1,087 research outputs found

    Comparison of five assays for DNA extraction from bacterial cells in human faecal samples

    Get PDF
    Aim To determine the most effective DNA extraction method for bacteria in faecal samples. Materials and Results This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. Conclusion From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. Significance and Impact of the Study The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose

    Intestinal microbiome landscaping : insight in community assemblage and implications for microbial modulation strategies

    Get PDF
    High individuality, large complexity and limited understanding of the mechanisms underlying human intestinal microbiome function remain the major challenges for designing beneficial modulation strategies. Exemplified by the analysis of intestinal bacteria in a thousand Western adults, we discuss key concepts of the human intestinal microbiome landscape, i.e. the compositional and functional 'core', the presence of community types and the existence of alternative stable states. Genomic investigation of core taxa revealed functional redundancy, which is expected to stabilize the ecosystem, as well as taxa with specialized functions that have the potential to shape the microbiome landscape. The contrast between Prevotella-and Bacteroides-dominated systems has been well described. However, less known is the effect of not so abundant bacteria, for example, Dialister spp. that have been proposed to exhibit distinct bistable dynamics. Studies employing time-series analysis have highlighted the dynamical variation in the microbiome landscape with and without the effect of defined perturbations, such as the use of antibiotics or dietary changes. We incorporate ecosystem-level observations of the human intestinal microbiota and its keystone species to suggest avenues for designing microbiome modulation strategies to improve host health.Peer reviewe

    Methods for exploring the faecal microbiome of premature infants: a review

    Get PDF
    The premature infant gut microbiome plays an important part in infant health and development, and recognition of the implications of microbial dysbiosis in premature infants has prompted significant research into these issues. The approaches to designing investigations into microbial populations are many and varied, each with its own benefits and limitations. The technique used can influence results, contributing to heterogeneity across studies. This review aimed to describe the most common techniques used in researching the preterm infant microbiome, detailing their various limitations. The objective was to provide those entering the field with a broad understanding of available methodologies, so that the likely effects of their use can be factored into literature interpretation and future study design. We found that although many techniques are used for characterising the premature infant microbiome, 16S rRNA short amplicon sequencing is the most common. 16S rRNA short amplicon sequencing has several benefits, including high accuracy, discoverability and high throughput capacity. However, this technique has limitations. Each stage of the protocol offers opportunities for the injection of bias. Bias can contribute to variability between studies using 16S rRNA high throughout sequencing. Thus, we recommend that the interpretation of previous results and future study design be given careful consideration

    Exploring the faecal microbiome associated with preterm birth

    Get PDF
    Jacob Westaway investigated the gut microbiome of preterm infants born in North Queensland, Australia. He observed unique associations between the gut microbiome and the unfavourable covariates, as well as acute modulation in association with probiotic prophylaxis. This supports expansion of the probiotic supplementation criteria and highlights the importance of the microbiome in health

    Contribution of diet to the composition of the human gut microbiota

    Get PDF
    This paper is part of the Proceedings from the 2013 ENGIHR Conference in Valencia, Spain. More papers from this supplement can be found at http://www.microbecolhealthdis.net Microbial Ecology in Health & Disease 2015. © 2015 Daniela Graf et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ACKNOWLEDGEMENTS The authors acknowledge the support of the European Science Foundation (ESF), in the framework of the Research Networking Programe, The European Network for Gastrointestinal Health Research.Peer reviewedPublisher PD

    Effects of diets with different phosporus availability on the intestinal microbiota of chickens and pigs

    Get PDF
    In the research works of the present thesis, 16S rRNA gene sequencing and metaproteomics were employed to investigate the gut microbiota of chickens and pigs kept at experimental diets with varying amount of calcium-phosphorus (CaP) and supplemented MP. This represents a valuable approach to investigate the bacterial specimens involved in the P absorption, allowing for a comprehensive understanding of how the intestinal bacteria adapt to a new diet and which metabolic routes are affected by changing levels of supplemented P and/or MP. Two major experimental trials were performed during the investigation. The first one was conducted on chickens operating a modulation in the dietary levels of Ca, P and MP. This trial highlighted a shift in the composition of the crop and ceca-associated microbial community depending on the composition of the diet fed. Also, investigated protein inventory revealed that the stress condition due to the reduced P availability is mirrored in the gastrointestinal tract (GIT)-associated microbiota. Marked differences were observed in the functions of the bacterial community in the case of P-available diets versus P-deficient ones. Protein repertoire of the first case draws a thriving microbial community focused on complex and anabolic functions. Contrariwise, the bacterial community in the case of P-lacking diets appears to deal with catabolic functions and stress response. The second trial was conducted on pigs and attempts to define the dynamics featuring the microbiota adaptation to a new challenging diet composed of different protein sources and varying levels of Ca and P. Statistical evidences reveal a stepwise adaptation of the fecal microbiota to the experimental diets fed. Both DNA-based approach and metaproteomics independently reveal three main adaptation phases: -before the feeding of the experimental trial (i.e. Zero), -the response of the microbial community to the challenging factor (i.e. MA) and, finally, - the newly achieved homeostatic balance (i.e. EQ). As observed in the first trial, feeding of the experimental diets impairs the overall fecal microbiota composition, stimulating the presence of phase-specific bacterial specimens and a characteristic relative abundance of the shared ones. Bacterial families responsible for the phase-specific architecture of the fecal microbiota are also active in the biochemical pathways driving the functional peculiarities of each adaptation phase. A deeper investigation of the identified protein repertoire revealed that the observed statistical differences among the adaptation phases are uniquely due to the Ca and P composition of the diets fed. None of the observed effects can be attributed to the diverse protein sources supplemented with the diets. Functional categorization of the identified protein inventory depicts three diverse functional assets of the microbial community. Specifically, prior the feeding of the experimental diets, bacteria are hypothesized to live under homeostatic condition, since they appear to be involved in complex and highly-specialized functions. Following the administration of the experimental diets microbial community changes its functional priority and reduce the expression of highly specialized functions to focus on more essential ones. Proteins involved in complex functions such as widening the substrates array and facing complex sugars tend to increase in abundance while the new homeostatic balance is achieved. Altogether, data from both trials provide useful information for future studies aimed to design effective breeding strategies finalized to reduce the P supplementation in the routinely breeding of livestock and maintain a balanced microbial activity in the animal GIT. Investigation of the dynamics of the porcine microbiota provides instructions on the minimal exposure time required from the intestinal microbiota to adapt to a new dietary composition. This is of fundamental importance for the design of future studies aimed to confirm and/or continue our results. Moreover, the anatomical and physiological similarities occurring between humans and pigs, make our findings of interest for future human nutritional studies, where the mechanisms and lasts of the microbiota adaptation process is still object of discussion.In den Forschungsprojekten der vorliegenden Arbeit wurden die 16S rRNA Gensequenzierung und Metaproteomik zur Untersuchung der Darmmikrobiota von Hühnern und Schweinen verwendet. Dabei wurde der Einfluss von Versuchsdiäten mit unterschiedlichen Mengen an Calcium-Phosphor (CaP) und ergänzten MP untersucht. Dies ist ein wertvoller Ansatz zur Identifizierung von Bakterienspezies, welche an der P-Absorption beteiligt sind, und ermöglicht es ein umfassendes Verständnis darüber zugewinnen, wie sich die Darmbakterien an eine neue Ernährung und an Stoffwechselwege, die von sich verändernden Mengen an ergänztem P und / oder MP beeinflusst werden, anpassen können. Im Rahmen dieser Arbeit wurden zwei große experimentelle Studien durchgeführt. Die erste Studie wurde an Hühnern durchgeführt und basierte auf der Verabreichung unterschiedlicher Mengen an Ca, P und MP in der Nahrung. Die Ergebnisse dieser Studie zeigten eine Nahrungs-abhängige Verschiebung in der Zusammensetzung der mikrobiellen Gemeinschaft in Kropf und Blinddarm. Außerdem zeigte das untersuchte Proteinrepertoire, dass der Stresszustand, aufgrund der reduzierten P-Verfügbarkeit, sich in der Mikrobiota im Gastrointestinaltrakt (GIT) widergespiegelt. Der Vergleich von Diäten mit verfügbarem P mit P-armen Diäten zeigte deutliche Unterschiede in den Funktionen der Bakteriengemeinschaft. Bei einer Diät mit verfügbarem P in der Nahrung, deutet die Analyse des Proteinrepertoires auf eine florierende mikrobielle Gemeinschaft hin, in der komplexe und anabole Funktionen vorherrschen. Im Gegensatz dazu scheinen bei Diäten mit P-Mangel katabole Funktionen und Stressreaktionen in der bakteriellen Gemeinschaft zu überwiegen. Die zweite Studie wurde an Schweinen durchgeführt und versuchte die Dynamik zu definieren, mit der sich die Mikrobiota an eine neue, herausfordernde, aus verschiedenen Proteinquellen und unterschiedlichen Mengen an Ca und P bestehende Nahrung, anpasst. Statistische Beweise zeigen eine stufenweise Anpassung der fäkalen Mikrobiota an die verabreichten Versuchsnahrung. Sowohl DNA- als auch metaproteomische Analysen, die getrennt voneinander durchgeführt wurden, zeigen drei Hauptanpassungsphasen, welche die folgenden experimentalen Adaptationsgruppen definieren: - vor der Verabreichung der Versuchsdiät (d.h. Null), - die Reaktion der mikrobiellen Gemeinschaft auf den herausfordernden Faktor (d.h. MA) und schließlich, - das Stadium des neu erreichten homöostatischen Gleichgewichts (d.h. EQ). Wie im ersten Versuch beobachtet wurde, beeinträchtigt die Fütterung der Versuchsnahrung die gesamte Zusammensetzung der fäkalen Mikrobiota, sie stimuliert das Vorhandensein von phasenspezifischen Bakterienarten und eine charakteristische relative Häufigkeit der phasenunspezifischen Bakterien. Bakterielle Familien, die für die phasenspezifische Struktur der fäkalen Mikrobiota verantwortlich sind, sind auch in den biochemischen Pfaden aktiv und sind im Wesentlichen für die funktionellen Besonderheiten jeder Anpassungsphase verantwortlich. Eine tiefere Untersuchung des identifizierten Proteinrepertoires ergab, dass die beobachteten statistischen Unterschiede zwischen den Adaptationsphasen eindeutig auf die Ca- und P-Gehalte der gefütterten Diäten zurückzuführen sind. Keine der beobachteten Effekten kann den verschiedenen gleichzeitig verabreichten Proteinquellen zugeschrieben werden. Die funktionelle Kategorisierung des identifizierten Proteinbestands zeigt drei verschiedene funktionelle Aspekte der mikrobiellen Gemeinschaft. Insbesondere wird angenommen, dass vor dem Füttern der experimentellen Diäten, die Bakterien unter homöostatischen Bedingungen leben, da sie in komplexe und hochspezialisierte Funktionen involviert zu sein scheinen. Nach der Verabreichung der experimentellen Diäten ändert die mikrobielle Gemeinschaft ihre funktionelle Priorität und reduziert die Ausübung hochspezialisierter Funktionen, um sich auf die wesentlichen zu konzentrieren. Während das neue homöostatische Gleichgewicht erreicht wird, nimmt die Menge an Proteinen, die an komplexen Funktionen beteiligt sind, wie z. B. die Verarbeitung einer großen Substratvielfalt sowie die Metabolisierung von komplexen Zuckern, zu. Zusammengefasst, liefern die aus beiden Experimente gewonnenen Daten nützliche Informationen für zukünftige Studien mit dem Ziel, effektive Zuchtstrategien zu entwickeln, um die P-Supplementierung bei der routinemäßigen Viehzucht zu reduzieren und eine ausgewogene mikrobielle Aktivität im Gastrointestinaltrakt des Tieres aufrechtzuerhalten. Die Untersuchung der Anpassungsdynamik der Schweinemikrobiota liefert genaue Informationen darüber, wie lange die intestinale Mikrobiota benötigt, um sich an eine neue Nahrungszusammensetzung anzupassen. Dies ist von zentraler Bedeutung für die Gestaltung zukünftiger experimenteller Studien, die darauf abzielen, unsere Ergebnisse zu bestätigen und/oder unsere Forschung fortzusetzen. Aufgrund der anatomischen und physiologischen Ähnlichkeiten, die zwischen Menschen und Schweinen auftreten, sind die erhaltenen Befunde auch für zukünftige Ernährungsstudien am Menschen von Interesse, bei denen die Mechanismen und die Dauer der Adaptationsprozesse der Mikrobiota noch untersucht werden

    Characterization of the Human Host Gut Microbiome with an Integrated Genomics / Proteomics Approach

    Get PDF
    The new field of ‘omics’ has spawned the development of metaproteomics, an approach that has the ability to identify and decipher the metabolic functions of a proteome derived from a microbial community that is largely uncultivable. With the development and availabilities of high throughput proteomics, high performance liquid chromatography coupled to mass spectrometry (MS) has been leading the field for metaproteomics. MS-based metaproteomics has been successful in its’ investigations of complex microbial communities from soils to the human body. Like the environment, the human body is host to a multitude of microorganisms that reside within the skin, oral cavity, vagina, and gastrointestinal tract, referred to as the human microbiome. The human microbiome is made up of trillions of bacteria that outnumber human genes by several orders of magnitude. These microbes are essential for human survival with a significant dependence on the microbes to encode and carryout metabolic functions that humans have not evolved on their own. Recently, metaproteomics has emerged as the primary technology to understand the metabolic functional signature of the human microbiome. Using a newly developed integrated approach that combines metagenomics and metaproteomics, we attempted to address the following questions: i) do humans share a core functional microbiome and ii) how do microbial communities change in response to disease. This resulted in a comprehensive identification and characterization of the metaproteome from two healthy human gut microbiomes. These analyses have resulted in an extended application to characterize how Crohn’s disease affects the functional signature of the microbiota. Contrary to measuring highly complex and representative gut metaproteomes is a less complex, controlled human-derived microbial community present in the gut of gnotobiotic mice. This human gut model system enhanced the capability to directly monitor fundamental interactions between two dominant phyla, Bacteroides and Firmicutes, in gut microbiomes colonized with two or more phylotypes. These analyses revealed membership abundance and functional differences between phylotypes when present in either a binary or 12-member consortia. This dissertation aims to characterize host microbial interactions and develop MS-based methods that can provide a better understanding of the human gut microbiota composition and function using both approaches

    Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

    Get PDF
    peer-reviewedMicroorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i) the identification of enzymes with desirable technological properties, capable of catalyzing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii) the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii) the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.Science Foundation Ireland(SFI)Grant Number 13/SIRG/215
    corecore