MR DNA is Unwrapping the microbiome 16sRNA Gene ITS fungi and more Where Microbes Are Our Passion!

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

Aug 20.

 

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).

 

Author information:

(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 itoma@gwu.edu

msiegel@mfa.gwu.edu. (2)Division of Infectious Diseases, The George Washington

University School of Medicine and Health Sciences, Washington, DC, USA

itoma@gwu.edu msiegel@mfa.gwu.edu. (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.

 

DOI: 10.1128/JCM.01678-14

PMCID: PMC4313225

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

Directions.

 

Jervis Bardy J(1), Psaltis AJ(2).

 

Author information:

(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 alkis.psaltis@adelaide.edu.au.

 

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

targets.

 

© The Author(s) 2016.

 

DOI: 10.1177/0003489416641429

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.

 

Author information:

(1)Department of Neonatology, Shenzhen Bao'an Maternal and Child Health Hospital,

Shenzhen, Guangdong 518133, China. lugj1111@aliyun.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

respectively.

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

over time.

 

 

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).

 

Author information:

(1)Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn,

Aberdeen, AB21 9SB, UK. john.wallace@abdn.ac.uk. (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.

n.mckain@abdn.ac.uk. (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.

mick.watson@roslin.ed.ac.uk. (9)SRUC, West Mains Road, Edinburgh, EH9 3JG, UK.

rainer.roehe@sruc.ac.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.

 

DOI: 10.1186/s12864-015-2032-0

PMCID: PMC4619255

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).

 

Author information:

(1)Department of Food and Nutrition, Hakodate Junior College, 52-1, Takaoka-cho,

Hakodate 042-0955, Japan. Electronic address: sawabe@hakodate-jc.ac.jp.

(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

041-0821, Japan.

 

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.

 

DOI: 10.1016/j.jiph.2015.11.008

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).

 

Author information:

(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

metagenomics.

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.

 

DOI: 10.1038/ajg.2015.357

PMCID: PMC4697132

PMID: 26526081  [PubMed - indexed for MEDLINE]

 

 

5. Microbiology. 2012 Nov;158(Pt 11):2789-95. doi: 10.1099/mic.0.057943-0. Epub 2012

Aug 17.

 

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.

 

Author information:

(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.

 

DOI: 10.1099/mic.0.057943-0

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).

 

Author information:

(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/.

CONTACT: kasshau@gwdg.de

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics

online.

 

© The Author 2015. Published by Oxford University Press.

 

DOI: 10.1093/bioinformatics/btv287

PMCID: PMC4547618

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).

 

Author information:

(1)U. S. Food and Drug Administration, Center for Food Safety and Applied

Nutrition, OARSA, Laurel, MD, USA. karen.jarvis@fda.hhs.gov. (2)Oak Ridge

Institute for Science and Technology, Oak Ridge, TN, USA.

james.dna.white@gmail.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

samples.

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.

 

DOI: 10.1186/s12866-015-0497-2

PMCID: PMC4534111

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

DNA mixtures.

 

Boers SA(1), Hays JP(1), Jansen R(2).

 

Author information:

(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.

longitudinal surveys).

 

DOI: 10.1038/srep14181

PMCID: PMC4570986

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

Drosophila melanogaster.

 

Chaston JM(1), Newell PD(2), Douglas AE.

 

Author information:

(1)Department of Entomology, Cornell University, Ithaca, New York, USA

john_chaston@byu.edu. (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

host health.

 

Copyright © 2014 Chaston et al.

 

DOI: 10.1128/mBio.01631-14

PMCID: PMC4196228

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.

 

Author information:

(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.

 

DOI: 10.1016/j.jbiotec.2013.07.021

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).

 

Author information:

(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: sjpark@jejunu.ac.kr.

 

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.

 

DOI: 10.1016/j.anaerobe.2015.01.004

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.

 

Author information:

(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

metagenomes.

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.

 

DOI: 10.1186/1471-2180-13-116

PMCID: PMC3679945

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).

 

Author information:

(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 [2]. (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.

 

DOI: 10.1038/srep05100

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).

 

Author information:

(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 [2] 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.

 

DOI: 10.1038/ismej.2013.174

PMCID: PMC3930316

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.

 

Author information:

(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.

 

DOI: 10.1142/S0219720014410030

PMID: 24712530  [PubMed - indexed for MEDLINE]

 

 

16. PLoS One. 2016 Jan 19;11(1):e0146939. doi: 10.1371/journal.pone.0146939.

eCollection 2016.

 

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),

Watson CH(1).

 

Author information:

(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.

 

DOI: 10.1371/journal.pone.0146939

PMCID: PMC4718623

PMID: 26784944  [PubMed - indexed for MEDLINE]

 

 

17. PLoS One. 2014 Apr 8;9(4):e93827. doi: 10.1371/journal.pone.0093827. eCollection

2014.

 

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).

 

Author information:

(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.

 

DOI: 10.1371/journal.pone.0093827

PMCID: PMC3979728

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.

 

DOI: 10.1016/j.jbspin.2013.02.005

PMID: 23806346  [PubMed - indexed for MEDLINE]

 

 

19. Front Microbiol. 2016 Aug 23;7:1297. doi: 10.3389/fmicb.2016.01297. eCollection

2016.

 

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).

 

Author information:

(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

GbRKiel, Germany.

 

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.

 

DOI: 10.3389/fmicb.2016.01297

PMCID: PMC4994424

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.

 

Author information:

(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.

 

DOI: 10.1111/j.1462-2920.2012.02774.x

PMCID: PMC3655542

PMID: 22568606  [PubMed - indexed for MEDLINE]

 

 

 

 

18. PLoS One. 2016 May 26;11(5):e0154090. doi: 10.1371/journal.pone.0154090.

eCollection 2016.

 

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).

 

Author information:

(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

diseases.

 

DOI: 10.1371/journal.pone.0154090

PMCID: PMC4881937

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

steers.

 

Myer PR(1), Kim M(2), Freetly HC(3), Smith TP(4).

 

Author information:

(1)Department of Animal Science, University of Tennesse Institute of Agriculture,

University of Tennessee, Knoxville, TN 37996. Electronic address: pmyer@utk.edu.

(2)USDA-ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933(1).

Electronic address: mkim2276@korea.kr. (3)USDA-ARS, U.S. Meat Animal Research

Center, Clay Center, NE 68933(1). Electronic address:

harvey.freetly@ars.usda.gov. (4)USDA-ARS, U.S. Meat Animal Research Center, Clay

Center, NE 68933(1). Electronic address: tim.smith@ars.usda.gov.

 

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.

 

DOI: 10.1016/j.mimet.2016.06.004

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

disease.

 

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).

 

Author information:

(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: richard.flavell@yale.edu.

 

Comment in

    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.

 

DOI: 10.1016/j.cell.2014.08.006

PMCID: PMC4174347

PMID: 25171403  [PubMed - indexed for MEDLINE]

 

 

 

603.

J Clin Microbiol. 2002 Apr;40(4):1333-8.

rpoB gene sequence-based identification of Staphylococcus species.

Drancourt M1, Raoult D.

Author information

Abstract

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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604.

Vet Res. 2001 Nov-Dec;32(6):611-6.

Possible misidentification of Bacteroides sp., probably B. ureolyticus as Taylorella equigenitalis: implications for the laboratory diagnosis of CEM.

Moore JE1, Millar BC, Xu J, Buckley TC.

Author information

Abstract

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.