificação , Lipase/genética , Lipase/isolamento & purificação , Dados de Sequência Molecular , Filogenia
16.
Curr Microbiol ; 58(5): 464-71, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19159975

RESUMO

An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.


Assuntos
Bactérias/genética , Trato Gastrointestinal/microbiologia , Biologia Molecular/métodos , Reação em Cadeia da Polimerase/métodos , RNA Bacteriano/isolamento & purificação , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Análise por Conglomerados , DNA Complementar/química , DNA Complementar/genética , Fezes/microbiologia , Camundongos , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Rúmen/microbiologia , Ruminantes , Análise de Sequência de DNA
17.
Microbiol Resour Announc ; 8(13)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30923245

RESUMO

We report the 3.7-Mb genome sequence of Oribacterium sp. strain C9, isolated from the rumen of a steer grazing on Rhodes grass in Rockhampton, Queensland, Australia. This draft genome consists of 3,720,024 bp with a 42.8% G+C content, 3,130 predicted coding DNA sequences (CDSs), and 67 RNAs.

18.
Microbiol Resour Announc ; 8(30)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346024

RESUMO

We report the genome sequence of Sporanaerobacter acetigenes strain F-12, isolated from the rumen of a steer grazing on Rhodes grass in Townsville (Lansdown Research Station), Queensland, Australia. This draft genome consists of 2,866,191 bp, with 31.23% G+C content and 2,889 predicted coding sequences.

19.
Front Genet ; 9: 62, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29535763

RESUMO

Dairy heifers were subjected to a non-life-threatening challenge designed to induce ruminal acidosis by feeding grain and sugar. Large among animal variation in clinical signs of acidosis, rumen metabolite concentrations, and the rumen microbiome occurred. This exploratory study investigates sources of the variation by examining associations between the genome, metabolome, and microbiome, albeit with a limited population. The broader objective is to provide a rationale for a larger field study to identify markers for susceptibility to ruminal acidosis. Initially, heifers (n = 40) allocated to five feed additive groups were fed 20-days pre-challenge with a total mixed ration and additives. Fructose (0.1% of bodyweight/day) was added for the last 10 days pre-challenge. On day 21 heifers were challenged with 1.0% of bodyweight dry matter wheat + 0.2% of bodyweight fructose + additives. Rumen samples were collected via stomach tube weekly (day 0, 7, and 14) and at five times over 3.6 h after challenge and analyzed for pH and volatile fatty acid, ammonia, D-, and L-lactate concentrations. Relative abundance of bacteria and archaea were determined using Illumina MiSeq. Genotyping was undertaken using a 150K Illumina SNPchip. Genome-wide association was performed for metabolite and microbiome measures (n = 33). Few genome associations occurred with rumen pH, concentration of acetate, propionate, total volatile fatty acids, or ammonia, or the relative abundance of the Firmicutes, Bacteroidetes, and Spirochaetes phyla. Metabolites and microbial phyla that had markers associated and quantitative trait loci (QTL) were: acetate to propionate ratio (A:P), D-, L-, and total lactate, butyrate, acidosis eigenvalue, Actinobacteria, Chloroflexi, Euryarchaeota, Fibrobacteres, Planctomycetes, Proteobacteria, and Tenericutes. A putative genomic region overlapped for Actinobacteria, Euryarchaeota, and Fibrobacteres and covered the region that codes for matrix extracellular phosphoglycoprotein (MEPE). Other overlapping regions were: (1) Chloroflexi, Tenericutes, and A:P, (2) L- and total lactate and Actinobacteria, and (3) Actinobacteria, Euryarchaeota, Fibrobacteres, and A:P. Genome-wide associations with the metabolome and microbiome occurred despite the small population, suggesting that markers for ruminal acidosis susceptibility exist. The findings may explain some of the variation in metabolomic and microbial data and provide a rationale for a larger study with a population that has variation in acidosis.

20.
Artigo em Inglês | MEDLINE | ID: mdl-28674607

RESUMO

Fluoroacetate producing plants grow worldwide and it is believed they produce this toxic compound as a defence mechanism against grazing by herbivores. Ingestion by livestock often results in fatal poisonings, which causes significant economic problems to commercial farmers in many countries such as Australia, Brazil and South Africa. Several approaches have been adopted to protect livestock from the toxicity with limited success including fencing, toxic plant eradication and agents that bind the toxin. Genetically modified bacteria capable of degrading fluoroacetate have been able to protect ruminants from fluoroacetate toxicity under experimental conditions but concerns over the release of these microbes into the environment have prevented the application of this technology. Recently, a native bacterium from an Australian bovine rumen was isolated which can degrade fluoroacetate. This bacterium, strain MFA1, which belongs to the Synergistetes phylum degrades fluoroacetate to fluoride ions and acetate. The discovery and isolation of this bacterium provides a new opportunity to detoxify fluoroacetate in the rumen. This review focuses on fluoroacetate toxicity in ruminant livestock, the mechanism of fluoroacetate toxicity, tolerance of some animals to fluoroaceate, previous attempts to mitigate toxicity, aerobic and anaerobic microbial degradation of fluoroacetate, and future directions to overcome fluoroacetate toxicity.

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