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68. Anim Health Res Rev. 2012 Jun;13(1):78-88. doi: 10.1017/S1466252312000059. Epub

2012 May 30.


Current state of knowledge: the canine gastrointestinal microbiome.


Hooda S(1), Minamoto Y, Suchodolski JS, Swanson KS.


Author information:

(1)Department of Animal Sciences, University of Illinois, 1207 West Gregory

Drive, Urbana, IL 61801, USA.


Gastrointestinal (GI) microbes have important roles in the nutritional,

immunological, and physiologic processes of the host. Traditional cultivation

techniques have revealed bacterial density ranges from 10(4) to 10(5) colony

forming units (CFU)/g in the stomach, from 10(5) to 10(7) CFU/g in the small

intestine, and from 10(9) to 10(11) CFU/g in the colon of healthy dogs. As a

small number of bacterial species can be grown and studied in culture, however,

progress was limited until the recent emergence of DNA-based techniques. In

recent years, DNA sequencing technology and bioinformatics have allowed for

better phylogenetic and functional/metabolic characterization of the canine gut

microbiome. Predominant phyla include Firmicutes, Bacteroidetes, Fusobacteria,

Proteobacteria, and Actinobacteria. Studies using 16S ribosomal RNA (rRNA) gene

pyrosequencing have demonstrated spatial differences along the GI tract and among

microbes adhered to the GI mucosa compared to those in intestinal contents or

feces. Similar to humans, GI microbiome dysbiosis is common in canine GI diseases

such as chronic diarrhea and inflammatory bowel diseases. DNA-based assays have

also identified key pathogens contributing to such conditions, including various

Clostridium, Campylobacter, Salmonella, and Escherichia spp. Moreover,

nutritionists have applied DNA-based techniques to study the effects of dietary

interventions such as dietary fiber, prebiotics, and probiotics on the canine GI

microbiome and associated health indices. Despite recent advances in the field,

the canine GI microbiome is far from being fully characterized and a deeper

characterization of the phylogenetic and functional/metabolic capacity of the GI

microbiome in health and disease is needed. This paper provides an overview of

recent studies performed to characterize the canine GI microbiome.


DOI: 10.1017/S1466252312000059

PMID: 22647637  [PubMed - indexed for MEDLINE]



69. Clin Chem. 2009 May;55(5):856-66. doi: 10.1373/clinchem.2008.107565. Epub 2009

Mar 5.


Metagenomic pyrosequencing and microbial identification.


Petrosino JF(1), Highlander S, Luna RA, Gibbs RA, Versalovic J.


Author information:

(1)Department of Molecular Virology and Microbiology, Baylor College of Medicine,

Houston, TX, USA.


BACKGROUND: The Human Microbiome Project has ushered in a new era for human

metagenomics and high-throughput next-generation sequencing strategies.

CONTENT: This review describes evolving strategies in metagenomics, with a

special emphasis on the core technology of DNA pyrosequencing. The challenges of

microbial identification in the context of microbial populations are discussed.

The development of next-generation pyrosequencing strategies and the technical

hurdles confronting these methodologies are addressed. Bioinformatics-related

topics include taxonomic systems, sequence databases, sequence-alignment tools,

and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is

summarized with respect to potential pyrosequencing applications.

SUMMARY: Both the approach of 16S rDNA amplicon sequencing and the whole-genome

sequencing approach may be useful for human metagenomics, and numerous

bioinformatics tools are being deployed to tackle such vast amounts of

microbiological sequence diversity. Metagenomics, or genetic studies of microbial

communities, may ultimately contribute to a more comprehensive understanding of

human health, disease susceptibilities, and the pathophysiology of infectious and

immune-mediated diseases.


DOI: 10.1373/clinchem.2008.107565

PMCID: PMC2892905

PMID: 19264858  [PubMed - indexed for MEDLINE]



70. Genome Res. 2009 Apr;19(4):636-43. doi: 10.1101/gr.084616.108. Epub 2009 Feb 27.


Global diversity in the human salivary microbiome.


Nasidze I(1), Li J, Quinque D, Tang K, Stoneking M.


Author information:

(1)Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany.


The human salivary microbiome may play a role in diseases of the oral cavity and

interact with microbiomes from other parts of the human body (in particular, the

intestinal tract), but little is known about normal variation in the salivary

microbiome. We analyzed 14,115 partial ( approximately 500 bp) 16S ribosomal RNA

(rRNA) sequences from saliva samples from 120 healthy individuals (10 individuals

from each of 12 worldwide locations). These sequences could be assigned to 101

known bacterial genera, of which 39 were not previously reported from the human

oral cavity; phylogenetic analysis suggests that an additional 64 unknown genera

are present. There is high diversity in the salivary microbiome within and

between individuals, but little geographic structure. Overall, approximately

13.5% of the total variance in the composition of genera is due to differences

among individuals, which is remarkably similar to the fraction of the total

variance in neutral genetic markers that can be attributed to differences among

human populations. Investigation of some environmental variables revealed a

significant association between the genetic distances among locations and the

distance of each location from the equator. Further characterization of the

enormous diversity revealed here in the human salivary microbiome will aid in

elucidating the role it plays in human health and disease, and in the

identification of potentially informative species for studies of human population



DOI: 10.1101/gr.084616.108

PMCID: PMC2665782

PMID: 19251737  [PubMed - indexed for MEDLINE]



71. FEMS Microbiol Lett. 2013 Feb;339(1):57-65. doi: 10.1111/1574-6968.12053. Epub

2012 Dec 20.


Longitudinal analysis of the lung microbiome in lung transplantation.


Borewicz K(1), Pragman AA, Kim HB, Hertz M, Wendt C, Isaacson RE.


Author information:

(1)Department of Veterinary and Biomedical Sciences, University of Minnesota, St.

Paul, MN 55108, USA.


Lung transplant recipients experience poor long-term survival, largely due to

chronic rejection. The pathogenesis of chronic rejection is incompletely

understood, but bacterial colonization of the lung is associated with chronic

rejection, while antibiotic use slows its progression. The lung harbors a

bacterial community, termed the microbiome, which is present both in health and

disease. We hypothesize that the lung microbiome will change following

transplantation, and these changes may correspond to the development of

rejection. Twelve bronchoalveolar lavage fluid (BALF) samples were obtained from

four patients at three time points after transplantation, and two BALF samples

were obtained from healthy, nontransplant controls. The microbiome of each sample

was determined by pyrosequencing the 16S rRNA gene hypervariable 3 region. The

data were analyzed using mothur, Ribosomal Database Project Classifier, Fast

UniFrac, and Metastats. Transplanted lungs contained more bacterial sequences and

demonstrated more microbial diversity than did control lungs. Bacteria in the

phyla Proteobacteria (class Betaproteobacteria) predominated in the transplant

samples. In contrast, the microbiome of the healthy lung consisted of the phyla

Proteobacteria (class Gammaproteobacteria) and Firmicutes. The microbiome of the

transplanted lung is vastly different from that of healthy lungs, mainly due to

the presence of the family Burkholderiaceae in transplant samples.


© 2012 Federation of European Microbiological Societies. Published by Blackwell

Publishing Ltd. All rights reserved.


DOI: 10.1111/1574-6968.12053

PMCID: PMC3546157

PMID: 23173619  [PubMed - indexed for MEDLINE]



72. Appl Environ Microbiol. 1999 Nov;65(11):4799-807.


Direct analysis of genes encoding 16S rRNA from complex communities reveals many

novel molecular species within the human gut.


Suau A(1), Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins MD, Doré J.


Author information:

(1)Laboratoire d'Ecologie et Physiologie du Système Digestif, Institut National

de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France.



The human intestinal tract harbors a complex microbial ecosystem which plays a

key role in nutrition and health. Although this microbiota has been studied in

great detail by culture techniques, microscopic counts on human feces suggest

that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative

analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the

bacterial diversity (both cultivated and noncultivated bacteria) within an

adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were

classified into 82 molecular species (at least 98% similarity). Three

phylogenetic groups contained 95% of the clones: the Bacteroides group, the

Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining

clones were distributed among a variety of phylogenetic clusters. Only 24% of the

molecular species recovered corresponded to described organisms (those whose

sequences were available in public databases), and all of these were established

members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron,

Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of

generated rDNA sequences (76%) did not correspond to known organisms and clearly

derived from hitherto unknown species within this human gut microflora.




PMID: 10543789  [PubMed - indexed for MEDLINE]



73. PLoS One. 2012;7(9):e42938. doi: 10.1371/journal.pone.0042938. Epub 2012 Sep 5.


Novel device to sample the esophageal microbiome--the esophageal string test.


Fillon SA(1), Harris JK, Wagner BD, Kelly CJ, Stevens MJ, Moore W, Fang R,

Schroeder S, Masterson JC, Robertson CE, Pace NR, Ackerman SJ, Furuta GT.


Author information:

(1)Department of Pediatrics, Section of Gastroenterology, Hepatology and

Nutrition, Digestive Health Institute, Children's Hospital Colorado, University

of Colorado Denver, School of Medicine, Aurora, Colorado, United States of

America. sophie.fillon@ucdenver.edu


A growing number of studies implicate the microbiome in the pathogenesis of

intestinal inflammation. Previous work has shown that adults with esophagitis

related to gastroesophageal reflux disease have altered esophageal microbiota

compared to those who do not have esophagitis. In these studies, sampling of the

esophageal microbiome was accomplished by isolating DNA from esophageal biopsies

obtained at the time of upper endoscopy. The aim of the current study was to

identify the esophageal microbiome in pediatric individuals with normal

esophageal mucosa using a minimally invasive, capsule-based string technology,

the Enterotest™. We used the proximal segment of the Enterotest string to sample

the esophagus, and term this the "Esophageal String Test" (EST). We hypothesized

that the less invasive EST would capture mucosal adherent bacteria present in the

esophagus in a similar fashion as mucosal biopsy. EST samples and mucosal

biopsies were collected from children with no esophageal inflammation (n = 15)

and their microbiome composition determined by 16S rRNA gene sequencing.

Microbiota from esophageal biopsies and ESTs produced nearly identical profiles

of bacterial genera and were different from the bacterial contents of samples

collected from the nasal and oral cavity. We conclude that the minimally invasive

EST can serve as a useful device for study of the esophageal microbiome.


DOI: 10.1371/journal.pone.0042938

PMCID: PMC3434161

PMID: 22957025  [PubMed - indexed for MEDLINE]



74. ISME J. 2011 Sep;5(9):1426-37. doi: 10.1038/ismej.2011.25. Epub 2011 Mar 24.


Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome.


Calvo-Bado LA(1), Oakley BB, Dowd SE, Green LE, Medley GF, Ul-Hassan A, Bateman

V, Gaze W, Witcomb L, Grogono-Thomas R, Kaler J, Russell CL, Wellington EM.


Author information:

(1)Department of Biological Sciences, School of Life Sciences, University of

Warwick, Coventry, UK. l.a.calvo-bado@warwick.ac.uk


We report the first study of the bacterial microbiome of ovine interdigital skin

based on 16S rRNA by pyrosequencing and conventional cloning with

Sanger-sequencing. Three flocks were selected, one a flock with no signs of

footrot or interdigital dermatitis, a second flock with interdigital dermatitis

alone and a third flock with both interdigital dermatitis and footrot. The sheep

were classified as having either healthy interdigital skin (H) and interdigital

dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial

community varied significantly by flock and clinical condition. The diversity and

richness of operational taxonomic units was greater in tissue from sheep with ID

than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and

Proteobacteria were the most abundant phyla comprising 25 genera.

Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID

and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of

ovine footrot, were not amplified because of mismatches in the 16S rRNA universal

forward primer (27F). A specific real-time PCR assay was used to demonstrate the

presence of D. nodosus, which was detected in all samples including the flock

with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D.

nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet.


DOI: 10.1038/ismej.2011.25

PMCID: PMC3160683

PMID: 21430786  [PubMed - indexed for MEDLINE]



PLoS One. 2016 Sep 19;11(9):e0163178. doi: 10.1371/journal.pone.0163178. eCollection 2016.


Effects of Elevated Tropospheric Ozone Concentration on the Bacterial Community in the Phyllosphere and Rhizoplane of Rice.

Ueda Y1, Frindte K2, Knief C2, Ashrafuzzaman M1, Frei M1.

Author information


Microbes constitute a vital part of the plant holobiont. They establish plant-microbe or microbe-microbe associations, forming a unique microbiota with each plant species and under different environmental conditions. These microbial communities have to adapt to diverse environmental conditions, such as geographical location, climate conditions and soil types, and are subjected to changes in their surrounding environment. Elevated ozone concentration is one of the most important aspects of global change, but its effect on microbial communities living on plant surfaces has barely been investigated. In the current study, we aimed at elucidating the potential effect of elevated ozone concentrations on the phyllosphere (aerial part of the plant) and rhizoplane (surface of the root) microbiota by adopting next-generation 16S rRNA amplicon sequencing. A standard japonica rice cultivar Nipponbare and an ozone-tolerant breeding line L81 (Nipponbare background) were pre-grown in a greenhouse for 10 weeks and then exposed to ozone at 85 ppb for 7 h daily for 30 days in open top chambers. Microbial cells were collected from the phyllosphere and rhizoplane separately. The treatment or different genotypes did not affect various diversity indices. On the other hand, the relative abundance of some bacterial taxa were significantly affected in the rhizoplane community of ozone-treated plants. A significant effect of ozone was detected by homogeneity of molecular variance analysis in the phyllosphere, meaning that the community from ozone-treated phyllosphere samples was more variable than those from control plants. In addition, a weak treatment effect was observed by clustering samples based on the Yue and Clayton and weighted UniFrac distance matrices among samples. We therefore conclude that the elevated ozone concentrations affected the bacterial community structure of the phyllosphere and the rhizosplane as a whole, even though this effect was rather weak and did not lead to changes of the function of the communities.

PMID: 27643794 DOI: 10.1371/journal.pone.0163178

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BMC Infect Dis. 2016 Sep 19;16:493. doi: 10.1186/s12879-016-1838-y.


First case report of infection due to Cupriavidus gilardii in a patient without immunodeficiency: a case report.

Kobayashi T1,2, Nakamura I3, Fujita H1,2, Tsukimori A1,2, Sato A1, Fukushima S1, Ohkusu K2, Matsumoto T2.

Author information



Cupriavidus gilardii is an aerobic, Gram-negative, glucose-nonfermenting rod that was first identified in 1999. Because of the difficulty in accurate species identification of C. gilardii, there are few case reports of infection caused by this organism. In previous reports, C. gilardii has been characterized as an organism with low pathogenicity that causes opportunistic infections.


We encountered a case of pacemaker-associated bloodstream infection caused by C. gilardii in a 90-year old woman without obvious immunodeficiency. We identified the isolates as C. gilardii by sequencing of the 16S rRNA gene. The patient was treated with removal of the lead and administration of antimicrobial agents. Because of the acquisition of antibiotic resistance during antibiotic treatment, the antimicrobial agent was changed during the course of treatment.


To our knowledge, this is the first report of an infection caused by this organism in a patient without obvious immunodeficiency. Although the true pathogenicity of C. gilardii is unclear, the possibility that it exerts pathogenicity not only in persons with immunodeficiency but also in immunocompetent persons is suggested.


Antimicrobial resistance; Case report; Cupriavidus gilardii; Glucose non-fermenting gram-negative rod; Immunocompetent patient; Pacemaker-associated bloodstream infection; Sequencing analysis of the 16S rRNA gene

PMID: 27643790 DOI: 10.1186/s12879-016-1838-y

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PLoS One. 2016 Sep 19;11(9):e0163148. doi: 10.1371/journal.pone.0163148. eCollection 2016.


Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.

Gill C1, van de Wijgert JH1, Blow F2, Darby AC2.

Author information



Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit.


After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering.


An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies. However, we recommend that the same method is used on all samples within a particular study.

PMID: 27643503 DOI: 10.1371/journal.pone.0163148

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J Hypertens. 2016 Sep;34 Suppl 1:e187. doi: 10.1097/01.hjh.0000500405.96430.2e.



Lee MS1.

Author information


Low-grade systemic inflammation in adipose tissues or liver, is an important etiologic factor in insulin resistance. LPS is an important element causing such metabolic inflammation, and intestinal flora is considered a major source of systemic LPS. We studied changes of intestinal microbiota associated with high-fat diet (HFD) that causes insulin resistance and metabolic stress. 16S rRNA gene sequencing showed that HFD significantly decreased the abundance of a mucin-degrading bacterium Akkermansia muciniphila compared to control diet. Reduced abundance of Akkermansia in HFD-fed mice was reversed by metformin, a widely prescribed anti-diabetic medicine. Oral administration of Akkermansia to HFD-fed mice significantly enhanced insulin sensitivity and attenuated adipose tissue inflammation probably by inducing Foxp3 regulatory T cells (Tregs) in the visceral adipose tissue. We also studied Paneth cells, specialized intestinal epithelial cells that produce antimicrobial peptides (AMP) and shape gut microbial milieu. We studied whether high-fat diet (HFD) impairs the function of Paneth cells and interferes with intestinal microbial homeostasis, potentially contributing to the development of metabolic syndrome. HFD-fed mice exhibited reduced expressions of some AMP and defective antimicrobial activity, which led to loss of epithelial integrity and a significantly higher content of LPS in the liver and serum of HFD-fed mice compared to chow-fed mice. Antibiotic treatment reduced translocation of bacterial components, steatosis, and inflammation in the liver of HFD-fed mice, which in turn improved systemic glucose metabolism. HFD-fed mice were also more susceptible to dextran sodium sulfate-induced colitis, probably due to impaired Paneth cell function. Our data suggest that Paneth cell dysfunction may be one of the underlying mechanisms of glucose intolerance and inflammatory bowel disease associated with western diet. These results suggest that high-fat diet could be a risk factor not only for metabolic syndrome but also for colitis, and modulation of the gut microbiota or Paneth cell function could be a new therapeutic modality against metabolic syndrome or colitis associated with western diet.

PMID: 27642901 DOI: 10.1097/01.hjh.0000500405.96430.2e


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