Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray ...designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearson's correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.
Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive ...technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.
Alterations in intestinal microbiota composition are associated with several chronic conditions, including obesity and inflammatory diseases. The microbiota of older people displays greater ...inter-individual variation than that of younger adults. Here we show that the faecal microbiota composition from 178 elderly subjects formed groups, correlating with residence location in the community, day-hospital, rehabilitation or in long-term residential care. However, clustering of subjects by diet separated them by the same residence location and microbiota groupings. The separation of microbiota composition significantly correlated with measures of frailty, co-morbidity, nutritional status, markers of inflammation and with metabolites in faecal water. The individual microbiota of people in long-stay care was significantly less diverse than that of community dwellers. Loss of community-associated microbiota correlated with increased frailty. Collectively, the data support a relationship between diet, microbiota and health status, and indicate a role for diet-driven microbiota alterations in varying rates of health decline upon ageing. PUBLICATION ABSTRACT
Alterations in intestinal microbiota composition are associated with several chronic conditions, including obesity and inflammatory diseases. The microbiota of older people displays greater ...inter-individual variation than that of younger adults. Here we show that the faecal microbiota composition from 178 elderly subjects formed groups, correlating with residence location in the community, day-hospital, rehabilitation or in long-term residential care. However, clustering of subjects by diet separated them by the same residence location and microbiota groupings. The separation of microbiota composition significantly correlated with measures of frailty, co-morbidity, nutritional status, markers of inflammation and with metabolites in faecal water. The individual microbiota of people in long-stay care was significantly less diverse than that of community dwellers. Loss of community-associated microbiota correlated with increased frailty. Collectively, the data support a relationship between diet, microbiota and health status, and indicate a role for diet-driven microbiota alterations in varying rates of health decline upon ageing.
The genus Lactobacillus Claesson, Marcus J; van Sinderen, Douwe; O'Toole, Paul W
FEMS microbiology letters,
04/2007, Volume:
269, Issue:
1
Journal Article
Peer reviewed
The genus Lactobacillus is a diverse group that includes many species used in food production and preservation. Some lactobacilli are considered probiotic, conferring health benefits upon the host. ...The heterogeneity of this genus poses challenges and opportunities when characterizing or exploiting individual strains. To date, 10 Lactobacillus genome sequences have been published, and at least 11 more sequencing projects are ongoing. These studies will dramatically improve one's understanding of metabolic processes, bioprocessing capabilities and potential roles in health and well-being of the Lactobacilli. This review describes the current status of Lactobacillus genome sequence projects, highlights the major findings and summarizes functional genomics or comparative genomics studies. The genomic basis for the unusual diversity of this genus is discussed, and the potential for comparative genomics to rigorously extend phylogenetic analysis of the Lactobacilli is described.
Alterations in the human intestinal microbiota are linked to conditions including inflammatory bowel disease, irritable bowel syndrome, and obesity. The microbiota also undergoes substantial changes ...at the extremes of life, in infants and older people, the ramifications of which are still being explored. We applied pyrosequencing of over 40,000 16S rRNA gene V4 region amplicons per subject to characterize the fecal microbiota in 161 subjects aged 65 y and older and 9 younger control subjects. The microbiota of each individual subject constituted a unique profile that was separable from all others. In 68% of the individuals, the microbiota was dominated by phylum Bacteroides, with an average proportion of 57% across all 161 baseline samples. Phylum Firmicutes had an average proportion of 40%. The proportions of some phyla and genera associated with disease or health also varied dramatically, including Proteobacteria, Actinobacteria, and Faecalibacteria. The core microbiota of elderly subjects was distinct from that previously established for younger adults, with a greater proportion of Bacteroides spp. and distinct abundance patterns of Clostridium groups. Analyses of 26 fecal microbiota datasets from 3-month follow-up samples indicated that in 85% of the subjects, the microbiota composition was more like the corresponding time-0 sample than any other dataset. We conclude that the fecal microbiota of the elderly shows temporal stability over limited time in the majority of subjects but is characterized by unusual phylum proportions and extreme variability. PUBLICATION ABSTRACT
Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in ...addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.
Genome watch: GI genomes Cerdeño-Tárraga, Ana; Claesson, Marcus J; Sebaihia, Mohammed ...
Nature reviews. Microbiology,
05/2005, Volume:
3, Issue:
5
Journal Article
Peer reviewed
This month's Genome Watch delves deep into the human gastrointestinal tract, describing the genome sequences of two major constituents of the gut microflora: Lactobacillus acidophilus, which is both ...commercially and medically important for its role as a probiotic, and Bacteroides fragilis, which, although a major constituent of the natural gut flora, is also an opportunistic pathogen that can cause an array of infections.
ABSTRACT
Lactobacillus
gasseri
ATCC 33323,
Lactobacillus salivarius
subsp.
salivarius
UCC 118, and
Lactobacillus casei
ATCC 334
contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1)
...distinguishable prophage sequences, respectively. Sequence analysis
revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group
of Sfi11-like
pac
site and
cos
site
Siphoviridae
, respectively. Phylogenetic investigation of
these newly described prophage sequences revealed that they have not
followed an evolutionary development similar to that of their bacterial
hosts and that they show a high degree of diversity, even within a
species. The attachment sites were determined for all these prophage
elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage
Sal2 integrates in a predicted arginino-succinate lyase-encoding gene.
In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are
integrated in noncoding regions in the
L. casei
ATCC 334 and
L. salivarius
UCC 118 genomes. Northern analysis showed that
large parts of the prophage genomes are transcriptionally silent and
that transcription is limited to genome segments located near the
attachment site. Finally, pulsed-field gel electrophoresis followed by
Southern blot hybridization with specific prophage probes indicates
that these prophage sequences are narrowly distributed within
lactobacilli.
The adsorption characteristics of three proteins bovine serum albumin (BSA), myoglobin (Mb), and cytochrome c (CytC) onto self-assembled monolayers of mercaptoundecanoic acid (MUA) on both gold ...nanoparticles (AuNP) and gold surfaces (Au) are described. The combination of quartz crystal microbalance measurements with dissipation (QCM-D) and pH titrations of the ζ-potential provide information on layer structure, surface coverage, and potential. All three proteins formed adsorption layers consisting of an irreversibly adsorbed fraction and a reversibly adsorbed fraction. BSA showed the highest affinity for the MUA/Au, forming an irreversibly adsorbed rigid monolayer with a side-down orientation and packing close to that expected in the jamming limit. In addition, BSA showed a large change in the adsorbed mass due to reversibly bound protein. The data indicate that the irreversibly adsorbed fraction of CytC is a monolayer structure, whereas the irreversibly adsorbed Mb is present in form of a bilayer. The observation of stable BSA complexes on MUA/AuNPs at the isoelectric point by ζ-potential measurements demonstrates that BSA can sterically stabilize MUA/AuNP. On the other hand, MUA/AuNP coated with either Mb or CytC formed a reversible flocculated state at the isoelectric point. The colloidal stability differences may be correlated with weaker binding in the reversibly bound overlayer in the case of Mb and CytC as compared to BSA.
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