low cost MR DNA 16s sequencing microbiome and metagenome low prices
16s rRNA Sequencing with MR DNA
16S ribosomal (rRNA) sequencing using next generation sequencing is a method used to identify and compare bacteria and archaea present within almost any type of sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.
21. J Microbiol Methods. 2015 Dec;119:239-42. doi: 10.1016/j.mimet.2015.11.001. Epub
2015 Nov 10.
Changes in 16s RNA Gene Microbial Community Profiling by Concentration of
Glassing A(1), Dowd SE(2), Galandiuk S(3), Davis B(4), Jorden JR(3), Chiodini
(1)Department of Biological and Physical Sciences, Montana State
University-Billings, Billings, Montana, United States. (2)Molecular Research (Mr.
DNA), Shallowater, Texas, United States. (3)Department of Surgery, University of
Louisville, Louisville, Kentucky, United States. (4)Department of Surgery, Paul
L. Foster School of Medicine, Texas Tech University Health Sciences Center, El
Paso, Texas, United States. (5)St. Vincent Healthcare, Sisters of Charity of
Leavenworth Health System, Billings, Montana, United States; Department of
Biological and Physical Sciences, Montana State University-Billings, Billings,
Montana, United States. Electronic address: firstname.lastname@example.org.
Microbial metagenomics are hindered in clinical tissue samples as a result of the
large relative amount of human DNA in relation to microbial DNA acting as
competitive inhibitors of downstream applications. We evaluated the LOOXSTER®
Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal
intestinal tissue samples having a low microbial biomass and to determine the
effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit
reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold
increase in the number of 16s bacterial gene sequences detected on the Illumina
MiSeq platform. This increase was accompanied by the detection of 41 additional
bacterial genera and 94 tentative species. The additional bacterial taxa detected
accounted for as much as 25% of the total bacterial population that significantly
altered the relative prevalence and composition of the intestinal microbiota. The
ability to reduce the competitive inhibition created by human DNA and the
concentration of bacterial DNA may allow metagenomics to be performed on complex
tissues containing a low bacterial biomass.
Copyright © 2015 Elsevier B.V. All rights reserved.
PMID: 26569458 [PubMed - indexed for MEDLINE]
22. Microbiome. 2014 Jul 11;2:24. doi: 10.1186/2049-2618-2-24. eCollection 2014.
Effect of chemotherapy on the microbiota and metabolome of human milk, a case
Urbaniak C(1), McMillan A(1), Angelini M(2), Gloor GB(3), Sumarah M(4), Burton
JP(1), Reid G(1).
(1)Lawson Health Research Institute, 268 Grosvenor Street, London, ON N6A 4V2,
Canada ; Department of Microbiology & Immunology, Western University, London, ON
N6A 5C1, Canada. (2)Perinatal and Women's Health, London Health Sciences Centre,
London, ON N6A 4L6, Canada. (3)Department of Biochemistry, Western University,
London, ON N6A 5C1, Canada. (4)Department of Chemistry, Western University,
London, ON N6A 5C1, Canada ; Agriculture and Agri-Food Canada, London, ON N5V
BACKGROUND: Human milk is an important source of bacteria for the developing
infant and has been shown to influence the bacterial composition of the neonatal
gut, which in turn can affect disease risk later in life. Human milk is also an
important source of nutrients, influencing bacterial composition but also
directly affecting the host. While recent studies have emphasized the adverse
effects of antibiotic therapy on the infant microbiota, the effects of maternal
chemotherapy have not been previously studied. Here we report the effects of drug
administration on the microbiota and metabolome of human milk.
METHODS: Mature milk was collected every two weeks over a four month period from
a lactating woman undergoing chemotherapy for Hodgkin's lymphoma. Mature milk was
also collected from healthy lactating women for comparison. Microbial profiles
were analyzed by 16S sequencing and the metabolome by gas chromatography-mass
FINDINGS: Chemotherapy caused a significant deviation from a healthy microbial
and metabolomic profile, with depletion of genera Bifidobacterium, Eubacterium,
Staphylococcus and Cloacibacterium in favor of Acinetobacter, Xanthomonadaceae
and Stenotrophomonas. The metabolites docosahexaenoic acid and inositol known for
their beneficial effects were also decreased.
CONCLUSION: With milk contents being critical for shaping infant immunity and
development, consideration needs to be given to the impact of drugs administered
to the mother and the long-term potential consequences for the health of the
PMID: 25061513 [PubMed]
23. Hepatology. 2016 Sep 17. doi: 10.1002/hep.28829. [Epub ahead of print]
Changes in blood microbiota profiles associated with liver fibrosis in obese
patients: A pilot analysis.
Lelouvier B(1), Servant F(2), Païssé S(2), Brunet AC(2,)(3), Benyahya S(2),
Serino M(4), Valle C(2), Rosa Ortiz M(5), Puig J(6), Courtney M(2), Federici
M(7), Manuel Fernández-Real J(5), Burcelin R(2,)(4), Amar J(8,)(9,)(10).
(1)Vaiomer SAS, Labège, France. email@example.com. (2)Vaiomer SAS,
Labège, France. (3)IMT, Université Paul Sabatier, Toulouse, France. (4)INSERM
U1048, I2MC, Toulouse, France. (5)Department of Diabetes, Endocrinology, and
Nutrition, IDIBGI, Girona, Spain. (6)Department of Radiology, IDI, IDIBGI,
Girona, Spain. (7)Department of Systems Medicine and Center for Atherosclerosis,
University of Rome "Tor Vergata", Rome, Italy. (8)Vaiomer SAS, Labège, France.
firstname.lastname@example.org. (9)INSERM U1048, I2MC, Toulouse, France.
email@example.com. (10)Department of Therapeutics, Rangueil Hospital,
Toulouse, France. firstname.lastname@example.org.
OBJECTIVE: The early detection of liver fibrosis among patients with nonalcoholic
fatty liver disease (NAFLD) is an important clinical need. In view of the
suggested role played by bacterial translocation in liver disease and obesity, we
sought to investigate the relationship between blood microbiota and liver
fibrosis in European cohorts of patients with severe obesity.
DESIGN: We carried out a cross-sectional study of obese patients,
well-characterized with respect to the severity of the NAFLD, in the cohort
FLORINASH. This cohort has been divided into a discovery cohort comprising 50
Spanish patients and then in a validation cohort of 71 Italian patients. Blood
bacterial DNA was analyzed both quantitatively by 16S rDNA qPCR and qualitatively
by 16S rDNA targeted metagenomic sequencing and functional metagenome prediction.
Spanish plasma bile acid contents were analyzed by LC/MS.
RESULTS: The 16S rDNA concentration was significantly higher in patients of the
discovery cohort with liver fibrosis. By 16S sequencing, we found specific
differences in the proportion of several bacterial taxa in both blood and feces
that correlate with the presence of liver fibrosis thus defining a specific
signature of the liver disease. Several secondary/primary bile acids ratios were
also decreased with liver fibrosis in the discovery cohort. We confirmed in the
validation cohort the correlation between blood 16S rDNA concentration and liver
fibrosis whereas we did not confirm the specific bacterial taxa signature,
despite a similar trend in patients with more severe fibrosis.
CONCLUSIONS: We have shown that changes in blood microbiota are associated with
liver fibrosis in obese patients. Blood microbiota analysis provides potential
biomarkers for the detection of liver fibrosis in this population. This article
is protected by copyright. All rights reserved.
© 2016 by the American Association for the Study of Liver Diseases.
PMID: 27639192 [PubMed - as supplied by publisher]
81. Microbiome. 2015 Nov 26;3:56. doi: 10.1186/s40168-015-0118-1.
Metagenomic analysis of the microbiota in the highly compartmented hindguts of
six wood- or soil-feeding higher termites.
Rossmassler K(1,)(2), Dietrich C(1), Thompson C(1), Mikaelyan A(1,)(3), Nonoh
JO(1), Scheffrahn RH(4), Sillam-Dussès D(5,)(6), Brune A(7,)(8).
(1)Department of Biogeochemistry, Max Planck Institute for Terrestrial
Microbiology, Marburg, Germany. (2)Present address: Department of Civil and
Environmental Engineering, Colorado State University, Fort Collins, CO, USA.
(3)LOEWE Center for Synthetic Microbiology (Synmikro), Philipps-Universität
Marburg, Marburg, Germany. (4)Fort Lauderdale Research and Education Center,
University of Florida, Davie, FL, USA. (5)Laboratory of Experimental and
Comparative Ethology, University Paris 13, EA4443, Sorbonne Paris Cité,
Villetaneuse, France. (6)Institute of Research for Development-Sorbonne
Universités, Institute of Ecology and Environmental Sciences, U242, Bondy,
France. (7)Department of Biogeochemistry, Max Planck Institute for Terrestrial
Microbiology, Marburg, Germany. email@example.com. (8)LOEWE Center for
Synthetic Microbiology (Synmikro), Philipps-Universität Marburg, Marburg,
BACKGROUND: Termites are important contributors to carbon and nitrogen cycling in
tropical ecosystems. Higher termites digest lignocellulose in various stages of
humification with the help of an entirely prokaryotic microbiota housed in their
compartmented intestinal tract. Previous studies revealed fundamental differences
in community structure between compartments, but the functional roles of
individual lineages in symbiotic digestion are mostly unknown.
RESULTS: Here, we conducted a highly resolved analysis of the gut microbiota in
six species of higher termites that feed on plant material at different levels of
humification. Combining amplicon sequencing and metagenomics, we assessed
similarities in community structure and functional potential between the major
hindgut compartments (P1, P3, and P4). Cluster analysis of the relative
abundances of orthologous gene clusters (COGs) revealed high similarities among
wood- and litter-feeding termites and strong differences to humivorous species.
However, abundance estimates of bacterial phyla based on 16S rRNA genes greatly
differed from those based on protein-coding genes.
CONCLUSION: Community structure and functional potential of the microbiota in
individual gut compartments are clearly driven by the digestive strategy of the
host. The metagenomics libraries obtained in this study provide the basis for
future studies that elucidate the fundamental differences in the
symbiont-mediated breakdown of lignocellulose and humus by termites of different
feeding groups. The high proportion of uncultured bacterial lineages in all
samples calls for a reference-independent approach for the correct taxonomic
assignment of protein-coding genes.
PMID: 26607965 [PubMed - indexed for MEDLINE]
82. Appl Environ Microbiol. 2013 Oct;79(20):6196-206. doi: 10.1128/AEM.01641-13. Epub
2013 Aug 2.
Metagenome survey of a multispecies and alga-associated biofilm revealed key
elements of bacterial-algal interactions in photobioreactors.
Krohn-Molt I(1), Wemheuer B, Alawi M, Poehlein A, Güllert S, Schmeisser C,
Pommerening-Röser A, Grundhoff A, Daniel R, Hanelt D, Streit WR.
(1)University of Hamburg, Biocenter Klein Flottbek, Microbiology and
Biotechnology, Hamburg, Germany.
Photobioreactors (PBRs) are very attractive for sunlight-driven production of
biofuels and capturing of anthropogenic CO2. One major problem associated with
PBRs however, is that the bacteria usually associated with microalgae in
nonaxenic cultures can lead to biofouling and thereby affect algal productivity.
Here, we report on a phylogenetic, metagenome, and functional analysis of a
mixed-species bacterial biofilm associated with the microalgae Chlorella vulgaris
and Scenedesmus obliquus in a PBR. The biofilm diversity and population dynamics
were examined through 16S rRNA phylogeny. Overall, the diversity was rather
limited, with approximately 30 bacterial species associated with the algae. The
majority of the observed microorganisms were affiliated with Alphaproteobacteria,
Betaproteobacteria, and Bacteroidetes. A combined approach of sequencing via GS
FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall
production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger
contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high
metabolic diversity with respect to the use of polymers and aromatic and
nonaromatic compounds. Genes associated with the biosynthesis of essential B
vitamins were highly redundant and functional. Moreover, a relatively high number
of predicted and functional lipase and esterase genes indicated that the
alga-associated bacteria are possibly a major sink for lipids and fatty acids
produced by the microalgae. This is the first metagenome study of microalga- and
PBR-associated biofilm bacteria, and it gives new clues for improved biofuel
production in PBRs.
PMID: 23913425 [PubMed - indexed for MEDLINE]
83. Vet Microbiol. 2013 Mar 23;162(2-4):891-8. doi: 10.1016/j.vetmic.2012.11.018.
Epub 2012 Nov 20.
Metagenomic analysis of the canine oral cavity as revealed by high-throughput
pyrosequencing of the 16S rRNA gene.
Sturgeon A(1), Stull JW, Costa MC, Weese JS.
(1)Department of Pathobiology, Ontario Veterinary College, University of Guelph,
Guelph, Ontario N1G 2W1, Canada. firstname.lastname@example.org
Efficient characterization of the canine oral microbiome is critical for
understanding the role of oral bacteria in health and for providing insight into
early diagnosis and treatment strategies against periodontal disease. To date,
characterization has been limited to cloning-based sequencing and conventional
culture-based studies, which generally underestimate community diversity as a
result of inherent biases in their methodologies. Pyrosequencing, a cloning- and
culture-independent sequencing approach, eliminates these elements of bias from
the analysis and enables extensive sequencing of microbial populations. In this
report, pyrosequencing of the 16S rRNA gene was used to examine oral samples from
six healthy dogs in an effort to determine community membership, diversity, and
zoonotic implications. Pyrosequencing revealed a mean (SD) of 226 (59)
operational taxonomic units (OTUs, 97% similarity), representing 181 genera from
13 bacterial phyla. The phyla Bacteroidetes (60.2%), Proteobacteria (20.8%),
Firmicutes (11.4%), Fusobacteria (4.7%), and Spirochaetes (1.7%) predominated.
The most commonly identified genera were Porphyromonas (39.2% of sequences),
Fusobacterium (4.5%), Capnocytophaga (3.8%), Derxia (3.7%), Moraxella (3.3%), and
Bergeyella (2.7%). Fifty-six OTUs, corresponding to 38 unique genus-level
identifications, were present in all samples, which supports the concept of a
stable core microbiome existing between healthy dogs. Potentially zoonotic and
periodontal bacteria were detected in all dogs, and highlights the zoonotic and
disease potential of the oral microflora. Results suggest that the canine oral
cavity harbors a rich and diverse bacterial community, and exceeds estimates by
previous culture- and cloning-based studies.
Copyright © 2012 Elsevier B.V. All rights reserved.
PMID: 23228621 [PubMed - indexed for MEDLINE]
84. Antonie Van Leeuwenhoek. 2012 Feb;101(2):205-15. doi: 10.1007/s10482-011-9624-8.
Epub 2011 Jul 27.
Taxonomic and functional assignment of cloned sequences from high Andean forest
Montaña JS(1), Jiménez DJ, Hernández M, Angel T, Baena S.
(1)Department of Microbiology, Pontificia Universidad Javeriana, Carrera 7 No.
43-82, Building 53, P.O. Box 5671, Bogotá, DC, Colombia.
Total metagenomic DNA was isolated from high Andean forest soil and subjected to
taxonomical and functional composition analyses by means of clone library
generation and sequencing. The obtained yield of 1.7 μg of DNA/g of soil was used
to construct a metagenomic library of approximately 20,000 clones (in the plasmid
p-Bluescript II SK+) with an average insert size of 4 Kb, covering 80 Mb of the
total metagenomic DNA. Metagenomic sequences near the plasmid cloning site were
sequenced and them trimmed and assembled, obtaining 299 reads and 31 contigs
(0.3 Mb). Taxonomic assignment of total sequences was performed by BLASTX,
resulting in 68.8, 44.8 and 24.5% classification into taxonomic groups using the
metagenomic RAST server v2.0, WebCARMA v1.0 online system and MetaGenome Analyzer
v3.8 software, respectively. Most clone sequences were classified as Bacteria
belonging to phlya Actinobacteria, Proteobacteria and Acidobacteria. Among the
most represented orders were Actinomycetales (34% average), Rhizobiales,
Burkholderiales and Myxococcales and with a greater number of sequences in the
genus Mycobacterium (7% average), Frankia, Streptomyces and Bradyrhizobium. The
vast majority of sequences were associated with the metabolism of carbohydrates,
proteins, lipids and catalytic functions, such as phosphatases,
glycosyltransferases, dehydrogenases, methyltransferases, dehydratases and
epoxide hydrolases. In this study we compared different methods of taxonomic and
functional assignment of metagenomic clone sequences to evaluate microbial
diversity in an unexplored soil ecosystem, searching for putative enzymes of
biotechnological interest and generating important information for further
functional screening of clone libraries.
PMID: 21792685 [PubMed - indexed for MEDLINE]
85. PLoS One. 2015 Apr 13;10(4):e0123990. doi: 10.1371/journal.pone.0123990.
Bacterial community associated with the intestinal tract of Chinese mitten crab
(Eriocheir sinensis) farmed in Lake Tai, China.
Chen X(1), Di P(1), Wang H(1), Li B(2), Pan Y(3), Yan S(4), Wang Y(3).
(1)Laboratory of Quality and Safety Risk Assessment for Aquatic Products on
Storage & Preservation, Ministry of Agriculture, Shanghai, China; College of Food
Science and Technology, Shanghai Ocean University, Shanghai, China. (2)College of
Food Science and Technology, Shanghai Ocean University, Shanghai, China.
(3)Laboratory of Quality and Safety Risk Assessment for Aquatic Products on
Storage & Preservation, Ministry of Agriculture, Shanghai, China; Shanghai
Engineering Research Center of Aquatic-Product Processing & Preservation,
Shanghai, China; College of Food Science and Technology, Shanghai Ocean
University, Shanghai, China. (4)Institute of Biochemistry and Molecular Cell
Biology, University of Goettingen, Goettingen, Germany.
Chinese mitten crab (CMC, Eriocheir sinensis) is an economically valuable species
in South-East Asia that has been widely farmed in China. Characterization of the
intestinal bacterial diversity of CMC will provide insights into the
aquaculturing of CMCs. Based on the analysis of cloned 16S rRNA genes from
culture-independent CMC gut bacteria, 124 out of 128 different clones reveal ＞95%
nucleotide similarity to the species belonging to the four phyla of Tenericutes,
Bacteroidetes, Firmicutes and Proteobacteria; one clone shows 91% sequence
similarity to the member of TM7 (a candidate phylum without cultured
representatives). Fluorescent in situ hybridization also reveals the abundance of
Bacteroidetes in crab intestine. Electron micrographs show that spherical and
filamentous bacteria are closely associated with the microvillus brush border of
the midgut epithelium and are often inserted into the space between the
microvilli using a stalk-like cell appendage. In contrast, the predominant
rod-shaped bacteria in the hindgut are tightly attached to the epithelium surface
by an unusual pili-like structure. Both 16S rRNA gene denaturing gel gradient
electrophoresis and metagenome library indicate that the CMC Mollicutes group 2
appears to be present in both the midgut and hindgut with no significant
difference in abundance. The CMC Mollicutes group 1, however, was found mostly in
the midgut of CMCs. The CMC gut Mollicutes phylotypes appear to be most closely
related to Mollicutes symbionts detected in the gut of isopods (Crustacea:
Isopoda). Overall, the results suggest that CMCs harbor diverse, novel and
specific gut bacteria, which are likely to live in close relationships with the
PMID: 25875449 [PubMed - indexed for MEDLINE]
86. Appl Environ Microbiol. 2015 Nov;81(22):7893-904. doi: 10.1128/AEM.02294-15. Epub
2015 Sep 4.
Coexistence of Lactic Acid Bacteria and Potential Spoilage Microbiota in a Dairy
Stellato G(1), De Filippis F(1), La Storia A(1), Ercolini D(2).
(1)Department of Agricultural Sciences, Division of Microbiology, University of
Naples Federico II, Portici, Italy. (2)Department of Agricultural Sciences,
Division of Microbiology, University of Naples Federico II, Portici, Italy
Microbial contamination in food processing plants can play a fundamental role in
food quality and safety. In this study, the microbiota in a dairy plant was
studied by both 16S rRNA- and 26S rRNA-based culture-independent high-throughput
amplicon sequencing. Environmental samples from surfaces and tools were studied
along with the different types of cheese produced in the same plant. The
microbiota of environmental swabs was very complex, including more than 200
operational taxonomic units with extremely variable relative abundances (0.01 to
99%) depending on the species and sample. A core microbiota shared by 70% of the
samples indicated a coexistence of lactic acid bacteria with a remarkable level
of Streptococcus thermophilus and possible spoilage-associated bacteria,
including Pseudomonas, Acinetobacter, and Psychrobacter, with a relative
abundance above 50%. The most abundant yeasts were Kluyveromyces marxianus,
Yamadazyma triangularis, Trichosporon faecale, and Debaryomyces hansenii.
Beta-diversity analyses showed a clear separation of environmental and cheese
samples based on both yeast and bacterial community structure. In addition,
predicted metagenomes also indicated differential distribution of metabolic
pathways between the two categories of samples. Cooccurrence and coexclusion
pattern analyses indicated that the occurrence of potential spoilers was excluded
by lactic acid bacteria. In addition, their persistence in the environment can be
helpful to counter the development of potential spoilers that may contaminate the
cheeses, with possible negative effects on their microbiological quality.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PMID: 26341209 [PubMed - indexed for MEDLINE]
87. Sci Rep. 2015 Apr 8;5:9456. doi: 10.1038/srep09456.
A pyrosequencing insight into sprawling bacterial diversity and community
dynamics in decaying deadwood logs of Fagus sylvatica and Picea abies.
Hoppe B(1), Krger K(2), Kahl T(3), Arnstadt T(4), Buscot F(5), Bauhus J(3), Wubet
(1)1] Department of Soil Ecology, UFZ - Helmholtz Centre for Environmental
Research, Halle (Saale), Germany  Chair of Silviculture, Faculty of
Environment and Natural Resources, University of Freiburg, Freiburg i. Brsg.,
Germany. (2)Department of Soil Ecology, UFZ - Helmholtz Centre for Environmental
Research, Halle (Saale), Germany. (3)Chair of Silviculture, Faculty of
Environment and Natural Resources, University of Freiburg, Freiburg i. Brsg.,
Germany. (4)Department of Bio- and Environmental Sciences, TU Dresden,
International Institute Zittau, Zittau, Germany. (5)1] Department of Soil
Ecology, UFZ - Helmholtz Centre for Environmental Research, Halle (Saale),
Germany  The German Centre for Integrative Biodiversity Research (iDiv),
University Leipzig, Leipzig, Germany.
Deadwood is an important biodiversity hotspot in forest ecosystems. While
saproxylic insects and wood-inhabiting fungi have been studied extensively,
little is known about deadwood-inhabiting bacteria. The study we present is among
the first to compare bacterial diversity and community structure of deadwood
under field conditions. We therefore compared deadwood logs of two temperate
forest tree species Fagus sylvatica and Picea abies using 16S rDNA pyrosequencing
to identify changes in bacterial diversity and community structure at different
stages of decay in forest plots under different management regimes.
Alphaproteobacteria, Acidobacteria and Actinobacteria were the dominant taxonomic
groups in both tree species. There were no differences in bacterial OTU richness
between deadwood of Fagus sylvatica and Picea abies. Bacteria from the order
Rhizobiales became more abundant during the intermediate and advanced stages of
decay, accounting for up to 25% of the entire bacterial community in such logs.
The most dominant OTU was taxonomically assigned to the genus Methylovirgula,
which was recently described in a woodblock experiment of Fagus sylvatica.
Besides tree species we were able to demonstrate that deadwood physico-chemical
properties, in particular remaining mass, relative wood moisture, pH, and C/N
ratio serve as drivers of community composition of deadwood-inhabiting bacteria.
PMID: 25851097 [PubMed - indexed for MEDLINE]
88. World J Gastroenterol. 2015 Mar 7;21(9):2759-69. doi: 10.3748/wjg.v21.i9.2759.
Fecal microbes, short chain fatty acids, and colorectal cancer across
Hester CM(1), Jala VR(1), Langille MG(1), Umar S(1), Greiner KA(1), Haribabu
(1)Christina M Hester, K Allen Greiner, Department of Family Medicine Research
Division, University of Kansas Medical Center, Kansas City, KS 66160, United
AIM: To investigate differences in microbes and short chain fatty acid (SCFA)
levels in stool samples from Hispanic and non-Hispanic African American, American
Indian, and White participants.
METHODS: Stool samples from twenty participants were subjected to analysis for
relative levels of viable bacteria and for SCFA levels. Additionally, the samples
were subjected to 16S rRNA gene pyrosequencing for identification of bacteria
present in the stool. We used a metagenome functional prediction technique to
analyze genome copy numbers and estimate the abundance of butyrate kinase in all
RESULTS: We found that African Americans had significantly lower levels of
acetate, butyrate, and total SCFAs than all other racial/ethnic groups. We also
found that participant microbial profiles differed by racial/ethnic group.
African Americans had significantly more Firmicutes than Whites, with enriched
Ruminococcaceae. The Firmicutes/Bacteroidetes ratio was also significantly higher
for African Americans than for Whites (P = 0.049). We found Clostridium levels to
be significantly and inversely related to total SCFA levels (P = 0.019) and we
found Bacteroides to be positively associated (P = 0.027) and Clostridium to be
negatively associated (P = 0.012) with levels of butyrate. We also identified a
correlation between copy number for a butyrate kinase predicted from 16S rRNA
gene abundance and levels of butyrate in stool.
CONCLUSION: The identified differences in gut flora and SCFA levels may relate to
colorectal cancer mortality differentials and may be useful as targets for future
clinical and behavioral interventions.
PMID: 25759547 [PubMed - indexed for MEDLINE]
89. J Infect Dis. 2013 Apr;207(7):1105-14. doi: 10.1093/infdis/jit005. Epub 2013 Jan
Quantitation and composition of cutaneous microbiota in diabetic and nondiabetic
Redel H(1), Gao Z, Li H, Alekseyenko AV, Zhou Y, Perez-Perez GI, Weinstock G,
Sodergren E, Blaser MJ.
(1)Department of Medicine, New York University School of Medicine, New York, NY
10010, USA. email@example.com
BACKGROUND: Diabetic foot infections are a leading cause of lower extremity
amputations. Our study examines the microbiota of diabetic skin prior to ulcer
development or infection.
METHODS: In a case-control study, outpatient males were recruited at a veterans
hospital. Subjects were swabbed at 4 cutaneous sites, 1 on the forearm and 3 on
the foot. Quantitative polymerase chain reaction (qPCR) with primers and probes
specific for bacteria, Staphylococcus species, Staphylococcus aureus, and fungi
were performed on all samples. High-throughput 16S ribosomal RNA (rRNA)
sequencing was performed on samples from the forearm and the plantar aspect of
RESULTS: qPCR analysis of swab specimens from 30 diabetic subjects and 30 control
subjects showed no differences in total numbers of bacteria or fungi at any
sampled site. Increased log concentrations of Staphylococcus aureus, quantified
by the number of nuc gene copies, were present in diabetic men on the plantar
aspect of the foot. High-throughput 16S rRNA sequencing found that, on the foot,
the microbiota in controls (n = 24) was dominated by Staphylococcus species,
whereas the microbiota in diabetics (n = 23) was more diverse at the genus level.
The forearm microbiota had similar diversity in diabetic and control groups.
CONCLUSIONS: The feet of diabetic men had decreased populations of Staphylococcus
species, increased populations of S. aureus, and increased bacterial diversity,
compared with the feet of controls. These ecologic changes may affect the risk
for wound infections.
PMID: 23300163 [PubMed - indexed for MEDLINE]
90. BMC Genomics. 2013;14 Suppl 5:S16. doi: 10.1186/1471-2164-14-S5-S16. Epub 2013
Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and
SOLiD shotgun sequencing.
Mitra S, Förster-Fromme K, Damms-Machado A, Scheurenbrand T, Biskup S, Huson DH,
BACKGROUND: Metagenomics seeks to understand microbial communities and
assemblages by DNA sequencing. Technological advances in next generation
sequencing technologies are fuelling a rapid growth in the number and scope of
projects aiming to analyze complex microbial environments such as marine, soil or
the gut. Recent improvements in longer read lengths and paired-sequencing allow
better resolution in profiling microbial communities. While both 454 sequencing
and Illumina sequencing have been used in numerous metagenomic studies, SOLiD
sequencing is not commonly used in this area, as it is believed to be more
suitable in the context of reference-guided projects.
RESULTS: To investigate the performance of SOLiD sequencing in a metagenomic
context, we compared taxonomic profiles of SOLiD mate-pair sequencing reads with
Sanger paired reads and 454 single reads. All sequences were obtained from the
bacterial 16S rRNA gene, which was amplified from microbial DNA extracted from a
human fecal sample. Additionally, from the same fecal sample, complete genomic
microbial DNA was extracted and shotgun sequenced using SOLiD sequencing to study
the composition of the intestinal microbiota and the existing microbial
metabolism. We found that the microbiota composition of 16S rRNA gene sequences
obtained using Sanger, 454 and SOLiD sequencing provide results comparable to the
result based on shotgun sequencing. Moreover, with SOLiD sequences we obtained
more resolution down to the species level. In addition, the shotgun data allowed
us to determine a functional profile using the databases SEED and KEGG.
CONCLUSIONS: This study shows that SOLiD mate-pair sequencing is a viable and
cost-efficient option for analyzing a complex microbiome. To the best of our
knowledge, this is the first time that SOLiD sequencing has been used in a human
PMID: 24564472 [PubMed - indexed for MEDLINE]
91. Biofouling. 2014;30(10):1211-23. doi: 10.1080/08927014.2014.977267.
Integrated metagenomic and metaproteomic analyses of marine biofilm communities.
Leary DH(1), Li RW, Hamdan LJ, Hervey WJ 4th, Lebedev N, Wang Z, Deschamps JR,
Kusterbeck AW, Vora GJ.
(1)a Center for Bio/Molecular Science and Engineering , US Naval Research
Laboratory , Washington , DC , USA.
Metagenomic and metaproteomic analyses were utilized to determine the composition
and function of complex air-water interface biofilms sampled from the hulls of
two US Navy destroyers. Prokaryotic community analyses using PhyloChip-based 16S
rDNA profiling revealed two significantly different and taxonomically rich
biofilm communities (6,942 taxa) in which the majority of unique taxa were
ascribed to members of the Gammaproteobacteria, Alphaproteobacteria and
Clostridia. Although metagenomic sequencing indicated that both biofilms were
dominated by prokaryotic sequence reads (> 91%) with the majority of the
bacterial reads belonging to the Alphaproteobacteria, the Ship-1 metagenome
harbored greater organismal and functional diversity and was comparatively
enriched for sequences from Cyanobacteria, Bacteroidetes and macroscopic
eukaryotes, whereas the Ship-2 metagenome was enriched for sequences from
Proteobacteria and microscopic photosynthetic eukaryotes. Qualitative liquid
chromatography-tandem mass spectrometry metaproteome analyses identified 678
unique proteins, revealed little overlap in species and protein composition
between the ships and contrasted with the metagenomic data in that ~80% of
classified and annotated proteins were of eukaryotic origin and dominated by
members of the Bacillariophyta, Cnidaria, Chordata and Arthropoda (data deposited
to the ProteomeXchange, identifier PXD000961). Within the shared metaproteome,
quantitative (18)O and iTRAQ analyses demonstrated a significantly greater
abundance of structural proteins from macroscopic eukaryotes on Ship-1 and diatom
photosynthesis proteins on Ship-2. Photosynthetic pigment composition and
elemental analyses confirmed that both biofilms were dominated by phototrophic
processes. These data begin to provide a better understanding of the complex
organismal and biomolecular composition of marine biofilms while highlighting
caveats in the interpretation of stand-alone environmental '-omics' datasets.
PMID: 25407927 [PubMed - indexed for MEDLINE]
92. PLoS One. 2013 Oct 22;8(10):e76724. doi: 10.1371/journal.pone.0076724.
Ultradeep 16S rRNA sequencing analysis of geographically similar but diverse
unexplored marine samples reveal varied bacterial community composition.
Aravindraja C(1), Viszwapriya D, Karutha Pandian S.
(1)Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu,
BACKGROUND: Bacterial community composition in the marine environment differs
from one geographical location to another. Reports that delineate the bacterial
diversity of different marine samples from geographically similar location are
limited. The present study aims to understand whether the bacterial community
compositions from different marine samples harbour similar bacterial diversity
since these are geographically related to each other.
METHODS AND PRINCIPAL FINDINGS: In the present study, 16S rRNA deep sequencing
analysis targeting V3 region was performed using Illumina bar coded sequencing. A
total of 22.44 million paired end reads were obtained from the metagenomic DNA of
Marine sediment, Rhizosphere sediment, Seawater and the epibacterial DNA of
Seaweed and Seagrass. Diversity index analysis revealed that Marine sediment has
the highest bacterial diversity and the least bacterial diversity was observed in
Rhizosphere sediment. Proteobacteria, Actinobacteria and Bacteroidetes were the
dominant taxa present in all the marine samples. Nearly 62-71% of rare species
were identified in all the samples and most of these rare species were unique to
a particular sample. Further taxonomic assignment at the phylum and genus level
revealed that the bacterial community compositions differ among the samples.
CONCLUSION: This is the first report that supports the fact that, bacterial
community composition is specific for specific samples irrespective of its
similar geographical location. Existence of specific bacterial community for each
sample may drive overall difference in bacterial structural composition of each
sample. Further studies like whole metagenomic sequencing will throw more
insights to the key stone players and its interconnecting metabolic pathways. In
addition, this is one of the very few reports that depicts the unexplored
bacterial diversity of marine samples (Marine sediment, Rhizosphere sediment,
Seawater) and the host associated marine samples (Seaweed and Seagrass) at higher
depths from uncharacterised coastal region of Palk Bay, India using next
generation sequencing technology.
PMID: 24167548 [PubMed - indexed for MEDLINE]
93. Nucleic Acids Res. 2013 Jan 7;41(1):e1. doi: 10.1093/nar/gks808. Epub 2012 Aug
Evaluation of general 16S ribosomal RNA gene PCR primers for classical and
next-generation sequencing-based diversity studies.
Klindworth A(1), Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO.
(1)Max Planck Institute for Marine Microbiology, Microbial Genomics and
Bioinformatics Research Group, Celsiusstr 1, 28359 Bremen, Germany.
16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for
the cultivation-independent investigation of microbial diversity. The accuracy of
these analyses depends strongly on the choice of primers. The overall coverage
and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico
with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef
108 NR). Based on this evaluation a selection of 'best available' primer pairs
for Bacteria and Archaea for three amplicon size classes (100-400, 400-1000, ≥
1000 bp) is provided. The most promising bacterial primer pair
(S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was
experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA
amplicons with 16S rDNA fragments from directly sequenced metagenomes. The
results of this study may be used as a guideline for selecting primer pairs with
the best overall coverage and phylum spectrum for specific applications,
therefore reducing the bias in PCR-based microbial diversity studies.
PMID: 22933715 [PubMed - indexed for MEDLINE]
94. PLoS One. 2013;8(3):e57782. doi: 10.1371/journal.pone.0057782. Epub 2013 Mar 4.
Detection of transient bacteraemia following dental extractions by 16S rDNA
pyrosequencing: a pilot study.
Benítez-Páez A(1), Álvarez M, Belda-Ferre P, Rubido S, Mira A, Tomás I.
(1)Genomics and Health Department, Centre for Advanced Research in Public Health,
CSISP, Valencia, Spain.
OBJECTIVE: The current manuscript aims to determine the prevalence, duration and
bacterial diversity of bacteraemia following dental extractions using
conventional culture-dependent methods and 16S rDNA pyrosequencing.
METHODS: The study group included 8 patients undergoing dental extractions under
general anaesthesia. Peripheral venous blood samples were collected at baseline,
30 seconds and 15 minutes after the dental extractions. Blood samples were
analysed for bacteraemia applying conventional microbiological cultures under
aerobic and anaerobic conditions as well as pyrosequencing using universal
bacterial primers that target the 16S ribosomal DNA gene.
RESULTS: Transient bacteremia was detected by culture-based methods in one sample
at baseline time, in eight samples at 30 seconds, and in six samples at 15
minutes after surgical procedure; whereas bacteraemia was detected only in five
blood samples at 30 seconds after dental extraction by using pyrosequencing. By
applying conventional microbiological methods, a single microbial species was
detected in six patients, and Streptococcus viridans was the most frequently
cultured identified bacterium. By using pyrosequencing approaches however, the
estimated blood microbial diversity after dental extractions was 13.4±1.7
bacterial families and 22.8±1.1 genera per sample.
CONCLUSION: The application of 16S rDNA pyrosequencing underestimated the
prevalence and duration of bacteraemia following dental extractions, presumably
due to not reaching the minimum DNA required for PCR amplification. However, this
molecular technique, unlike conventional culture-dependent methods, revealed an
extraordinarily high bacterial diversity of post-extraction bacteraemia. We
propose that microorganisms recovered by culture may be only the tip of an
iceberg of a really diverse microbiota whose viability and potential
pathogenicity should be further studied.
PMID: 23469240 [PubMed - indexed for MEDLINE]
95. ISME J. 2015 Sep;9(9):2028-45. doi: 10.1038/ismej.2015.22. Epub 2015 Mar 3.
Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading
enrichment cultures with 41 environmental samples.
Tan B(1), Fowler SJ(2), Abu Laban N(3), Dong X(4), Sensen CW(4), Foght J(1), Gieg
(1)Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada. (2)Department of Biological Sciences, University of Calgary, Calgary,
Alberta, Canada. (3)1] Department of Biological Sciences, University of Alberta,
Edmonton, Alberta, Canada  Department of Biological Sciences, University of
Calgary, Calgary, Alberta, Canada. (4)Visual Genomics Centre, Faculty of
Medicine, Calgary, Alberta, Canada.
Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs
and hydrocarbon-contaminated environments and thus warrants greater understanding
to improve current technologies for fossil fuel extraction and bioremediation. In
this study, three hydrocarbon-degrading methanogenic cultures established from
two geographically distinct environments and incubated with different hydrocarbon
substrates (added as single hydrocarbons or as mixtures) were subjected to
metagenomic and 16S rRNA gene pyrosequencing to test whether these differences
affect the genetic potential and composition of the communities. Enrichment of
different putative hydrocarbon-degrading bacteria in each culture appeared to be
substrate dependent, though all cultures contained both acetate- and H2-utilizing
methanogens. Despite differing hydrocarbon substrates and inoculum sources, all
three cultures harbored genes for hydrocarbon activation by fumarate addition
(bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for
associated downstream pathways (bbs, bcr, bam), though the cultures incubated
with hydrocarbon mixtures contained a broader diversity of fumarate addition
genes. A comparative metagenomic analysis of the three cultures showed that they
were functionally redundant despite their enrichment backgrounds, sharing
multiple features associated with syntrophic hydrocarbon conversion to methane.
In addition, a comparative analysis of the culture metagenomes with those of 41
environmental samples (containing varying proportions of methanogens) showed that
the three cultures were functionally most similar to each other but distinct from
other environments, including hydrocarbon-impacted environments (for example, oil
sands tailings ponds and oil-affected marine sediments). This study provides a
basis for understanding key functions and environmental selection in methanogenic
PMID: 25734684 [PubMed - indexed for MEDLINE]
96. Microbiologyopen. 2013 Apr;2(2):276-83. doi: 10.1002/mbo3.73. Epub 2013 Feb 17.
Analysis of the bacterial communities associated with two ant-plant symbioses.
Seipke RF(1), Barke J, Heavens D, Yu DW, Hutchings MI.
(1)School of Biological Sciences, University of East Anglia, Norwich Research
Park, Norwich NR4 7TJ, United Kingdom. firstname.lastname@example.org
Insect fungiculture is practiced by ants, termites, beetles, and gall midges and
it has been suggested to be widespread among plant-ants. Some of the insects
engaged in fungiculture, including attine ants and bark beetles, are known to use
symbiotic antibiotic-producing actinobacteria to protect themselves and their
fungal cultivars against infection. In this study, we analyze the bacterial
communities on the cuticles of the plant-ant genera Allomerus and Tetraponera
using deep sequencing of 16S rRNA. Allomerus ants cultivate fungus as a building
material to strengthen traps for prey, while Tetraponera ants cultivate fungus as
a food source. We report that Allomerus and Tetraponera microbiomes contain >75%
Proteobacteria and remarkably the bacterial phyla that dominate their cuticular
microbiomes are very similar despite their geographic separation (South America
and Africa, respectively). Notably, antibiotic-producing actinomycete bacteria
represent a tiny fraction of the cuticular microbiomes of both Allomerus and
Tetraponera spp. and instead they are dominated by γ-proteobacteria Erwinia and
Serratia spp. Both these phyla are known to contain antibiotic-producing species
which might therefore play a protective role in these ant-plant systems.
© 2013 The Authors. Published by Blackwell Publishing Ltd.
PMID: 23417898 [PubMed - indexed for MEDLINE]
97. ISME J. 2014 Feb;8(2):478-91. doi: 10.1038/ismej.2013.159. Epub 2013 Sep 12.
Making a living while starving in the dark: metagenomic insights into the energy
dynamics of a carbonate cave.
Ortiz M(1), Legatzki A(1), Neilson JW(1), Fryslie B(2), Nelson WM(3), Wing RA(4),
Soderlund CA(3), Pryor BM(4), Maier RM(1).
(1)Department of Soil, Water and Environmental Science, University of Arizona,
Tucson, AZ, USA. (2)Department of Computer Science, University of Arizona,
Tucson, AZ, USA. (3)BIO5 Institute, Tucson, AZ, USA. (4)Department of Plant
Sciences, University of Arizona, Tucson, AZ, USA.
Carbonate caves represent subterranean ecosystems that are largely devoid of
phototrophic primary production. In semiarid and arid regions, allochthonous
organic carbon inputs entering caves with vadose-zone drip water are minimal,
creating highly oligotrophic conditions; however, past research indicates that
carbonate speleothem surfaces in these caves support diverse, predominantly
heterotrophic prokaryotic communities. The current study applied a metagenomic
approach to elucidate the community structure and potential energy dynamics of
microbial communities, colonizing speleothem surfaces in Kartchner Caverns, a
carbonate cave in semiarid, southeastern Arizona, USA. Manual inspection of a
speleothem metagenome revealed a community genetically adapted to low-nutrient
conditions with indications that a nitrogen-based primary production strategy is
probable, including contributions from both Archaea and Bacteria. Genes for all
six known CO2-fixation pathways were detected in the metagenome and RuBisCo genes
representative of the Calvin-Benson-Bassham cycle were over-represented in
Kartchner speleothem metagenomes relative to bulk soil, rhizosphere soil and
deep-ocean communities. Intriguingly, quantitative PCR found Archaea to be
significantly more abundant in the cave communities than in soils above the cave.
MEtaGenome ANalyzer (MEGAN) analysis of speleothem metagenome sequence reads
found Thaumarchaeota to be the third most abundant phylum in the community, and
identified taxonomic associations to this phylum for indicator genes
representative of multiple CO2-fixation pathways. The results revealed that this
oligotrophic subterranean environment supports a unique chemoautotrophic
microbial community with potentially novel nutrient cycling strategies. These
strategies may provide key insights into other ecosystems dominated by
oligotrophy, including aphotic subsurface soils or aquifers and photic systems
such as arid deserts.
PMID: 24030597 [PubMed - indexed for MEDLINE]
98. Arch Dis Child Fetal Neonatal Ed. 2013 Jul;98(4):F334-40. doi:
10.1136/archdischild-2012-303035. Epub 2013 Jan 8.
The individual-specific and diverse nature of the preterm infant microbiota.
Barrett E(1), Kerr C, Murphy K, O'Sullivan O, Ryan CA, Dempsey EM, Murphy BP,
O'Toole PW, Cotter PD, Fitzgerald GF, Ross RP, Stanton C.
(1)Food Biosciences Department, Teagasc Food Research Centre, Moorepark, Fermoy,
County Cork, Ireland.
OBJECTIVE: To examine the composition of the evolving microbiota of preterm
infants at weeks 2 and 4 of life.
SETTINGS: The paediatric intensive care unit of the Cork University Maternity
METHODS: The microbial diversity of faecal samples from 10 preterm infants was
determined using 16S rRNA amplicon pyrosequencing technology.
RESULTS: In total, 452 863 sequences were obtained from 20 faecal samples
collected from 10 preterm infants, allowing a level of analysis not previously
reported. The preterm infant microbiota samples were dominated by Proteobacteria
(46%), followed by Firmicutes (45%), while the phyla Actinobacteria (2%) and
Bacteroidetes (7%) were detected at much lower levels at week 2 of life. This
colonisation pattern was similar at week 4 of life. At the family level,
Enterobacteriaceae were detected at 50% and 58% at weeks 2 and 4, respectively.
The preterm infants were characterised by a lack of detectable Bifidobacterium
and Lactobacillus genera commonly associated with the infant gut. In addition to
the dominance of the Proteobacteria, a high level of interindividual variation
was observed, indeed the relative proportions of different phyla, families and
genera in different infants ranged from <1% to >90%.
CONCLUSIONS: The results indicate that in addition to an uncharacteristic
microbiota relative to that reported for healthy term infants, there was a large
interindividual variation in the faecal microbiota diversity of preterm infants
suggesting that the preterm microbiota is individual-specific and does not
display a uniformity among infants.
PMID: 23303303 [PubMed - indexed for MEDLINE]
99. Appl Microbiol Biotechnol. 2013 Mar;97(6):2681-90. doi:
10.1007/s00253-012-4082-4. Epub 2012 May 5.
Bacterial communities in different sections of a municipal wastewater treatment
plant revealed by 16S rDNA 454 pyrosequencing.
Ye L(1), Zhang T.
(1)Environmental Biotechnology Lab, Department of Civil Engineering, The
University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
In this study, we successfully demonstrated that 454 pyrosequencing was a
powerful approach for investigating the bacterial communities in the activated
sludge, digestion sludge, influent, and effluent samples of a full scale
wastewater treatment plant treating saline sewage. For each sample, 18,808
effective sequences were selected and utilized to do the bacterial diversity and
abundance analysis. In total, 2,455, 794, 1,667, and 1,932 operational taxonomic
units were obtained at 3 % distance cutoff in the activated sludge, digestion
sludge, influent, and effluent samples, respectively. The corresponding most
dominant classes in the four samples are Alphaproteobacteria, Thermotogae,
Deltaproteobacteria, and Gammaproteobacteria. About 67 % sequences in the
digestion sludge sample were found to be affiliated with the Thermotogales order.
Also, these sequences were assigned into a recently proposed genus Kosmotoga by
the Ribosomal Database Project classifier. In the effluent sample, we found high
abundance of Mycobacterium and Vibrio, which are genera containing pathogenic
bacteria. Moreover, in this study, we proposed a method to differentiate the
"gene percentage" and "cell percentage" by using Ribosomal RNA Operon Copy Number
PMID: 22555912 [PubMed - indexed for MEDLINE]
100. Sci Rep. 2015 Sep 22;5:14266. doi: 10.1038/srep14266.
An integrated insight into the response of sedimentary microbial communities to
heavy metal contamination.
Yin H(1,)(2), Niu J(1,)(2), Ren Y(3), Cong J(1,)(2), Zhang X(4,)(5), Fan
F(6,)(5), Xiao Y(1,)(2), Zhang X(1,)(2), Deng J(7), Xie M(7), He Z(7), Zhou
J(7,)(8,)(9), Liang Y(1,)(2), Liu X(1,)(2).
(1)School of Minerals Processing and Bioengineering, Central South University,
Changsha 410083, China. (2)Key laboratory of Biometallurgy, Ministry of
Education, Changsha 410083, China. (3)College of Food Science and Technology,
Hunan Agricultural University, Changsha 410083, China. (4)Key Laboratory of
Microbial Resources Collection and Preservation, Ministry of Agriculture, Beijing
100081, China. (5)Institute of Agricultural Resources and Regional Planning,
Chinese Academy of Agricultural Sciences, Beijing 100081, China. (6)Key
Laboratory of Plant Nutrition and Fertilizer, Beijing 100081, China. (7)Institute
for Environmental Genomics and Department of Botany and Microbiology, University
of Oklahoma, Norman 73019, USA. (8)School of Environment, Tsinghua University,
Beijing 100084, China. (9)Earth Sciences Division, Lawrence Berkeley National
Laboratory, Berkeley 94710, USA.
Response of biological communities to environmental stresses is a critical issue
in ecology, but how microbial communities shift across heavy metal gradients
remain unclear. To explore the microbial response to heavy metal contamination
(e.g., Cr, Mn, Zn), the composition, structure and functional potential of
sedimentary microbial community were investigated by sequencing of 16S rRNA gene
amplicons and a functional gene microarray. Analysis of 16S rRNA sequences
revealed that the composition and structure of sedimentary microbial communities
changed significantly across a gradient of heavy metal contamination, and the
relative abundances were higher for Firmicutes, Chloroflexi and Crenarchaeota,
but lower for Proteobacteria and Actinobacteria in highly contaminated samples.
Also, molecular ecological network analysis of sequencing data indicated that
their possible interactions might be enhanced in highly contaminated communities.
Correspondently, key functional genes involved in metal homeostasis (e.g., chrR,
metC, merB), carbon metabolism, and organic remediation showed a higher abundance
in highly contaminated samples, indicating that bacterial communities in
contaminated areas may modulate their energy consumption and organic remediation
ability. This study indicated that the sedimentary indigenous microbial community
may shift the composition and structure as well as function priority and
interaction network to increase their adaptability and/or resistance to
PMID: 26391875 [PubMed - indexed for MEDLINE]
36. Front Microbiol. 2016 Jul 19;7:1122. doi: 10.3389/fmicb.2016.01122. eCollection
Methane Inhibition Alters the Microbial Community, Hydrogen Flow, and
Fermentation Response in the Rumen of Cattle.
Martinez-Fernandez G(1), Denman SE(1), Yang C(2), Cheung J(1), Mitsumori M(3),
(1)CSIRO, Agriculture and Food, Queensland Bioscience Precinct St Lucia, QLD,
Australia. (2)Institute of Dairy Science, MoE Key Laboratory of Molecular Animal
Nutrition, College of Animal Sciences, Zhejiang University Hangzhou, China.
(3)NARO Institute of Livestock and Grassland Science Tsukuba, Japan.
Management of metabolic hydrogen ([H]) in the rumen has been identified as an
important consideration when reducing ruminant CH4 emissions. However, little is
known about hydrogen flux and microbial rumen population responses to CH4
inhibition when animals are fed with slowly degradable diets. The effects of the
anti-methanogenic compound, chloroform, on rumen fermentation, microbial ecology,
and H2/CH4 production were investigated in vivo. Eight rumen fistulated Brahman
steers were fed a roughage hay diet (Rhode grass hay) or roughage hay:concentrate
diet (60:40) with increasing levels (low, mid, and high) of chloroform in a
cyclodextrin matrix. The increasing levels of chloroform resulted in an increase
in H2 expelled as CH4 production decreased with no effect on dry matter intakes.
The amount of expelled H2 per mole of decreased methane, was lower for the hay
diet suggesting a more efficient redirection of hydrogen into other microbial
products compared with hay:concentrate diet. A shift in rumen fermentation toward
propionate and branched-chain fatty acids was observed for both diets. Animals
fed with the hay:concentrate diet had both higher formate concentration and H2
expelled than those fed only roughage hay. Metabolomic analyses revealed an
increase in the concentration of amino acids, organic, and nucleic acids in the
fluid phase for both diets when methanogenesis was inhibited. These changes in
the rumen metabolism were accompanied by a shift in the microbiota with an
increase in Bacteroidetes:Firmicutes ratio and a decrease in Archaea and
Synergistetes for both diets. Within the Bacteroidetes family, some OTUs assigned
to Prevotella were promoted under chloroform treatment. These bacteria may be
partly responsible for the increase in amino acids and propionate in the rumen.
No significant changes were observed for abundance of fibrolytic bacteria,
protozoa, and fungi, which suggests that fiber degradation was not impaired. The
observed 30% decrease in methanogenesis did not adversely affect rumen metabolism
and the rumen microbiota was able to adapt and redirect [H] into other microbial
end-products for both diets. However, it is also required dietary supplements or
microbial treatments to capture the additional H2 expelled by the animal to
further improve rumen digestive efficiency.
PMID: 27486452 [PubMed]
37. Elife. 2013 Nov 5;2:e01202. doi: 10.7554/eLife.01202.
Expansion of intestinal Prevotella copri correlates with enhanced susceptibility
Scher JU(1), Sczesnak A, Longman RS, Segata N, Ubeda C, Bielski C, Rostron T,
Cerundolo V, Pamer EG, Abramson SB, Huttenhower C, Littman DR.
(1)Department of Medicine, New York University School of Medicine and Hospital
for Joint Diseases, New York, United States.
Nat Rev Microbiol. 2014 Jan;12(1):5.
Nat Rev Rheumatol. 2014 Jan;10(1):2.
Rheumatoid arthritis (RA) is a prevalent systemic autoimmune disease, caused by a
combination of genetic and environmental factors. Animal models suggest a role
for intestinal bacteria in supporting the systemic immune response required for
joint inflammation. Here we performed 16S sequencing on 114 stool samples from
rheumatoid arthritis patients and controls, and shotgun sequencing on a subset of
44 such samples. We identified the presence of Prevotella copri as strongly
correlated with disease in new-onset untreated rheumatoid arthritis (NORA)
patients. Increases in Prevotella abundance correlated with a reduction in
Bacteroides and a loss of reportedly beneficial microbes in NORA subjects. We
also identified unique Prevotella genes that correlated with disease. Further,
colonization of mice revealed the ability of P. copri to dominate the intestinal
microbiota and resulted in an increased sensitivity to chemically induced
colitis. This work identifies a potential role for P. copri in the pathogenesis
of RA. DOI: http://dx.doi.org/10.7554/eLife.01202.001.
PMID: 24192039 [PubMed - indexed for MEDLINE]
38. Gigascience. 2016 Aug 2;5(1):34. doi: 10.1186/s13742-016-0140-7.
INC-Seq: accurate single molecule reads using nanopore sequencing.
Li C(1,)(2), Chng KR(1), Boey EJ(1), Ng AH(1), Wilm A(1), Nagarajan N(3,)(4).
(1)Genome Institute of Singapore, Singapore, 138672, Singapore. (2)Department of
Computer Science, National University of Singapore, Singapore, 117417, Singapore.
(3)Genome Institute of Singapore, Singapore, 138672, Singapore.
email@example.com. (4)Department of Computer Science, National
University of Singapore, Singapore, 117417, Singapore.
BACKGROUND: Nanopore sequencing provides a rapid, cheap and portable real-time
sequencing platform with the potential to revolutionize genomics. However,
several applications are limited by relatively high single-read error rates
(>10 %), including RNA-seq, haplotype sequencing and 16S sequencing.
RESULTS: We developed the Intramolecular-ligated Nanopore Consensus Sequencing
(INC-Seq) as a strategy for obtaining long and accurate nanopore reads, starting
with low input DNA. Applying INC-Seq for 16S rRNA-based bacterial profiling
generated full-length amplicon sequences with a median accuracy >97 %.
CONCLUSIONS: INC-Seq reads enabled accurate species-level classification,
identification of species at 0.1 % abundance and robust quantification of
relative abundances, providing a cheap and effective approach for pathogen
detection and microbiome profiling on the MinION system.
PMID: 27485345 [PubMed - in process]
39. Ecology. 2016 May;97(5):1307-18.
Climate and edaphic controllers influence rhizosphere community assembly for a
wild annual grass.
Nuccio EE, Anderson-Furgeson J, Estera KY, Pett-Ridge J, De Valpine P, Brodie EL,
The interface between roots and soil, known as the rhizosphere, is a dynamic
habitat in the soil ecosystem. Unraveling the factors that control rhizosphere
community assembly is a key starting point for understanding the diversity of
plant-microbial interactions that occur in soil. The goals of this study were to
determine how environmental factors shape rhizosphere microbial communities, such
as local soil characteristics and the regional climate, and to determine the
relative influence of the rhizosphere on microbial community assembly compared to
the pressures imposed by the local and regional environment. We identified the
bacteria present in the soil immediately adjacent to the roots of wild oat (A
vena spp.) in three California grasslands using deep Illumina 16S sequencing.
Rhizosphere communities were more similar to each other than to the surrounding
soil communities from which they were derived, despite the fact that the
grasslands studied were separated by hundreds of kilometers. The rhizosphere was
the dominant factor structuring bacterial community composition (38% variance
explained), and was comparable in magnitude to the combined local and regional
effects (22% and 21%, respectively). Rhizosphere communities were most influenced
by factors related to the regional climate (soil moisture and temperature), while
background soil communities were more influenced by soil characteristics (pH,
CEC, exchangeable cations, clay content). The Avena core microbiome was strongly
phylogenetically clustered according to the metrics NRI and NTI, which indicates
that selective processes likely shaped these communities. Furthermore, 17% of
these taxa were not detectable in the background soil, even with a robust
sequencing depth of approximately 70,000 sequences per sample. These results
support the hypothesis that roots select less abundant or possibly rare
populations in the soil microbial community, which appear to be lineages of
bacteria that have made a physiological tradeoff for rhizosphere competence at
the expense of their competitiveness in non-rhizosphere soil.
PMID: 27349106 [PubMed - indexed for MEDLINE]
40. Anaerobe. 2016 Apr;38:130-7. doi: 10.1016/j.anaerobe.2015.10.006. Epub 2015 Oct
Comparison of culture-dependent and independent approaches to characterize fecal
bifidobacteria and lactobacilli.
Quartieri A(1), Simone M(1), Gozzoli C(1), Popovic M(1), D'Auria G(2), Amaretti
A(1), Raimondi S(1), Rossi M(3).
(1)Department of Life Sciences, University of Modena and Reggio Emilia, Modena,
Italy. (2)Genómica y Salud, Fundación para el Fomento de la Investigación
Sanitaria y Biomédica de la Comunidad Valenciana, Valencia, Spain. (3)Department
of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
Electronic address: firstname.lastname@example.org.
Different culture-dependent and independent methods were applied to investigate
the population of bifidobacteria and lactobacilli in the feces of five healthy
subjects. Bacteria were isolated on MRS, a complex medium supporting growth of
lactobacilli and bifidobacteria, and on three selective media for bifidobacteria
and two for lactobacilli. Taxonomic characterization of the isolates was carried
out by RAPD-PCR and partial 16S sequencing. The selectivity of genus-specific
media was also investigated by challenging colonies from MRS plates to grow onto
each medium. In parallel, a quantitative and qualitative description of
bifidobacteria and lactic acid bacteria was obtained by FISH, qPCR, TRFLP, and
16S rRNA gene sequencing. Bifidobacteria did not fail to grow on their specific
media and were easily isolated and enumerated, showing comparable quantitative
data among culture-dependent and -independent techniques. The Bifidobacterium
species identified on plates and those extracted from TRFLP and 16S rRNA gene
sequencing were mostly overlapping. Selective media for lactobacilli gave
unsuitable results, being too stringent or too permissive. The quantification of
lactobacilli through selective plates, qPCR, FISH, and 16S rRNA gene sequencing
gave unreliable results. Therefore, unlike bifidobacteria, intestinal
lactobacilli are still problematic in terms of quantification and accurate
profiling at level of species and possibly of strains by both culture-dependent
and culture-independent techniques.
Copyright © 2015 Elsevier Ltd. All rights reserved.
PMID: 26481833 [PubMed - in process]
16s sequencing illumina or PGM low cost prices with MR DNA
Environ Sci Pollut Res Int. 2016 Jul;23(13):13245-54. doi: 10.1007/s11356-016-6474-y. Epub 2016 Mar 29.
Influence of zinc nanoparticles on survival of worms Eisenia fetida and taxonomic diversity of the gut microflora.
Yausheva Е1, Sizova Е2,3, Lebedev S2,3, Skalny A2, Miroshnikov S3, Plotnikov A4, Khlopko Y4, Gogoleva N5, Cherkasov S4.
The study was conducted to examine the effect of zinc nanoparticles on survival of worms Eisenia fetida and composition of the gut microflora. Analysis of the survival data has shown that the introduction of high doses of the nanoparticles causes death of worms in the second group with 35 % mortality rate and activates protective mechanisms realized as mucous film. DNA from the worm guts was extracted and 16S metagenomic sequencing was fulfilled using MiSeq (Illumina). Regarding the gut microflora of worms in the control group, high diversity of microorganisms (303 OTUs) was noted. Most of those belong to the taxa Firmicutes (51.9 % of the total high-quality united reads), Proteobacteria (24.1 % of the total), and Actinobacteria (13.3 % of the total), which were represented by numerous species of gen. Clostridium (C. saccharobutylicum, C. saccharoperbutylacetonicum, C. beijerinckii), gen. Pseudomonas (P. hydrogenovora, P. aeruginosa, and P. putida), gen. Bacillus (B. megaterium, B. silvestris), gen. Cellulomonas (B. megaterium, B. silvestris), and other numerically smaller genera. Adding of zinc nanoparticles to the substrate decreased the diversity of bacteria (78 OTUs) as well as percentage of bacteria belonging to the taxon Firmicutes (-41.6 %) and increased the proportion of Proteobacteria due to growth in abundance of gen. Verminephrobacter (+46 %) and gen. Ochrobactrum (+19.5 %).
16S metagenome; Eisenia fetida; High-throughput sequencing; Microcrystalline cellulose; Microflora; Nanoparticles; Taxa; Zinc
PMID: 27023811 DOI: 10.1007/s11356-016-6474-y
[PubMed - in process]
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5.Publication Types, MeSH Terms
Select item 26067561
Sci Rep. 2015 Jun 12;5:10044. doi: 10.1038/srep10044.
Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing.
Chao Y1, Mao Y2, Wang Z3, Zhang T2.
The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.
PMID: 26067561 PMCID: PMC4464384 DOI: 10.1038/srep10044
[PubMed - indexed for MEDLINE] Free PMC Article
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Select item 26035208
PLoS One. 2015 Jun 2;10(6):e0128711. doi: 10.1371/journal.pone.0128711. eCollection 2015.
Screening currency notes for microbial pathogens and antibiotic resistance genes using a shotgun metagenomic approach.
Jalali S1, Kohli S2, Latka C3, Bhatia S4, Vellarikal SK5, Sivasubbu S5, Scaria V1, Ramachandran S1.
Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents.
PMID: 26035208 PMCID: PMC4452720 DOI: 10.1371/journal.pone.0128711
[PubMed - indexed for MEDLINE] Free PMC Article
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Select item 25910603
Microb Ecol. 2015 Oct;70(3):701-9. doi: 10.1007/s00248-015-0611-x. Epub 2015 Apr 26.
Deciphering Cyanide-Degrading Potential of Bacterial Community Associated with the Coking Wastewater Treatment Plant with a Novel Draft Genome.
Wang Z1,2, Liu L1,3, Guo F1, Zhang T4.
Biotreatment processes fed with coking wastewater often encounter insufficient removal of pollutants, such as ammonia, phenols, and polycyclic aromatic hydrocarbons (PAHs), especially for cyanides. However, only a limited number of bacterial species in pure cultures have been confirmed to metabolize cyanides, which hinders the improvement of these processes. In this study, a microbial community of activated sludge enriched in a coking wastewater treatment plant was analyzed using 454 pyrosequencing and Illumina sequencing to characterize the potential cyanide-degrading bacteria. According to the classification of these pyro-tags, targeting V3/V4 regions of 16S rRNA gene, half of them were assigned to the family Xanthomonadaceae, implying that Xanthomonadaceae bacteria are well-adapted to coking wastewater. A nearly complete draft genome of the dominant bacterium was reconstructed from metagenome of this community to explore cyanide metabolism based on analysis of the genome. The assembled 16S rRNA gene from this draft genome showed that this bacterium was a novel species of Thermomonas within Xanthomonadaceae, which was further verified by comparative genomics. The annotation using KEGG and Pfam identified genes related to cyanide metabolism, including genes responsible for the iron-harvesting system, cyanide-insensitive terminal oxidase, cyanide hydrolase/nitrilase, and thiosulfate:cyanide transferase. Phylogenetic analysis showed that these genes had homologs in previously identified genomes of bacteria within Xanthomonadaceae and even presented similar gene cassettes, thus implying an inherent cyanide-decomposing potential. The findings of this study expand our knowledge about the bacterial degradation of cyanide compounds and will be helpful in the remediation of cyanides contamination.
Activated sludge; Coking wastewater; Cyanides; Metagenome; Thermomonas
PMID: 25910603 DOI: 10.1007/s00248-015-0611-x
[PubMed - indexed for MEDLINE]
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Microb Cell Fact. 2015 Mar 14;14:33. doi: 10.1186/s12934-015-0218-4.
Dissecting microbial community structure and methane-producing pathways of a full-scale anaerobic reactor digesting activated sludge from wastewater treatment by metagenomic sequencing.
Guo J1,2, Peng Y3, Ni BJ4, Han X5,6, Fan L7, Yuan Z8.
Anaerobic digestion has been widely applied to treat the waste activated sludge from biological wastewater treatment and produce methane for biofuel, which has been one of the most efficient solutions to both energy crisis and environmental pollution challenges. Anaerobic digestion sludge contains highly complex microbial communities, which play crucial roles in sludge treatment. However, traditional approaches based on 16S rRNA amplification or fluorescent in situ hybridization cannot completely reveal the whole microbial community structure due to the extremely high complexity of the involved communities. In this sense, the next-generation high-throughput sequencing provides a powerful tool for dissecting microbial community structure and methane-producing pathways in anaerobic digestion.
In this work, the metagenomic sequencing was used to characterize microbial community structure of the anaerobic digestion sludge from a full-scale municipal wastewater treatment plant. Over 3.0 gigabases of metagenomic sequence data were generated with the Illumina HiSeq 2000 platform. Taxonomic analysis by MG-RAST server indicated that overall bacteria were dominant (~93%) whereas a considerable abundance of archaea (~6%) were also detected in the anaerobic digestion sludge. The most abundant bacterial populations were found to be Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Key microorganisms and related pathways involved in methanogenesis were further revealed. The dominant proliferation of Methanosaeta and Methanosarcina, together with the functional affiliation of enzymes-encoding genes (acetate kinase (AckA), phosphate acetyltransferase (PTA), and acetyl-CoA synthetase (ACSS)), suggested that the acetoclastic methanogenesis is the dominant methanogenesis pathway in the full-scale anaerobic digester.
In short, the metagenomic sequencing study of this work successfully dissected the detail microbial community structure and the dominated methane-producing pathways of a full-scale anaerobic digester. The knowledge garnered would facilitate to develop more efficient full-scale anaerobic digestion systems to achieve high-rate waste sludge treatment and methane production.
PMID: 25880314 PMCID: PMC4381419 DOI: 10.1186/s12934-015-0218-4
[PubMed - indexed for MEDLINE] Free PMC Article
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Appl Biochem Biotechnol. 2015 Apr;175(7):3258-70. doi: 10.1007/s12010-015-1491-8. Epub 2015 Feb 10.
Metagenomic analysis of the sludge microbial community in a lab-scale denitrifying phosphorus removal reactor.
Lv XM1, Shao MF, Li J, Li CL.
Denitrifying phosphorus removal is an attractive wastewater treatment process due to its reduced carbon source demand and sludge minimization potential. In the present study, the metagenome of denitrifying phosphorus removal sludge from a lab-scale anaerobic-anoxic SBR was generated by Illumina sequencing to study the microbial community. Compared with the aerobic phosphorus removal sludge, the denitrifying phosphorus removal sludge demonstrated quite similar microbial community profile and microbial diversity with sludge from Aalborg East EBPR WWTP. Proteobacteria was the most dominant phylum; within Proteobacteria, β-Proteobacteria was the most dominant class, followed by α-, γ-, δ-, and ε-Proteobacteria. The genes involved in phosphate metabolism and biofilm formation reflected the selective pressure of the phosphorus removal process. Moreover, ppk sequence from DPAO was outside the Accumulibacter clusters, which suggested different core phosphorus removal bacteria in denitrifying and aerobic phosphorus removal systems. In a summary, putative DPAO might be a novel genus that is closely related between Accumulibacter and Dechloromonas within Rhodocyclus. The microbial community and metabolic profiles achieved in this study will eventually help to improve the understanding of key microorganisms and the entire community in order to improve the phosphorus removal efficiency of EBPR processes.
PMID: 25820294 DOI: 10.1007/s12010-015-1491-8
[PubMed - indexed for MEDLINE]
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16s rRNA Sequencing with MR DNA
16S ribosomal (rRNA) sequencing using next generation sequencing is a method used to identify and compare bacteria and archaea present within almost any type of sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.
16s sequencing illumina or PGM low cost prices with MR DNA