A Look Behind the Curtain of 16s Sequencing MR DNA
361. PLoS One. 2013 Nov 19;8(11):e80702. doi: 10.1371/journal.pone.0080702.
A modular organization of the human intestinal mucosal microbiota and its
association with inflammatory bowel disease.
Tong M(1), Li X, Wegener Parfrey L, Roth B, Ippoliti A, Wei B, Borneman J,
McGovern DP, Frank DN, Li E, Horvath S, Knight R, Braun J.
(1)Department of Molecular and Medical Pharmacology, David Geffen School of
Medicine, University of California Los Angeles, Los Angeles, California, United
States of America.
Abnormalities of the intestinal microbiota are implicated in the pathogenesis of
Crohn's disease (CD) and ulcerative colitis (UC), two spectra of inflammatory
bowel disease (IBD). However, the high complexity and low inter-individual
overlap of intestinal microbial composition are formidable barriers to
identifying microbial taxa representing this dysbiosis. These difficulties might
be overcome by an ecologic analytic strategy to identify modules of interacting
bacteria (rather than individual bacteria) as quantitative reproducible features
of microbial composition in normal and IBD mucosa. We sequenced 16S ribosomal RNA
genes from 179 endoscopic lavage samples from different intestinal regions in 64
subjects (32 controls, 16 CD and 16 UC patients in clinical remission). CD and UC
patients showed a reduction in phylogenetic diversity and shifts in microbial
composition, comparable to previous studies using conventional mucosal biopsies.
Analysis of weighted co-occurrence network revealed 5 microbial modules. These
modules were unprecedented, as they were detectable in all individuals, and their
composition and abundance was recapitulated in an independent, biopsy-based
mucosal dataset 2 modules were associated with healthy, CD, or UC disease states.
Imputed metagenome analysis indicated that these modules displayed distinct
metabolic functionality, specifically the enrichment of oxidative response and
glycan metabolism pathways relevant to host-pathogen interaction in the
disease-associated modules. The highly preserved microbial modules accurately
classified IBD status of individual patients during disease quiescence,
suggesting that microbial dysbiosis in IBD may be an underlying disorder
independent of disease activity. Microbial modules thus provide an integrative
view of microbial ecology relevant to IBD.
PMID: 24260458 [PubMed - indexed for MEDLINE]
362. BMC Genomics. 2013 Sep 22;14:641. doi: 10.1186/1471-2164-14-641.
Alignment-free supervised classification of metagenomes by recursive SVM.
Cui H(1), Zhang X.
(1)Department of Automation, Bioinformatics Division/Center for Synthetic &
Systems Biology, TNLIST, MOE Key Laboratory of Bioinformatics, Tsinghua
University, Beijing 100084, China. email@example.com.
BACKGROUND: Comparison and classification of metagenome samples is one of the
major tasks in the study of microbial communities of natural environments or
niches on human bodies. Bioinformatics methods play important roles on this task,
including 16S rRNA gene analysis and some alignment-based or alignment-free
methods on metagenomic data. Alignment-free methods have the advantage of not
depending on known genome annotations and therefore have high potential in
studying complicated microbiomes. However, the existing alignment-free methods
are all based on unsupervised learning strategy (e.g., PCA or hierarchical
clustering). These types of methods are powerful in revealing major similarities
and grouping relations between microbiome samples, but cannot be applied for
discriminating predefined classes of interest which might not be the dominating
assortment in the data. Supervised classification is needed in the latter
scenario, with the goal of classifying samples into predefined classes and
finding the features that can discriminate the classes. The effectiveness of
supervised classification with alignment-based features on metagenomic data have
been shown in some recent studies. The application of alignment-free supervised
classification methods on metagenome data has not been well explored yet.
RESULTS: We developed a method for this task using k-tuple frequencies as
features counted directly from metagenome short reads and the R-SVM (Recursive
SVM) for feature selection and classification. We tested our method on a
simulation dataset, a real dataset composed of several known genomes, and a real
metagenome NGS short reads dataset. Experiments on simulated data showed that the
method can classify the classes almost perfectly and can recover major sequence
signatures that distinguish the two classes. On the real human gut metagenome
data, the method can discriminate samples of inflammatory bowel disease (IBD)
patients from control samples with high accuracy, which cannot be separated when
comparing the samples with unsupervised clustering approaches.
CONCLUSIONS: The proposed alignment-free supervised classification method can
perform well in discriminating of metagenomic samples of predefined classes and
in selecting characteristic sequence features for the discrimination. This study
shows as an example on the feasibility of using metagenome sequence features of
microbiomes on human bodies to study specific human health conditions using
supervised machine learning methods.
PMID: 24053649 [PubMed - indexed for MEDLINE]
363. PLoS One. 2012;7(3):e32219. doi: 10.1371/journal.pone.0032219. Epub 2012 Mar 5.
Identifying low pH active and lactate-utilizing taxa within oral microbiome
communities from healthy children using stable isotope probing techniques.
McLean JS(1), Fansler SJ, Majors PD, McAteer K, Allen LZ, Shirtliff ME, Lux R,
(1)Microbial and Environmental Genomics, The J Craig Venter Institute, San Diego,
California, United States of America. firstname.lastname@example.org
BACKGROUND: Many human microbial infectious diseases including dental caries are
polymicrobial in nature. How these complex multi-species communities evolve from
a healthy to a diseased state is not well understood. Although many health- or
disease-associated oral bacteria have been characterized in vitro, their
physiology within the complex oral microbiome is difficult to determine with
current approaches. In addition, about half of these species remain uncultivated
to date with little known besides their 16S rRNA sequence. Lacking culture-based
physiological analyses, the functional roles of uncultivated species will remain
enigmatic despite their apparent disease correlation. To start addressing these
knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS)
with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities
from healthy children for in vitro temporal monitoring of metabolites and
identification of metabolically active and inactive bacterial species.
METHODOLOGY/PRINCIPAL FINDINGS: Supragingival plaque samples from caries-free
children incubated with (13)C-substrates under imposed healthy (buffered, pH 7)
and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic
acid from glucose metabolism. Rapid lactate utilization upon glucose depletion
was observed under pH 7 conditions. SIP analyses revealed a number of genera
containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5.
The diversity of active species decreased significantly at pH 4.5 and was
dominated by Lactobacillus and Propionibacterium species, both of which have been
previously found within carious lesions from children.
CONCLUSIONS/SIGNIFICANCE: Our approach allowed for identification of species that
metabolize carbohydrates under different pH conditions and supports the
importance of Lactobacilli and Propionibacterium in the development of childhood
caries. Identification of species within healthy subjects that are active at low
pH can lead to a better understanding of oral caries onset and generate
appropriate targets for preventative measures in the early stages.
PMID: 22403637 [PubMed - indexed for MEDLINE]
364. PLoS One. 2011 Mar 8;6(3):e17447. doi: 10.1371/journal.pone.0017447.
Metatranscriptomic approach to analyze the functional human gut microbiota.
Gosalbes MJ(1), Durbán A, Pignatelli M, Abellan JJ, Jiménez-Hernández N,
Pérez-Cobas AE, Latorre A, Moya A.
(1)Unidad Mixta de Investigación en Genómica y Salud-Centro Superior
Investigación en Salud Pública (Generalitat Valenciana)/Instituto Cavanilles de
Biodiversidad y Biología Evolutiva (Universitat de València), València, Spain.
The human gut is the natural habitat for a large and dynamic bacterial community
that has a great relevance for health. Metagenomics is increasing our knowledge
of gene content as well as of functional and genetic variability in this
microbiome. However, little is known about the active bacteria and their
function(s) in the gastrointestinal tract. We performed a metatranscriptomic
study on ten healthy volunteers to elucidate the active members of the gut
microbiome and their functionality under conditions of health. First, the
microbial cDNAs obtained from each sample were sequenced using 454 technology.
The analysis of 16S transcripts showed the phylogenetic structure of the active
microbial community. Lachnospiraceae, Ruminococcaceae, Bacteroidaceae,
Prevotellaceae, and Rickenellaceae were the predominant families detected in the
active microbiota. The characterization of mRNAs revealed a uniform functional
pattern in healthy individuals. The main functional roles of the gut microbiota
were carbohydrate metabolism, energy production and synthesis of cellular
components. In contrast, housekeeping activities such as amino acid and lipid
metabolism were underrepresented in the metatranscriptome. Our results provide
new insights into the functionality of the complex gut microbiota in healthy
individuals. In this RNA-based survey, we also detected small RNAs, which are
important regulatory elements in prokaryotic physiology and pathogenicity.
PMID: 21408168 [PubMed - indexed for MEDLINE]
365. PLoS One. 2013;8(1):e54461. doi: 10.1371/journal.pone.0054461. Epub 2013 Jan 25.
Blood microbiota dysbiosis is associated with the onset of cardiovascular events
in a large general population: the D.E.S.I.R. study.
Amar J(1), Lange C, Payros G, Garret C, Chabo C, Lantieri O, Courtney M, Marre M,
Charles MA, Balkau B, Burcelin R; D.E.S.I.R. Study Group.
Collaborators: Balkau B, Ducimetière P, Eschwège E, Alhenc-Gelas F, Girault A,
Fumeron F, Marre M, Roussel R, Bonnet F, Cauchi S, Froguel P, Cogneau J, Born C,
Caces E, Copin N, Moreau JG, Lantieri O, Rakotozafy F, Tichet J, Vol S.
(1)INSERM U1048, Institute of Research on Metabolic and Cardiovascular Diseases,
CHU Toulouse, Toulouse, France. email@example.com
AIM: We recently described a human blood microbiome and a connection between this
microbiome and the onset of diabetes. The aim of the current study was to assess
the association between blood microbiota and incident cardiovascular disease.
METHODS AND RESULTS: D.E.S.I.R. is a longitudinal study with the primary aim of
describing the natural history of the metabolic syndrome and its complications.
Participants were evaluated at inclusion and at 3-, 6-, and 9-yearly follow-up
visits. The 16S ribosomal DNA bacterial gene sequence, that is common to the vast
majority of bacteria (Eubac) and a sequence that mostly represents Proteobacteria
(Pbac), were measured in blood collected at baseline from 3936 participants. 73
incident cases of acute cardiovascular events, including 30 myocardial
infarctions were recorded. Eubac was positively correlated with Pbac (r = 0.59;
P<0.0001). In those destined to have cardiovascular complications, Eubac was
lower (0.14±0.26 vs 0.12±0.29 ng/µl; P = 0.02) whereas a non significant increase
in Pbac was observed. In multivariate Cox analysis, Eubac was inversely
correlated with the onset of cardiovascular complications, (hazards ratio 0.50
95% CI 0.35-0.70) whereas Pbac (1.56, 95%CI 1.12-2.15) was directly correlated.
CONCLUSION: Pbac and Eubac were shown to be independent markers of the risk of
cardiovascular disease. This finding is evidence for the new concept of the role
played by blood microbiota dysbiosis on atherothrombotic disease. This concept
may help to elucidate the relation between bacteria and cardiovascular disease.
PMID: 23372728 [PubMed - indexed for MEDLINE]
366. J Med Entomol. 2013 Mar;50(2):404-14.
16S rRNA gene-based identification of Elizabethkingia meningoseptica
(Flavobacteriales: Flavobacteriaceae) as a dominant midgut bacterium of the Asian
malaria vector Anopheles stephensi (Dipteria: Culicidae) with antimicrobial
Ngwa CJ(1), Glöckner V, Abdelmohsen UR, Scheuermayer M, Fischer R, Hentschel U,
(1)University of Würzburg, Research Center for Infectious Diseases,
Josef-Schneider-Str. 2/D15, 97080 Würzburg, Germany.
Following their transmission from the human to the mosquito with the bloodmeal,
malaria parasites have to persevere in the mosquito midgut for approximately 1 d.
During this period the parasites are highly vulnerable to factors of the mosquito
midgut, including bacteria. We here aimed at determining the microbial diversity
of gut bacteria of the Asian malaria vector Anopheles stephensi (Liston) during
development and under different feeding regimes, including feeds on malaria
parasite-infected blood. 16S rRNA and denaturing gradient gel electrophoresis
analyses demonstrated an increasing reduction in the microbial diversity during
mosquito development from egg to adult and identified the gram-negative bacterium
Elizabethkingia meningoseptica King as the dominant species in the midgut of
lab-reared male and female mosquitoes. E. meningoseptica is transmitted between
generations and its predominance in the mosquito midgut was not altered by diet,
when the gut microbiota was compared between sugar-fed and blood-fed female
mosquitoes. Furthermore, feeds on blood infected with malaria parasites did not
impact the presence of E. meningoseptica in the gut. Extracts from cultured E.
meningoseptica were active against gram-positive and negative bacteria and yeast
and against the blood and gametocyte transmission stages of the malaria parasite
Plasmodium falciparum Welch. The antimicrobial and antiplasmodial activities of
E. meningoseptica may account for its dominance in the midgut of the malaria
PMID: 23540130 [PubMed - indexed for MEDLINE]
367. Int Endod J. 2012 Jun;45(6):530-41. doi: 10.1111/j.1365-2591.2011.02006.x. Epub
2012 Jan 17.
Ecology of the microbiome of the infected root canal system: a comparison between
apical and coronal root segments.
Ozok AR(1), Persoon IF, Huse SM, Keijser BJ, Wesselink PR, Crielaard W, Zaura E.
(1)Department of Conservative and Preventive Dentistry, Academic Centre for
Dentistry Amsterdam, University of Amsterdam and VU University, Amsterdam, The
AIM: To evaluate the microbial ecology of the coronal and apical segments of
infected root canal systems using a complete sampling technique and
METHODOLOGY: The roots of 23 extracted teeth with apical periodontitis were
sectioned in half, horizontally, and cryo-pulverized. Bacterial communities were
profiled using tagged 454 pyrosequencing of the 16S rDNA hypervariable V5-V6
RESULTS: The sequences were classified into 606 taxa (species or higher taxon),
representing 24 bacterial phyla or candidate divisions and one archaeal phylum.
Proteobacteria were more abundant in the apical samples (P < 0.05), whilst
Actinobacteria were in significantly higher proportions in the coronal samples.
The apical samples harboured statistically significantly more taxa than the
coronal samples (P = 0.01) and showed a higher microbial diversity. Several taxa
belonging to fastidious obligate anaerobes were significantly more abundant in
the apical segments of the roots compared with their coronal counterparts.
CONCLUSIONS: Endodontic infections are more complex than reported previously. The
apical part of the root canal system drives the selection of a more diverse and
more anaerobic community than the coronal part. The presence of a distinct
ecological niche in the apical region explains the difficulty of eradication of
the infection and emphasizes the need for new treatment approaches to be
© 2012 International Endodontic Journal.
PMID: 22251411 [PubMed - indexed for MEDLINE]
368. BMC Genomics. 2010 Sep 28;11:523. doi: 10.1186/1471-2164-11-523.
Study of inter- and intra-individual variations in the salivary microbiota.
Lazarevic V(1), Whiteson K, Hernandez D, François P, Schrenzel J.
(1)Genomic Research Laboratory, Geneva University Hospitals, Rue
Gabrielle-Perret-Gentil 4, CH-1211 Geneva 14, Switzerland.
BACKGROUND: Oral bacterial communities contain species that promote health and
others that have been implicated in oral and/or systemic diseases.
Culture-independent approaches provide the best means to assess the diversity of
oral bacteria because most of them remain uncultivable.
RESULTS: The salivary microbiota from five adults was analyzed at three
time-points by means of the 454 pyrosequencing technology. The V1-V3 region of
the bacterial 16S rRNA genes was amplified by PCR using saliva lysates and
broad-range primers. The bar-coded PCR products were pooled and sequenced
unidirectionally to cover the V3 hypervariable region. Of 50,708 obtained
sequences, 31,860 passed the quality control. Non-bacterial sequences (2.2%) were
removed leaving 31,170 reads. Samples were dominated by seven major phyla:
members of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and
candidate division TM7 were identified in all samples; Fusobacteria and
Spirochaetes were identified in all individuals, but not at all time-points. The
dataset was represented by 3,011 distinct sequences (100%-ID phylotypes) of ~215
nucleotides and 583 phylotypes defined at ≥97% identity (97%-ID phylotypes). We
compared saliva samples from different individuals in terms of the phylogeny of
their microbial communities. Based on the presence and absence of phylotypes
defined at 100% or 97% identity thresholds, samples from each subject formed
separate clusters. Among individual taxa, phylum Bacteroidetes and order
Clostridiales (Firmicutes) were the best indicators of intraindividual similarity
of the salivary flora over time. Fifteen out of 81 genera constituted 73 to 94%
of the total sequences present in different samples. Of these, 8 were shared by
all time points of all individuals, while 15-25 genera were present in all three
time-points of different individuals. Representatives of the class
Sphingobacteria, order Sphingobacteriales and family Clostridiaceae were found
only in one subject.
CONCLUSIONS: The salivary microbial community appeared to be stable over at least
5 days, allowing for subject-specific grouping using UniFrac. Inclusion of all
available samples from more distant time points (up to 29 days) confirmed this
observation. Samples taken at closer time intervals were not necessarily more
similar than samples obtained across longer sampling times. These results point
to the persistence of subject-specific taxa whose frequency fluctuates between
the time points. Genus Gemella, identified in all time-points of all individuals,
was not defined as a core-microbiome genus in previous studies of salivary
bacterial communities. Human oral microbiome studies are still in their infancy
and larger-scale projects are required to better define individual and universal
oral microbiome core.
PMID: 20920195 [PubMed - indexed for MEDLINE]
Moore JE1, McCalmont M, Xu J, Millar BC, Heaney N.
A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10(6) CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.
PMID: 12147519 PMCID: PMC123994
[PubMed - indexed for MEDLINE] Free PMC Article
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J Clin Microbiol. 2002 Apr;40(4):1511-3.
Peña JA1, McNeil K, Fox JG, Versalovic J.
One hundred twenty-six urease-negative gastric biopsy specimens were evaluated for the presence of Helicobacter genus-specific 16S ribosomal DNA (rDNA) and H. pylori-specific glmM DNA sequences by PCR. The species specificity of the glmM PCR assay was demonstrated, as H. pylori was the only Helicobacter species that yielded the expected glmM amplicon. Most urease-negative specimens (118 of 126 specimens) lacked Helicobacter DNA. However, 8 of 126 urease-negative specimens contained Helicobacter 16S rDNA. In order to identify the Helicobacter species present in urease-negative gastric biopsy specimens, 16S rDNA amplicons were cloned and sequenced. Sequence comparisons were performed by analyses of the sequences in public sequence databases. Two samples contained 16S rDNA that was identified as H. cinaedi with 100% identity and that spanned approximately 400 bp (398 and 398 bp, respectively). In contrast, multiple differences (97% identity; 390 of 398 bp) were observed with H. pylori 16S rDNA in this region. This finding was verified by sequencing an overlapping 537-bp fragment within the 5' portion of 16S rDNA. Although the clinical findings were consistent with H. pylori infection (e.g., duodenal ulcer disease), rapid urease testing and DNA sequence analyses suggested the presence of H. cinaedi organisms and the absence of H. pylori in two human antral biopsy specimens. This study represents the first report of an enteric urease-negative helicobacter in the human stomach. Although these organisms were previously associated with extragastric infections, the roles of these organisms in the pathogenesis of chronic gastritis or peptic ulcer disease remain unclear.
PMID: 11923384 PMCID: PMC140399
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J Clin Microbiol. 2002 Apr;40(4):1333-8.
Drancourt M1, Raoult D.
The complete sequence of rpoB, the gene encoding the beta subunit of RNA polymerase was determined for Staphylococcus saccharolyticus, Staphylococcus lugdunensis, S taphylococcus caprae, and Staphylococcus intermedius and partial sequences were obtained for an additional 27 Staphylococcus species. The complete rpoB sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial rpoB sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal rpoB sequence database we established enabled us to develop a molecular method for identifying Staphylococcus isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10 Staphylococcus isolates, and no positive results were obtained with 10 non-Staphylococcus gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the rpoB gene as a new tool for the accurate identification of Staphylococcus isolates.
PMID: 11923353 PMCID: PMC140360
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Vet Res. 2001 Nov-Dec;32(6):611-6.
Moore JE1, Millar BC, Xu J, Buckley TC.
A wild-type isolate with similar morphological and phenotypic properties to Taylorella equigenitalis, the causative bacterial agent of contagious equine metritis (CEM), was referred for molecular identification by PCR amplification of the 16S rRNA gene. A species-specific PCR failed to yield a product compatible with that of T. equigenitalis. The direct sequencing of the universal 16S rRNA PCR amplicon suggested the presence of a Bacteroides sp., probably Bacteroides ureolyticus, with no consequent effects on the movement and transportation of the animal. Adoption of such a molecular means of identification through sequencing may aid in the identification of the atypical forms of Taylorella equigenitalis, as recently described, as well as differentiating this species from Taylorella asinigenitalis.