A Look Behind the Curtain of 16s Sequencing MR DNA
1. J Clin Microbiol. 2014 Nov;52(11):3913-21. doi: 10.1128/JCM.01678-14. Epub 2014
Single-molecule long-read 16S sequencing to characterize the lung microbiome from
mechanically ventilated patients with suspected pneumonia.
Toma I(1), Siegel MO(2), Keiser J(3), Yakovleva A(4), Kim A(4), Davenport L(5),
Devaney J(5), Hoffman EP(5), Alsubail R(4), Crandall KA(6), Castro-Nallar E(6),
Pérez-Losada M(7), Hilton SK(6), Chawla LS(8), McCaffrey TA(9), Simon GL(10).
(1)Department of Medicine, Division of Genomic Medicine, The George Washington
University School of Medicine and Health Sciences, Washington, DC, USA Department
of Physical Therapy and Health Care Sciences, The George Washington University
School of Medicine and Health Sciences, Washington, DC, USA firstname.lastname@example.org
email@example.com. (2)Division of Infectious Diseases, The George Washington
University School of Medicine and Health Sciences, Washington, DC, USA
firstname.lastname@example.org email@example.com. (3)Department of Pathology, The George
Washington University School of Medicine and Health Sciences, Washington, DC,
USA. (4)Department of Medicine, Division of Genomic Medicine, The George
Washington University School of Medicine and Health Sciences, Washington, DC,
USA. (5)Children's National Medical Research Center, The George Washington
University School of Medicine and Health Sciences, Washington, DC, USA.
(6)Computational Biology Institute, The George Washington University School of
Medicine and Health Sciences, Washington, DC, USA. (7)Computational Biology
Institute, The George Washington University School of Medicine and Health
Sciences, Washington, DC, USA Centro de Investigação em Biodiversidade e Recursos
Genéticos (CIBIO), Vairão, Portugal. (8)Department of Anesthesiology, The George
Washington University School of Medicine and Health Sciences, Washington, DC,
USA. (9)Department of Medicine, Division of Genomic Medicine, The George
Washington University School of Medicine and Health Sciences, Washington, DC, USA
Department of Microbiology, Immunology, and Tropical Medicine, The George
Washington University School of Medicine and Health Sciences, Washington, DC,
USA. (10)Division of Infectious Diseases, The George Washington University School
of Medicine and Health Sciences, Washington, DC, USA.
In critically ill patients, the development of pneumonia results in significant
morbidity and mortality and additional health care costs. The accurate and rapid
identification of the microbial pathogens in patients with pulmonary infections
might lead to targeted antimicrobial therapy with potentially fewer adverse
effects and lower costs. Major advances in next-generation sequencing (NGS) allow
culture-independent identification of pathogens. The present study used NGS of
essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial
aspirates of intubated patients with suspected pneumonia. The results from 61
patients demonstrated that sufficient DNA was obtained from 72% of samples, 44%
of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27
sequenced samples, only 20 had bacterial culture growth, while the
microbiological and NGS identification of bacteria coincided in 17 (85%) of these
samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers
and were sequenced, the NGS identified a number of bacterial species in these
samples. Overall, a significant diversity of bacterial species was identified
from the same genus as the predominant cultured pathogens. The numbers of
NGS-identifiable bacterial genera were consistently higher than identified by
standard microbiological methods. As technical advances reduce the processing and
sequencing times, NGS-based methods will ultimately be able to provide clinicians
with rapid, precise, culture-independent identification of bacterial, fungal, and
viral pathogens and their antimicrobial sensitivity profiles.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID: 25143582 [PubMed - indexed for MEDLINE]
2. Ann Otol Rhinol Laryngol. 2016 Aug;125(8):613-21. doi: 10.1177/0003489416641429.
Epub 2016 Apr 7.
Next Generation Sequencing and the Microbiome of Chronic Rhinosinusitis: A Primer
for Clinicians and Review of Current Research, Its Limitations, and Future
Jervis Bardy J(1), Psaltis AJ(2).
(1)Department of Otolaryngology-Head & Neck Surgery, The Queen Elizabeth Hospital
and The University of Adelaide, Adelaide, South Australia. (2)Department of
Otolaryngology-Head & Neck Surgery, The Queen Elizabeth Hospital and The
University of Adelaide, Adelaide, South Australia firstname.lastname@example.org.
OBJECTIVE: Microbiomics in chronic diseases, including chronic rhinosinusitis
(CRS), have undergone rapid advances in recent times. The introduction of Next
Generation Sequencing (NGS) technology has produced significant clinical insights
regarding the bacteriology of these conditions. We review studies that have used
16S rRNA sequencing to specifically investigate the microbiota profiles of
patients with CRS in a variety of contexts.
METHODS: Literature review using the CINAHL, MEDLINE, PUBMED, and the Cochrane
databases. Papers utilizing 16S-sequencing technology on CRS specimens published
between January 1, 1995, and October 31, 2015, were included. Studies limited to
only healthy controls were excluded.
RESULTS: Consistent with published studies using non-NGS techniques, the main
genera commonly identified from the sinuses of CRS patients included
Staphylococcus, Propionibacterium, and Corynebacterium. The microbiome of CRS
patients had lower bacterial diversity compared to controls in a number of
studies. Also consistent with non-NGS-based studies, Staphylococcus was
implicated as an important genus, with highly colonized patients having worse
surgical outcomes. Conflicting reports of antibiotic effects on the CRS
microbiome were observed. Sampling methods were well investigated, many of the
studies reviewed failed to include important methodological detail.
CONCLUSION: While 16S sequencing is a novel microbiological laboratory method,
current studies have confirmed our existing understanding of bacteriology of CRS
without providing significant additional clinical insight. Complementing 16S
studies with more complex NGS methods while developing robust clinical studies
aimed at shifting the disrupted CRS microbiome will provide researches with the
opportunity to derive further clinical insight and develop new therapeutic
© The Author(s) 2016.
PMID: 27056556 [PubMed - in process]
1. Zhongguo Dang Dai Er Ke Za Zhi. 2014 Nov;16(11):1172-6.
[Dynamic changes of intestinal 16S rDNA metagenome in 5 infants].
[Article in Chinese]
Ma LY(1), Zhang M, Wang HL, Chen R, Huang Y, Liang XQ, Lu GJ.
(1)Department of Neonatology, Shenzhen Bao'an Maternal and Child Health Hospital,
Shenzhen, Guangdong 518133, China. email@example.com.
OBJECTIVE: To investigate the dynamic changes of intestinal 16S rDNA metagenome
in healthy infants.
METHODS: Seventeen fecal samples were collected at ages of 3 days, 1 month, 6
months and 1 year in 5 infants. Total bacterial DNAs were extracted and submitted
high throughout sequencing on the V6 viable region of 16S rDNA. Tags and
Operational Taxonomic Units (OTU) were then obtained and analysis of taxonomy,
abundance and alpha diversity were performed.
RESULTS: In total 2,190.66 Mbp raw data in 17 samples were produced. The OTU
numbers ranged from 36 to 308. The dominate phylum included Proteobacteria,
Firmicutes and Bacteroidetes and Actinobacteria. The bacterial families>1%
increased from only 2-4 per sample on day 3 to 7 at 1 or 6 months, 10 at 12
months. The average npShannon and Simpson index on day 3, at 1 month, 6 months
and 1 year were 1.117, 1.460, 2.088, 2.50 and 0.443, 0.408, 0.229, 0.143
CONCLUSIONS: Infants' intestines harbor abounding bacterial genomes. Distinct
individual differences exist in infants in terms of intestinal bacterial
abundance and composition. The abundance and diversity of gut bacteria increase
PMID: 25406568 [PubMed - indexed for MEDLINE]
2. BMC Genomics. 2015 Oct 23;16:839. doi: 10.1186/s12864-015-2032-0.
The rumen microbial metagenome associated with high methane production in cattle.
Wallace RJ(1), Rooke JA(2), McKain N(3), Duthie CA(4), Hyslop JJ(5), Ross DW(6),
Waterhouse A(7), Watson M(8), Roehe R(9).
(1)Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn,
Aberdeen, AB21 9SB, UK. firstname.lastname@example.org. (2)SRUC, West Mains Road,
Edinburgh, EH9 3JG, UK. John.Rooke@sruc.ac.uk. (3)Rowett Institute of Nutrition
and Health, University of Aberdeen, Bucksburn, Aberdeen, AB21 9SB, UK.
email@example.com. (4)SRUC, West Mains Road, Edinburgh, EH9 3JG, UK.
Carol-Anne.Duthie@sruc.ac.uk. (5)SRUC, West Mains Road, Edinburgh, EH9 3JG, UK.
Jimmy.Hyslop@sac.co.uk. (6)SRUC, West Mains Road, Edinburgh, EH9 3JG, UK.
Dave.Ross@sruc.ac.uk. (7)SRUC, West Mains Road, Edinburgh, EH9 3JG, UK.
Tony.Waterhouse@sruc.ac.uk. (8)Edinburgh Genomics, The Roslin Institute and
R(D)SVS, University of Edinburgh, Easter Bush, Edinburgh, EH25 9RG, UK.
firstname.lastname@example.org. (9)SRUC, West Mains Road, Edinburgh, EH9 3JG, UK.
BACKGROUND: Methane represents 16 % of total anthropogenic greenhouse gas
emissions. It has been estimated that ruminant livestock produce ca. 29 % of this
methane. As individual animals produce consistently different quantities of
methane, understanding the basis for these differences may lead to new
opportunities for mitigating ruminal methane emissions. Metagenomics is a
powerful new tool for understanding the composition and function of complex
microbial communities. Here we have applied metagenomics to the rumen microbial
community to identify differences in the microbiota and metagenome that lead to
high- and low-methane-emitting cattle phenotypes.
METHODS: Four pairs of beef cattle were selected for extreme high and low methane
emissions from 72 animals, matched for breed (Aberdeen-Angus or Limousin cross)
and diet (high or medium concentrate). Community analysis was carried out by qPCR
of 16S and 18S rRNA genes and by alignment of Illumina HiSeq reads to the
GREENGENES database. Total genomic reads were aligned to the KEGG genes
databasefor functional analysis.
RESULTS: Deep sequencing produced on average 11.3 Gb per sample. 16S rRNA gene
abundances indicated that archaea, predominantly Methanobrevibacter, were 2.5×
more numerous (P = 0.026) in high emitters, whereas among bacteria
Proteobacteria, predominantly Succinivibrionaceae, were 4-fold less abundant (2.7
vs. 11.2 %; P = 0.002). KEGG analysis revealed that archaeal genes leading
directly or indirectly to methane production were 2.7-fold more abundant in high
emitters. Genes less abundant in high emitters included acetate kinase, electron
transport complex proteins RnfC and RnfD and glucose-6-phosphate isomerase.
Sequence data were assembled de novo and over 1.5 million proteins were annotated
on the subsequent metagenome scaffolds. Less than half of the predicted genes
matched matched a domain within Pfam. Amongst 2774 identified proteins of the 20
KEGG orthologues that correlated with methane emissions, only 16 showed 100 %
identity with a publicly available protein sequence.
CONCLUSIONS: The abundance of archaeal genes in ruminal digesta correlated
strongly with differing methane emissions from individual animals, a finding
useful for genetic screening purposes. Lower emissions were accompanied by higher
Succinovibrionaceae abundance and changes in acetate and hydrogen production
leading to less methanogenesis, as similarly postulated for Australian macropods.
Large numbers of predicted protein sequences differed between high- and
low-methane-emitting cattle. Ninety-nine percent were unknown, indicating a
fertile area for future exploitation.
PMID: 26494241 [PubMed - indexed for MEDLINE]
3. J Infect Public Health. 2016 May-Jun;9(3):362-5. doi: 10.1016/j.jiph.2015.11.008.
Epub 2015 Dec 3.
First microbiota assessments of children's paddling pool waters evaluated using
16S rRNA gene-based metagenome analysis.
Sawabe T(1), Suda W(2), Ohshima K(3), Hattori M(4), Sawabe T(5).
(1)Department of Food and Nutrition, Hakodate Junior College, 52-1, Takaoka-cho,
Hakodate 042-0955, Japan. Electronic address: email@example.com.
(2)Laboratory of Metagenomics, Graduate School of Frontier Sciences, The
University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8561, Japan;
Department of Microbiology and Immunology, Keio University School of Medicine, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. (3)Laboratory of Metagenomics,
Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5,
Kashiwanoha, Kashiwa, Chiba 277-8561, Japan. (4)Laboratory of Metagenomics,
Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5,
Kashiwanoha, Kashiwa, Chiba 277-8561, Japan; Cooperative Major in Advanced Health
Science, Graduate School of Advanced Science and Engineering, Waseda University,
3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan. (5)Laboratory of Microbiology,
Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate
Insufficient chloric sterilization of children's paddling pool waters increases
the risk of diarrheal illness. Therefore, we investigated the microbiota changes
after children use pools. First, we applied 16S rRNA gene-based metagenome
analysis to understand the dynamics of microbiota in pool water, especially with
respect to the bio-contamination by potential pathogens. Proteobacteria were
major taxa detected in every pool water sample after children spent time in the
pool. In more detail, Gammaproteobacteria comprised the dominant class, which was
followed by Betaproteobacteria. Five phyla, Bacteroidetes, Firmicutes,
Actinobacteria and Deinococcus-Thermus phyla were minor groups. The pool water
microbiota are likely to be a consortium of intestinal and skin microbiota from
humans. Interestingly, the ratio of Gammaproteobacteria and Betaproteobacteria
differed according to the age of the children who used the pool, which means the
pool water was additionally contaminated by soil microbiota as a result of the
children's behavior. Furthermore, potential pathogens, such as Campylobacter
spp., Comamonas testosteroni and Burkholderia pseudomallei, were also found.
Considering the standard plate counts, the abundances of these human pathogens
are unlikely to be a sufficiently infectious dose. We suggest the importance of
sanitary measures in paddling pool waters to reduce bio-contamination from both
humans and the environment.
Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences.
Published by Elsevier Ltd. All rights reserved.
PMID: 26671497 [PubMed - in process]
4. Am J Gastroenterol. 2015 Dec;110(12):1718-29; quiz 1730. doi:
10.1038/ajg.2015.357. Epub 2015 Nov 3.
Extensive Modulation of the Fecal Metagenome in Children With Crohn's Disease
During Exclusive Enteral Nutrition.
Quince C(1), Ijaz UZ(2), Loman N(3), Eren AM(4), Saulnier D(5), Russell J(2),
Haig SJ(2), Calus ST(3), Quick J(3), Barclay A(6), Bertz M(5), Blaut M(5), Hansen
R(6), McGrogan P(6), Russell RK(6), Edwards CA(7), Gerasimidis K(7).
(1)Warwick Medical School, University of Warwick, Warwick, UK. (2)School of
Engineering, University of Glasgow, Glasgow, UK. (3)Institute of Microbiology and
Infection, University of Birmingham, Birmingham, UK. (4)Josephine Bay Paul Center
for Comparative Molecular Biology and Evolution, Marine Biological Laboratory,
Woods Hole, Massachusetts, USA. (5)Department of Gastrointestinal Microbiology,
German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee,
Nuthetal, Germany. (6)Department of Paediatric Gastroenterology, Hepatology and
Nutrition, Royal Hospital for Children, Glasgow, UK. (7)Human Nutrition, School
of Medicine, College of Medical, Veterinary and Life Sciences, University of
Glasgow, Glasgow Royal Infirmary, Glasgow, UK.
OBJECTIVES: Exploring associations between the gut microbiota and colonic
inflammation and assessing sequential changes during exclusive enteral nutrition
(EEN) may offer clues into the microbial origins of Crohn's disease (CD).
METHODS: Fecal samples (n=117) were collected from 23 CD and 21 healthy children.
From CD children fecal samples were collected before, during EEN, and when
patients returned to their habitual diets. Microbiota composition and functional
capacity were characterized using sequencing of the 16S rRNA gene and shotgun
RESULTS: Microbial diversity was lower in CD than controls before EEN (P=0.006);
differences were observed in 36 genera, 141 operational taxonomic units (OTUs),
and 44 oligotypes. During EEN, the microbial diversity of CD children further
decreased, and the community structure became even more dissimilar than that of
controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34
genera decreased and one increased during EEN. Fecal calprotectin correlated with
35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated
positively or negatively with calprotectin decreased during EEN. The microbiota
of CD patients had a broader functional capacity than healthy controls, but
diversity decreased with EEN. Genes involved in membrane transport, sulfur
reduction, and nutrient biosynthesis differed between patients and controls. The
abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis
decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis
(P=0.031), or the shikimate pathway (P=0.058), increased during EEN.
CONCLUSIONS: Disease improvement following treatment with EEN is associated with
extensive modulation of the gut microbiome.
PMID: 26526081 [PubMed - indexed for MEDLINE]
5. Microbiology. 2012 Nov;158(Pt 11):2789-95. doi: 10.1099/mic.0.057943-0. Epub 2012
Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA
genes in the viral metagenome of the marine sponge Hymeniacidon perlevis.
Harrington C(1), Del Casale A, Kennedy J, Neve H, Picton BE, Mooij MJ, O'Gara F,
Kulakov LA, Larkin MJ, Dobson AD.
(1)Marine Biotechnology Centre, Environmental Research Institute, University
College Cork, Cork, Ireland.
Marine sponges have never been directly examined with respect to the presence of
viruses or their potential involvement in horizontal gene transfer. Here we
demonstrate for the first time, to our knowledge, the presence of viruses in the
marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in
DNA isolated from these viruses, indicating that phage-derived transduction
appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial
16S rDNA isolated from sponge-derived viral and total DNA differed significantly,
indicating that not all species are equally involved in transduction.
PMID: 22902729 [PubMed - indexed for MEDLINE]
6. Bioinformatics. 2015 Sep 1;31(17):2882-4. doi: 10.1093/bioinformatics/btv287.
Epub 2015 May 7.
Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data.
Aßhauer KP(1), Wemheuer B(2), Daniel R(2), Meinicke P(1).
(1)Department of Bioinformatics and. (2)Department of Genomic and Applied
Microbiology and Göttingen Genomics Laboratory, Institute of Microbiology and
Genetics, Georg-August-University Göttingen, 37077 Göttingen, Germany.
MOTIVATION: The characterization of phylogenetic and functional diversity is a
key element in the analysis of microbial communities. Amplicon-based sequencing
of marker genes, such as 16S rRNA, is a powerful tool for assessing and comparing
the structure of microbial communities at a high phylogenetic resolution. Because
16S rRNA sequencing is more cost-effective than whole metagenome shotgun
sequencing, marker gene analysis is frequently used for broad studies that
involve a large number of different samples. However, in comparison to shotgun
sequencing approaches, insights into the functional capabilities of the community
get lost when restricting the analysis to taxonomic assignment of 16S rRNA data.
RESULTS: Tax4Fun is a software package that predicts the functional capabilities
of microbial communities based on 16S rRNA datasets. We evaluated Tax4Fun on a
range of paired metagenome/16S rRNA datasets to assess its performance. Our
results indicate that Tax4Fun provides a good approximation to functional
profiles obtained from metagenomic shotgun sequencing approaches.
AVAILABILITY AND IMPLEMENTATION: Tax4Fun is an open-source R package and
applicable to output as obtained from the SILVAngs web server or the application
of QIIME with a SILVA database extension. Tax4Fun is freely available for
download at http://tax4fun.gobics.de/.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
© The Author 2015. Published by Oxford University Press.
PMID: 25957349 [PubMed - indexed for MEDLINE]
7. BMC Microbiol. 2015 Aug 12;15:160. doi: 10.1186/s12866-015-0497-2.
Cilantro microbiome before and after nonselective pre-enrichment for Salmonella
using 16S rRNA and metagenomic sequencing.
Jarvis KG(1), White JR(2), Grim CJ(3,)(4), Ewing L(5), Ottesen AR(6), Beaubrun
JJ(7), Pettengill JB(8), Brown E(9), Hanes DE(10).
(1)U. S. Food and Drug Administration, Center for Food Safety and Applied
Nutrition, OARSA, Laurel, MD, USA. firstname.lastname@example.org. (2)Oak Ridge
Institute for Science and Technology, Oak Ridge, TN, USA.
email@example.com. (3)U. S. Food and Drug Administration, Center for Food
Safety and Applied Nutrition, OARSA, Laurel, MD, USA.
Christopher.Grim@fda.hhs.gov. (4)Oak Ridge Institute for Science and Technology,
Oak Ridge, TN, USA. Christopher.Grim@fda.hhs.gov. (5)U. S. Food and Drug
Administration, Center for Food Safety and Applied Nutrition, OARSA, Laurel, MD,
USA. Laura.Ewing-Peeples@fda.hhs.gov. (6)U. S. Food and Drug Administration,
Center for Food Safety and Applied Nutrition, ORS, College Park, MD, USA.
Andrea.Ottesen@fda.hhs.gov. (7)U. S. Food and Drug Administration, Center for
Food Safety and Applied Nutrition, OARSA, Laurel, MD, USA.
Junia.Jean-GillesBeaubrun@fda.hhs.gov. (8)U. S. Food and Drug Administration,
Center for Food Safety and Applied Nutrition, ORS, College Park, MD, USA.
James.Pettengill@fda.hhs.gov. (9)U. S. Food and Drug Administration, Center for
Food Safety and Applied Nutrition, ORS, College Park, MD, USA.
Eric.Brown@fda.hhs.gov. (10)U. S. Food and Drug Administration, Center for Food
Safety and Applied Nutrition, OARSA, Laurel, MD, USA. Darcy.Hanes@fda.hhs.gov.
BACKGROUND: Salmonella enterica is a common cause of foodborne gastroenteritis in
the United States and is associated with outbreaks in fresh produce such as
cilantro. Salmonella culture-based detection methods are complex and time
consuming, and improvments to increase detection sensitivity will benefit
consumers. In this study, we used 16S rRNA sequencing to determine the microbiome
of cilantro. We also investigated changes to the microbial community prior to and
after a 24-hour nonselective pre-enrichment culture step commonly used by
laboratory analysts to resuscitate microorganisms in foods suspected of
contamination with pathogens. Cilantro samples were processed for Salmonella
detection according to the method in the United States Food and Drug
Administration Bacteriological Analytical Manual. Genomic DNA was extracted from
culture supernatants prior to and after a 24-hour nonselective pre-enrichment
step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A
database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen
the libraries for Salmonella, as some samples were known to be culture positive.
Additionally, culture positive cilantro samples were examined for the presence of
Salmonella using shotgun metagenomics on the Illumina MiSeq.
RESULTS: Time zero uncultured samples had an abundance of Proteobacteria while
the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes.
Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples
revealed variable degrees of Salmonella contamination among the sequenced
CONCLUSIONS: Our cilantro study demonstrates the use of high-throughput
sequencing to reveal the microbiome of cilantro, and how the microbiome changes
during the culture-based protocols employed by food safety laboratories to detect
foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to
a predominance of Firmicutes suggests that changing our culture-based methods
will improve detection sensitivity for foodborne enteric pathogens.
PMID: 26264042 [PubMed - indexed for MEDLINE]
8. Sci Rep. 2015 Sep 16;5:14181. doi: 10.1038/srep14181.
Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial
Boers SA(1), Hays JP(1), Jansen R(2).
(1)Department of Medical Microbiology and Infectious Diseases, Erasmus University
Medical Centre, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands. (2)Department
of Molecular Biology, Regional Laboratory of Public Health Kennemerland,
Boerhaavelaan 26, 2035 RC, Haarlem, The Netherlands.
16S rRNA gene profiling has revolutionized the field of microbial ecology. Many
researchers in various fields have embraced this technology to investigate
bacterial compositions of samples derived from many different ecosystems.
However, it is important to acknowledge the current limitations and drawbacks of
16S rRNA gene profiling. Although sample handling, DNA extraction methods and the
choice of universal 16S rRNA gene PCR primers are well known factors that could
seriously affect the final results of microbiota profiling studies, inevitable
amplification artifacts, such as chimera formation and PCR competition, are
seldom appreciated. Here we report on a novel micelle based amplification
strategy, which overcomes these limitations via the clonal amplification of
targeted DNA molecules. Our results show that micelle PCR drastically reduces
chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional
PCR, resulting in improved microbial diversity estimates. In addition,
compartmentalization during micelle PCR prevents PCR competition due to unequal
amplification rates of different 16S template molecules, generating robust and
accurate 16S microbiota profiles required for comparative studies (e.g.
PMID: 26373611 [PubMed - indexed for MEDLINE]
9. MBio. 2014 Sep 30;5(5):e01631-14. doi: 10.1128/mBio.01631-14.
Metagenome-wide association of microbial determinants of host phenotype in
Chaston JM(1), Newell PD(2), Douglas AE.
(1)Department of Entomology, Cornell University, Ithaca, New York, USA
firstname.lastname@example.org. (2)Department of Entomology, Cornell University, Ithaca,
New York, USA.
Animal-associated bacteria (microbiota) affect host behaviors and physiological
traits. To identify bacterial genetic determinants of microbiota-responsive host
traits, we employed a metagenome-wide association (MGWA) approach in two steps.
First, we measured two microbiota-responsive host traits, development time and
triglyceride (TAG) content, in Drosophila melanogaster flies monoassociated with
each of 41 bacterial strains. The effects of monoassociation on host traits were
not confined to particular taxonomic groups. Second, we clustered protein-coding
sequences of the bacteria by sequence similarity de novo and statistically
associated the magnitude of the host trait with the bacterial gene contents. The
animals had been monoassociated with genome-sequenced bacteria, so the metagenome
content was unambiguous. This analysis showed significant effects of
pyrroloquinoline quinone biosynthesis genes on development time, confirming the
results of a published transposon mutagenesis screen, thereby validating the
MGWA; it also identified multiple genes predicted to affect host TAG content,
including extracellular glucose oxidation pathway components. To test the
validity of the statistical associations, we expressed candidate genes in a
strain that lacks them. Monoassociation with bacteria that ectopically expressed
a predicted oxidoreductase or gluconate dehydrogenase conferred reduced
Drosophila TAG contents relative to the TAG contents in empty vector controls.
Consistent with the prediction that glucose oxidation pathway gene expression
increased bacterial glucose utilization, the glucose content of the host diet was
reduced when flies were exposed to these strains. Our findings indicate that
microbiota affect host nutritional status through modulation of nutrient
acquisition. Together, these findings demonstrate the utility of MGWA for
identifying bacterial determinants of host traits and provide mechanistic insight
into how gut microbiota modulate the nutritional status of a model
host.IMPORTANCE: To understand how certain gut bacteria promote the health of
their animal hosts, we need to identify the bacterial genes that drive these
beneficial relationships. This task is challenging because the bacterial
communities can vary widely among different host individuals. To overcome this
difficulty, we quantified how well each of 41 bacterial species protected
Drosophila fruit flies from high fat content. The genomes of the chosen bacterial
strains were previously sequenced, so we could statistically associate specific
bacterial genes with bacterially mediated reduction in host fat content.
Bacterial genes that promote glucose utilization were strongly represented in the
association, and introducing these genes into the gut bacteria was sufficient to
lower the animal's fat content. Our method is applicable to the study of many
other host-microbe interactions as a way to uncover microbial genes important for
Copyright © 2014 Chaston et al.
PMID: 25271286 [PubMed - indexed for MEDLINE]
10. J Biotechnol. 2013 Sep 10;167(3):334-43. doi: 10.1016/j.jbiotec.2013.07.021. Epub
2013 Jul 20.
Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri
CD034 on the microbial community involved in grass ensiling.
Eikmeyer FG(1), Köfinger P, Poschenel A, Jünemann S, Zakrzewski M, Heinl S,
Mayrhuber E, Grabherr R, Pühler A, Schwab H, Schlüter A.
(1)Institute for Genome Research and Systems Biology, Center for Biotechnology,
Bielefeld University, D-33594 Bielefeld, Germany.
Silage is green fodder conserved by lactic acid fermentation performed by
epiphytic lactic acid bacteria under anaerobic conditions. To improve the
ensiling process and the quality of the resulting silage, starter cultures are
added to the fresh forage. A detailed analysis of the microbial community playing
a role in grass ensiling has been carried out by high throughput sequencing
technologies. Moreover, the influence of the inoculant Lactobacillus buchneri
CD034 on the microbial community composition was studied. For this purpose, grass
was ensiled untreated or inoculated with L. buchneri CD034. The fresh forage as
well as silages after 14 and 58 days of fermentation were characterized
physico-chemically. Characteristic silage conditions such as increased titers of
lactic acid bacteria and higher concentrations of acetic acid were observed in
the inoculated silage in comparison to the untreated samples. Taxonomic community
profiles deduced from 16S rDNA amplicon sequences indicated that the relative
abundance of Lactococci diminished in the course of fermentations and that the
proportion of bacteria belonging to the phyla Proteobacteria and Bacteroidetes
increased during the fermentation of untreated silage. In the inoculated silage,
members of these phyla were repressed due to an increased abundance of
Lactobacilli. In addition, metagenome analyses of silage samples confirmed
taxonomic profiles based on 16S rDNA amplicons. Moreover, Lactobacillus
plantarum, Lactobacillus brevis and Lactococcus lactis were found to be dominant
species within silages as analyzed by means of fragment recruitments of
metagenomic sequence reads on complete reference genome sequences. Fragment
recruitments also provided clear evidence for the competitiveness of the
inoculant strain L. buchneri CD034 during the fermentation of the inoculated
silage. The inoculation strain was able to outcompete other community members and
also affected physico-chemical characteristics of the silage.
Copyright © 2013 Elsevier B.V. All rights reserved.
PMID: 23880441 [PubMed - indexed for MEDLINE]
11. Anaerobe. 2015 Jun;33:1-7. doi: 10.1016/j.anaerobe.2015.01.004. Epub 2015 Jan 16.
High-throughput 16S rRNA gene sequencing reveals alterations of mouse intestinal
microbiota after radiotherapy.
Kim YS(1), Kim J(2), Park SJ(3).
(1)Department of Radiation Oncology, Jeju National University Hospital, Aran
13gil 15, Jeju 650-756, Republic of Korea. (2)Department of Anatomy, Jeju
National University School of Medicine, 102 Jejudaehak-ro, Jeju 650-756, Republic
of Korea; Department of Biomedicine & Drug Development, Jeju National University,
102 Jejudaehak-ro, Jeju 650-756, Republic of Korea. (3)Department of Biology,
Jeju National University, 102 Jejudaehak-ro, Jeju 650-756, Republic of Korea.
Electronic address: email@example.com.
The mammalian gastrointestinal tract harbors a highly complex microbial community
that comprises hundreds of different types of bacterial cells. The
gastrointestinal microbiota plays an important role in the function of the host
intestine. Most cancer patients undergoing pelvic irradiation experience side
effects such as diarrhea; however, little is currently known about the effects of
irradiation on the microorganisms colonizing the mucosal surfaces of the
gastrointestinal tract. The aim of this study was to investigate the effects of
gamma irradiation on the compositions of the large and small intestinal
microbiotas. The gut microbiotas in control mice and mice receiving irradiation
treatment were characterized by high-throughput sequencing of the bacterial 16S
rRNA gene. Irradiation treatment induced significant alterations in the bacterial
compositions of the large and small intestines at the genus level. Unexpectedly,
irradiation treatment increased the number of operational taxonomic units in the
small intestine but not the large intestine. In particular, irradiation treatment
increased the level of the genera Alistipes in the large intestine and increased
the level of the genus Corynebacterium in the small intestine. By contrast,
compared with that in the corresponding control group, the level of the genera
Prevotella was lower in the irradiated large intestine, and the level of the
genera Alistipes was lower in the irradiated small intestine. Overall, the data
presented here reveal the potential microbiological effects of pelvic irradiation
on the gastrointestinal tracts of cancer patients.
Copyright © 2015 Elsevier Ltd. All rights reserved.
PMID: 25600706 [PubMed - indexed for MEDLINE]
12. BMC Microbiol. 2013 May 25;13:116. doi: 10.1186/1471-2180-13-116.
Human milk metagenome: a functional capacity analysis.
Ward TL(1), Hosid S, Ioshikhes I, Altosaar I.
(1)Department of Biochemistry, Microbiology and Immunology, University of Ottawa,
Ottawa, ON K1H 8M5, Canada.
BACKGROUND: Human milk contains a diverse population of bacteria that likely
influences colonization of the infant gastrointestinal tract. Recent studies,
however, have been limited to characterization of this microbial community by 16S
rRNA analysis. In the present study, a metagenomic approach using Illumina
sequencing of a pooled milk sample (ten donors) was employed to determine the
genera of bacteria and the types of bacterial open reading frames in human milk
that may influence bacterial establishment and stability in this primal food
matrix. The human milk metagenome was also compared to that of breast-fed and
formula-fed infants' feces (n = 5, each) and mothers' feces (n = 3) at the phylum
level and at a functional level using open reading frame abundance. Additionally,
immune-modulatory bacterial-DNA motifs were also searched for within human milk.
RESULTS: The bacterial community in human milk contained over 360 prokaryotic
genera, with sequences aligning predominantly to the phyla of Proteobacteria
(65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus
(33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs,
30,128 open reading frames were annotated and assigned to functional categories.
When compared to the metagenome of infants' and mothers' feces, the human milk
metagenome was less diverse at the phylum level, and contained more open reading
frames associated with nitrogen metabolism, membrane transport and stress
response (P < 0.05). The human milk metagenome also contained a similar
occurrence of immune-modulatory DNA motifs to that of infants' and mothers' fecal
CONCLUSIONS: Our results further expand the complexity of the human milk
metagenome and enforce the benefits of human milk ingestion on the microbial
colonization of the infant gut and immunity. Discovery of immune-modulatory
motifs in the metagenome of human milk indicates more exhaustive analyses of the
functionality of the human milk metagenome are warranted.
PMID: 23705844 [PubMed - indexed for MEDLINE]
13. Sci Rep. 2014 May 29;4:5100. doi: 10.1038/srep05100.
Impact of treatment strategies on cephalosporin and tetracycline resistance gene
quantities in the bovine fecal metagenome.
Kanwar N(1), Scott HM(2), Norby B(3), Loneragan GH(4), Vinasco J(1), Cottell
JL(5), Chalmers G(5), Chengappa MM(1), Bai J(1), Boerlin P(5).
(1)Department of Diagnostic Medicine/Pathobiology, College of Veterinary
Medicine, Kansas State University, Manhattan, KS 66506, USA. (2)1] Department of
Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State
University, Manhattan, KS 66506, USA . (3)Department of Large Animal Clinical
Sciences, College of Veterinary Medicine, Michigan State University, East
Lansing, MI 48824, USA. (4)Department of Animal and Food Sciences, College of
Agriculture and Natural resources, Texas Tech University, Lubbock, TX 79409, USA.
(5)Department of Pathobiology, Ontario Veterinary College, University of Guelph,
Guelph, Ontario N1G 2W1, Canada.
The study objective was to determine the effects of two treatment regimens on
quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The
two regimens were ceftiofur crystalline-free acid (CCFA) administered to either
one or all steers within a pen and subsequent feeding/not feeding of therapeutic
doses of chlortetracycline. A 26-day randomized controlled field trial was
conducted on 176 steers. Real-time PCR was used to quantify bla(CMY-2),
bla(CTX-M), tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community
DNA. A significant increase in ceftiofur resistance and a decrease in
tetracycline resistance elements were observed among the treatment groups in
which all steers received CCFA treatment, expressed as gene copies/gram of feces.
Subsequent chlortetracycline administration led to rapid expansion of both
ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest
that chlortetracycline is contraindicated when attempting to avoid expansion of
resistance to critically important third-generation cephalosporins.
PMID: 24872333 [PubMed - indexed for MEDLINE]
14. ISME J. 2014 Mar;8(3):650-9. doi: 10.1038/ismej.2013.174. Epub 2013 Oct 10.
Genetic and functional properties of uncultivated MCG archaea assessed by
metagenome and gene expression analyses.
Meng J(1), Xu J(1), Qin D(1), He Y(1), Xiao X(1), Wang F(2).
(1)State Key Laboratory of Microbial Metabolism and State Key Laboratory of Ocean
Engineering, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
(2)1] State Key Laboratory of Microbial Metabolism and State Key Laboratory of
Ocean Engineering, Shanghai Jiao Tong University, Shanghai, People's Republic of
China  Key Laboratory of Systems Biomedicine, Ministry of Education, Shanghai
Jiao Tong University, Shanghai, People's Republic of China.
The Miscellaneous Crenarchaeota group (MCG) Archaea is one of the predominant
archaeal groups in anoxic environments and may have significant roles in the
global biogeochemical cycles. However, no isolate of MCG has been cultivated or
characterized to date. In this study, we investigated the genetic organization,
ecophysiological properties and evolutionary relationships of MCG archaea with
other archaeal members using metagenome information and the result of gene
expression experiments. A comparison of the gene organizations and similarities
around the 16S rRNA genes from all available MCG fosmid and cosmid clones
revealed no significant synteny among genomic fragments, demonstrating that there
are large genetic variations within members of the MCG. Phylogenetic analyses of
large-subunit+small-subunit rRNA, concatenated ribosomal protein genes and
topoisomerases IB gene (TopoIB) all demonstrate that MCG constituted a sister
lineage to the newly proposed archaeal phylum Aigarchaeota and Thaumarchaeota.
Genes involved in protocatechuate degradation and chemotaxis were found in a MCG
fosmid 75G8 genome fragment, suggesting that this MCG member may have a role in
the degradation of aromatic compounds. Moreover, the expression of a putative
4-carboxymuconolactone decarboxylase was observed when the sediment was
supplemented with protocatechuate, further supporting the hypothesis that this
MCG member degrades aromatic compounds.
PMID: 24108328 [PubMed - indexed for MEDLINE]
15. J Bioinform Comput Biol. 2014 Apr;12(2):1441003. doi: 10.1142/S0219720014410030.
Epub 2014 Mar 6.
Comparative metagenome analysis of an Alaskan glacier.
Choudhari S(1), Lohia R, Grigoriev A.
(1)Center for Computational and Integrative Biology, Rutgers University, Camden,
315 Penn St, Camden NJ 08102, USA.
The temperature in the Arctic region has been increasing in the recent past
accompanied by melting of its glaciers. We took a snapshot of the current
microbial inhabitation of an Alaskan glacier (which can be considered as one of
the simplest possible ecosystems) by using metagenomic sequencing of 16S rRNA
recovered from ice/snow samples. Somewhat contrary to our expectations and
earlier estimates, a rich and diverse microbial population of more than 2,500
species was revealed including several species of Archaea that has been
identified for the first time in the glaciers of the Northern hemisphere. The
most prominent bacterial groups found were Proteobacteria, Bacteroidetes, and
Firmicutes. Firmicutes were not reported in large numbers in a previously studied
Alpine glacier but were dominant in an Antarctic subglacial lake. Representatives
of Cyanobacteria, Actinobacteria and Planctomycetes were among the most numerous,
likely reflecting the dependence of the ecosystem on the energy obtained through
photosynthesis and close links with the microbial community of the soil.
Principal component analysis (PCA) of nucleotide word frequency revealed distinct
sequence clusters for different taxonomic groups in the Alaskan glacier community
and separate clusters for the glacial communities from other regions of the
world. Comparative analysis of the community composition and bacterial diversity
present in the Byron glacier in Alaska with other environments showed larger
overlap with an Arctic soil than with a high Arctic lake, indicating patterns of
community exchange and suggesting that these bacteria may play an important role
in soil development during glacial retreat.
PMID: 24712530 [PubMed - indexed for MEDLINE]
16. PLoS One. 2016 Jan 19;11(1):e0146939. doi: 10.1371/journal.pone.0146939.
Characterization of Bacterial Communities in Selected Smokeless Tobacco Products
Using 16S rDNA Analysis.
Tyx RE(1), Stanfill SB(1), Keong LM(1,)(2), Rivera AJ(1,)(3), Satten GA(4),
(1)Division of Laboratory Sciences at the Centers for Disease Control and
Prevention, Atlanta, GA, United States of America. (2)Battelle Analytical
Services, Atlanta, GA, United States of America. (3)Oak Ridge Institute of
Science and Education, Oak Ridge, TN, United States of America. (4)Division of
Reproductive Health, Center for Disease Control and Prevention, Atlanta, GA,
United States of America.
The bacterial communities present in smokeless tobacco (ST) products have not
previously reported. In this study, we used Next Generation Sequencing to study
the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak.
Sample diversity and taxonomic abundances were investigated in these products. A
total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes,
Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff
products contained a diverse distribution of all four phyla. Moist snuff products
were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and
Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program
PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of
Unobserved States) was used to impute the prevalence of genes encoding selected
bacterial toxins, antibiotic resistance genes and other pro-inflammatory
molecules. PICRUSt also predicted the presence of specific nitrate reductase
genes, whose products can contribute to the formation of carcinogenic
nitrosamines. Characterization of microbial community abundances and their
associated genomes gives us an indication of the presence or absence of pathways
of interest and can be used as a foundation for further investigation into the
unique microbiological and chemical environments of smokeless tobacco products.
PMID: 26784944 [PubMed - indexed for MEDLINE]
17. PLoS One. 2014 Apr 8;9(4):e93827. doi: 10.1371/journal.pone.0093827. eCollection
Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing
temporal microbial community dynamics.
Poretsky R(1), Rodriguez-R LM(2), Luo C(2), Tsementzi D(1), Konstantinidis KT(3).
(1)School of Civil and Environmental Engineering, Georgia Institute of
Technology, Atlanta, Georgia, United States of America. (2)School of Biology,
Georgia Institute of Technology, Atlanta, Georgia, United States of America;
Center for Bioinformatics Computational Genomics, Georgia Institute of
Technology, Atlanta, Georgia, United States of America. (3)School of Civil and
Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia,
United States of America; School of Biology, Georgia Institute of Technology,
Atlanta, Georgia, United States of America; Center for Bioinformatics
Computational Genomics, Georgia Institute of Technology, Atlanta, Georgia, United
States of America.
This study explored the short-term planktonic microbial community structure and
resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical
aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence
data. 16S rRNA gene amplicons generated from four temporally discrete samples
were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each
sample and representing ∼300 observed OTUs. Replicates obtained from the same
biological sample clustered together but several biases were observed, linked to
either the PCR or sequencing-preparation steps. In comparisons with companion
whole-community shotgun metagenome datasets, the estimated number of OTUs at each
timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera,
respectively, were identified in the metagenomes. Our analyses showed that the
16S rRNA gene captures broad shifts in community diversity over time, but with
limited resolution and lower sensitivity compared to metagenomic data. We also
identified OTUs that showed marked shifts in abundance over four close timepoints
separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA
amplicon data. A strong summer storm had less of an effect on community
composition than did seasonal mixing, which revealed a distinct succession of
organisms. This study provides insights into freshwater microbial communities and
advances the approaches for assessing community diversity and dynamics in situ.
PMID: 24714158 [PubMed - indexed for MEDLINE]
18. Joint Bone Spine. 2013 Jul;80(4):349-52. doi: 10.1016/j.jbspin.2013.02.005. Epub
2013 Jun 24.
Gut metagenome and spondyloarthritis.
Schaeverbeke T, Truchetet ME, Richez C.
PMID: 23806346 [PubMed - indexed for MEDLINE]
19. Front Microbiol. 2016 Aug 23;7:1297. doi: 10.3389/fmicb.2016.01297. eCollection
Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community
Structures Showed High Reproducibility within and Low Comparability between
Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.
Fischer MA(1), Güllert S(2), Neulinger SC(3), Streit WR(2), Schmitz RA(1).
(1)Department of Biology, Institute for General Microbiology,
Christian-Albrechts-Universität zu Kiel Kiel, Germany. (2)Biozentrum Klein
Flottbek, Institute of Microbiology & Biotechnology, Universität Hamburg Hamburg,
Germany. (3)Department of Biology, Institute for General Microbiology,
Christian-Albrechts-Universität zu KielKiel, Germany; omics2view.consulting
The application of next-generation sequencing technology in microbial community
analysis increased our knowledge and understanding of the complexity and
diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal
domain was often not particularly addressed in the analysis of microbial
communities. Consequently, established primers specifically amplifying the
archaeal 16S ribosomal gene region are scarce compared to the variety of primers
targeting bacterial sequences. In this study, we aimed to validate archaeal
primers suitable for high throughput next generation sequencing. Three archaeal
16S primer pairs as well as two bacterial and one general microbial 16S primer
pairs were comprehensively tested by in-silico evaluation and performing an
experimental analysis of a complex microbial community of a biogas reactor. The
results obtained clearly demonstrate that comparability of community profiles
established using different primer pairs is difficult. 16S rRNA gene data derived
from a shotgun metagenome of the same reactor sample added an additional
perspective on the community structure. Furthermore, in-silico evaluation of
primers, especially those for amplification of archaeal 16S rRNA gene regions,
does not necessarily reflect the results obtained in experimental approaches. In
the latter, archaeal primer pair ArchV34 showed the highest similarity to the
archaeal community structure compared to observed by the metagenomic approach and
thus appears to be the appropriate for analyzing archaeal communities in biogas
reactors. However, a disadvantage of this primer pair was its low specificity for
the archaeal domain in the experimental application leading to high amounts of
bacterial sequences within the dataset. Overall our results indicate a rather
limited comparability between community structures investigated and determined
using different primer pairs as well as between metagenome and 16S rRNA gene
amplicon based community structure analysis. This finding, previously shown for
Bacteria, was as well observed for the archaeal domain.
PMID: 27602022 [PubMed]
20. Environ Microbiol. 2013 May;15(5):1275-89. doi: 10.1111/j.1462-2920.2012.02774.x.
Epub 2012 May 9.
The metagenome of the marine anammox bacterium 'Candidatus Scalindua profunda'
illustrates the versatility of this globally important nitrogen cycle bacterium.
van de Vossenberg J(1), Woebken D, Maalcke WJ, Wessels HJ, Dutilh BE, Kartal B,
Janssen-Megens EM, Roeselers G, Yan J, Speth D, Gloerich J, Geerts W, van der
Biezen E, Pluk W, Francoijs KJ, Russ L, Lam P, Malfatti SA, Tringe SG, Haaijer
SC, Op den Camp HJ, Stunnenberg HG, Amann R, Kuypers MM, Jetten MS.
(1)Department of Microbiology, IWWR, Radboud University Nijmegen, 6525 AJ
Nijmegen, the Netherlands.
Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant
portion of the loss of fixed nitrogen from the oceans, making them important
players in the global nitrogen cycle. To date, marine anammox bacteria found in
marine water columns and sediments worldwide belong almost exclusively to the
'Candidatus Scalindua' species, but the molecular basis of their metabolism and
competitive fitness is presently unknown. We applied community sequencing of a
marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to
construct a genome assembly, which was subsequently used to analyse the most
abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes
were annotated, and only about half of them showed the highest identity to the
only other anammox bacterium of which a metagenome assembly had been constructed
so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016
genes of S. profunda could not be matched to the K. stuttgartiensis metagenome
assembly at all, and a similar number of genes in K.stuttgartiensis could not be
found in S. profunda. Most of these genes did not have a known function but 98
expressed genes could be attributed to oligopeptide transport, amino acid
metabolism, use of organic acids and electron transport. On the basis of the S.
profunda metagenome, and environmental metagenome data, we observed pronounced
differences in the gene organization and expression of important anammox enzymes,
such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic
nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation
of the ocean may include highly expressed ammonium, nitrite and oligopeptide
transport systems and pathways for the transport, oxidation, and assimilation of
small organic compounds that may allow a more versatile lifestyle contributing to
the competitive fitness of Scalindua in the marine realm.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.
PMID: 22568606 [PubMed - indexed for MEDLINE]
18. PLoS One. 2016 May 26;11(5):e0154090. doi: 10.1371/journal.pone.0154090.
Intestinal Microbiota Is Influenced by Gender and Body Mass Index.
Haro C(1,)(2), Rangel-Zúñiga OA(1,)(2), Alcalá-Díaz JF(1,)(2), Gómez-Delgado
F(1,)(2), Pérez-Martínez P(1,)(2), Delgado-Lista J(1,)(2), Quintana-Navarro
GM(1,)(2), Landa BB(3), Navas-Cortés JA(3), Tena-Sempere M(2,)(4), Clemente
JC(5,)(6), López-Miranda J(1,)(2), Pérez-Jiménez F(1,)(2), Camargo A(1,)(2).
(1)Lipids and Atherosclerosis Unit, GC9 Nutrigenomics. IMIBIC/Reina Sofia
University Hospital/University of Cordoba, Cordoba, Spain. (2)CIBER
Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos
III, Cordoba, Spain. (3)Instituto de Agricultura Sostenible (IAS), Consejo
Superior de Investigaciones Científicas (CSIC), Cordoba, Spain. (4)Department of
Cell Biology, Physiology, and Immunology, IMIBIC/Reina Sofia University
Hospital/University of Cordoba, Cordoba, Spain. (5)Department of Genetics and
Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029,
United States of America. (6)Immunology Institute, Icahn School of Medicine at
Mount Sinai, New York, NY 10029, United States of America.
Intestinal microbiota changes are associated with the development of obesity.
However, studies in humans have generated conflicting results due to high
inter-individual heterogeneity in terms of diet, age, and hormonal factors, and
the largely unexplored influence of gender. In this work, we aimed to identify
differential gut microbiota signatures associated with obesity, as a function of
gender and changes in body mass index (BMI). Differences in the bacterial
community structure were analyzed by 16S sequencing in 39 men and 36
post-menopausal women, who had similar dietary background, matched by age and
stratified according to the BMI. We observed that the abundance of the
Bacteroides genus was lower in men than in women (P<0.001, Q = 0.002) when BMI
was > 33. In fact, the abundance of this genus decreased in men with an increase
in BMI (P<0.001, Q<0.001). However, in women, it remained unchanged within the
different ranges of BMI. We observed a higher presence of Veillonella (84.6% vs.
47.2%; X2 test P = 0.001, Q = 0.019) and Methanobrevibacter genera (84.6% vs.
47.2%; X2 test P = 0.002, Q = 0.026) in fecal samples in men compared to women.
We also observed that the abundance of Bilophila was lower in men compared to
women regardless of BMI (P = 0.002, Q = 0.041). Additionally, after correcting
for age and sex, 66 bacterial taxa at the genus level were found to be associated
with BMI and plasma lipids. Microbiota explained at P = 0.001, 31.17% variation
in BMI, 29.04% in triglycerides, 33.70% in high-density lipoproteins, 46.86% in
low-density lipoproteins, and 28.55% in total cholesterol. Our results suggest
that gut microbiota may differ between men and women, and that these differences
may be influenced by the grade of obesity. The divergence in gut microbiota
observed between men and women might have a dominant role in the definition of
gender differences in the prevalence of metabolic and intestinal inflammatory
PMID: 27228093 [PubMed - in process]
19. J Microbiol Methods. 2016 Aug;127:132-40. doi: 10.1016/j.mimet.2016.06.004. Epub
2016 Jun 6.
Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing
technologies for phylogenetic analysis of the rumen bacterial community in
Myer PR(1), Kim M(2), Freetly HC(3), Smith TP(4).
(1)Department of Animal Science, University of Tennesse Institute of Agriculture,
University of Tennessee, Knoxville, TN 37996. Electronic address: firstname.lastname@example.org.
(2)USDA-ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933(1).
Electronic address: email@example.com. (3)USDA-ARS, U.S. Meat Animal Research
Center, Clay Center, NE 68933(1). Electronic address:
firstname.lastname@example.org. (4)USDA-ARS, U.S. Meat Animal Research Center, Clay
Center, NE 68933(1). Electronic address: email@example.com.
Next generation sequencing technologies have vastly changed the approach of
sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct
technologies are available for large-scale 16S sequencing. All three are subject
to biases introduced by sequencing error rates, amplification primer selection,
and read length, which can affect the apparent microbial community. In this
study, we compared short read 16S rRNA variable regions, V1-V3, with that of
near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial
communities, in order to examine the impact of technology selection on
phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform
were used to generate V1-V3 sequence, while long "circular consensus" reads from
the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two
platforms revealed similar microbial operational taxonomic units (OTUs), as well
as similar species richness, Good's coverage, and Shannon diversity metrics.
However, the V1-V8 amplified ruminal community resulted in significant increases
in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P <
0.05). Taxonomic classification accuracy was also greater in the near full-length
read. UniFrac distance matrices using jackknifed UPGMA clustering also noted
differences between the communities. These data support the consensus that longer
reads result in a finer phylogenetic resolution that may not be achieved by
shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community
demonstrates that utilizing near full-length 16S reads may be useful in
conducting a more thorough study, or for developing a niche-specific database to
use in analyzing data from shorter read technologies when budgetary constraints
preclude use of near-full length 16S sequencing.
Copyright © 2016 Elsevier B.V. All rights reserved.
PMID: 27282101 [PubMed - in process]
20. Cell. 2014 Aug 28;158(5):1000-10. doi: 10.1016/j.cell.2014.08.006.
Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel
Palm NW(1), de Zoete MR(2), Cullen TW(3), Barry NA(3), Stefanowski J(1), Hao
L(4), Degnan PH(3), Hu J(5), Peter I(5), Zhang W(6), Ruggiero E(6), Cho JH(6),
Goodman AL(3), Flavell RA(7).
(1)Department of Immunobiology, Yale University School of Medicine, New Haven, CT
06510, USA. (2)Department of Immunobiology, Yale University School of Medicine,
New Haven, CT 06510, USA; Howard Hughes Medical Institute, Yale University, New
Haven, CT 06510, USA. (3)Microbial Diversity Institute and Department of
Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06510,
USA. (4)Department of Pathology, Yale University School of Medicine, New Haven,
CT 06510, USA. (5)Department of Genetics and Genomic Sciences, Icahn School of
Medicine at Mount Sinai, New York, NY 10029, USA. (6)Departments of Medicine and
Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
(7)Department of Immunobiology, Yale University School of Medicine, New Haven, CT
06510, USA; Howard Hughes Medical Institute, Yale University, New Haven, CT
06510, USA. Electronic address: firstname.lastname@example.org.
Nat Rev Gastroenterol Hepatol. 2014 Nov;11(11):642.
Cell Host Microbe. 2014 Sep 10;16(3):265-7.
Z Gastroenterol. 2015 Jan;53(1):53.
Specific members of the intestinal microbiota dramatically affect inflammatory
bowel disease (IBD) in mice. In humans, however, identifying bacteria that
preferentially affect disease susceptibility and severity remains a major
challenge. Here, we used flow-cytometry-based bacterial cell sorting and 16S
sequencing to characterize taxa-specific coating of the intestinal microbiota
with immunoglobulin A (IgA-SEQ) and show that high IgA coating uniquely
identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven
colitis. We then used IgA-SEQ and extensive anaerobic culturing of fecal bacteria
from IBD patients to create personalized disease-associated gut microbiota
culture collections with predefined levels of IgA coating. Using these
collections, we found that intestinal bacteria selected on the basis of high
coating with IgA conferred dramatic susceptibility to colitis in germ-free mice.
Thus, our studies suggest that IgA coating identifies inflammatory commensals
that preferentially drive intestinal disease. Targeted elimination of such
bacteria may reduce, reverse, or even prevent disease development.
Copyright © 2014 Elsevier Inc. All rights reserved.
PMID: 25171403 [PubMed - indexed for MEDLINE]
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
[PubMed - indexed for MEDLINE] Free PMC Article
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Select item 11777011
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.