16-s.mobi A Global Perspective of Ribosomal Gene Sequencing
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61.

Front Med (Lausanne). 2016 Jun 13;3:22. doi: 10.3389/fmed.2016.00022. eCollection 2016.

Separating Putative Pathogens from Background Contamination with Principal Orthogonal Decomposition: Evidence for Leptospira in the Ugandan Neonatal Septisome.

Schiff SJ1, Kiwanuka J2, Riggio G3, Nguyen L4, Mu K3, Sproul E3, Bazira J5, Mwanga-Amumpaire J6, Tumusiime D2, Nyesigire E2, Lwanga N5, Bogale KT7, Kapur V8, Broach JR9, Morton SU10, Warf BC11, Poss M3.

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Abstract

Neonatal sepsis (NS) is responsible for over 1 million yearly deaths worldwide. In the developing world, NS is often treated without an identified microbial pathogen. Amplicon sequencing of the bacterial 16S rRNA gene can be used to identify organisms that are difficult to detect by routine microbiological methods. However, contaminating bacteria are ubiquitous in both hospital settings and research reagents and must be accounted for to make effective use of these data. In this study, we sequenced the bacterial 16S rRNA gene obtained from blood and cerebrospinal fluid (CSF) of 80 neonates presenting with NS to the Mbarara Regional Hospital in Uganda. Assuming that patterns of background contamination would be independent of pathogenic microorganism DNA, we applied a novel quantitative approach using principal orthogonal decomposition to separate background contamination from potential pathogens in sequencing data. We designed our quantitative approach contrasting blood, CSF, and control specimens and employed a variety of statistical random matrix bootstrap hypotheses to estimate statistical significance. These analyses demonstrate that Leptospira appears present in some infants presenting within 48 h of birth, indicative of infection in utero, and up to 28 days of age, suggesting environmental exposure. This organism cannot be cultured in routine bacteriological settings and is enzootic in the cattle that often live in close proximity to the rural peoples of western Uganda. Our findings demonstrate that statistical approaches to remove background organisms common in 16S sequence data can reveal putative pathogens in small volume biological samples from newborns. This computational analysis thus reveals an important medical finding that has the potential to alter therapy and prevention efforts in a critically ill population.

KEYWORDS:

16S rRNA; Leptospira; bacteria; neonatal sepsis; principal orthogonal decomposition; singular value decomposition

PMID: 27379237 PMCID: PMC4904006 DOI: 10.3389/fmed.2016.00022

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62.

Front Microbiol. 2016 Jun 17;7:919. doi: 10.3389/fmicb.2016.00919. eCollection 2016.

The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing.

Thiel V1, Wood JM2, Olsen MT2, Tank M1, Klatt CG3, Ward DM2, Bryant DA4.

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Abstract

Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus spp.

KEYWORDS:

extreme environments; hot spring; microbial community; microbial diversity; phototrophic bacteria

PMID: 27379049 PMCID: PMC4911352 DOI: 10.3389/fmicb.2016.00919

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63.

Front Microbiol. 2016 Jun 6;7:870. doi: 10.3389/fmicb.2016.00870. eCollection 2016.

Corrigendum: A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

Herbold CW1, Pelikan C1, Kuzyk O1, Hausmann B1, Angel R1, Berry D1, Loy A1.

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Abstract

[This corrects the article on p. 731 in vol. 6, PMID: 26236305.].

KEYWORDS:

16S rRNA; MiSeq; amoA; dsrA; dsrB; functional gene; nifH; nxrB

Erratum for

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes. [Front Microbiol. 2015]

PMID: 27375591 PMCID: PMC4893487 DOI: 10.3389/fmicb.2016.00870

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64.

Appl Environ Microbiol. 2016 Aug 30;82(18):5542-52. doi: 10.1128/AEM.01131-16. Print 2016 Sep 15.

Grapevine (Vitis vinifera) Crown Galls Host Distinct Microbiota.

Faist H1, Keller A2, Hentschel U3, Deeken R4.

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Abstract

Crown gall disease of grapevine is caused by virulent Agrobacterium strains and establishes a suitable habitat for agrobacteria and, potentially, other bacteria. The microbial community associated with grapevine plants has not been investigated with respect to this disease, which frequently results in monetary losses. This study compares the endophytic microbiota of organs from grapevine plants with or without crown gall disease and the surrounding vineyard soil over the growing seasons of 1 year. Amplicon-based community profiling revealed that the dominating factor causing differences between the grapevine microbiota is the sample site, not the crown gall disease. The soil showed the highest microbial diversity, which decreased with the distance from the soil over the root and the graft union of the trunk to the cane. Only the graft union microbiota was significantly affected by crown gall disease. The bacterial community of graft unions without a crown gall hosted transient microbiota, with the three most abundant bacterial species changing from season to season. In contrast, graft unions with a crown gall had a higher species richness, which in every season was dominated by the same three bacteria (Pseudomonas sp., Enterobacteriaceae sp., and Agrobacterium vitis). For in vitro-cultivated grapevine plantlets, A. vitis infection alone was sufficient to cause crown gall disease. Our data show that microbiota in crown galls is more stable over time than microbiota in healthy graft unions and that the microbial community is not essential for crown gall disease outbreak.

IMPORTANCE:

The characterization of bacterial populations in animal and human diseases using high-throughput deep-sequencing technologies, such as 16S amplicon sequencing, will ideally result in the identification of disease-specific microbiota. We analyzed the microbiota of the crown gall disease of grapevine, which is caused by infection with the bacterial pathogen Agrobacterium vitis. All other Agrobacterium species were found to be avirulent, even though they lived together with A. vitis in the same crown gall tumor. As has been reported for human cancer, the crown gall tumor also hosted opportunistic bacteria that are adapted to the tumor microenvironment. Characterization of the microbiota in various diseases using amplicon sequencing may help in early diagnosis, to serve as a preventative measure of disease in the future.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

PMID: 27371584 PMCID: PMC5007782 [Available on 2017-02-28] DOI: 10.1128/AEM.01131-16

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65.

FEMS Microbiol Ecol. 2016 Sep;92(9). pii: fiw146. doi: 10.1093/femsec/fiw146. Epub 2016 Jul 1.

The gut microbiome of the sea urchin, Lytechinus variegatus, from its natural habitat demonstrates selective attributes of microbial taxa and predictive metabolic profiles.

Hakim JA1, Koo H1, Kumar R2, Lefkowitz EJ3, Morrow CD4, Powell ML1, Watts SA5, Bej AK5.

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Abstract

In this paper, we describe the microbial composition and their predictive metabolic profile in the sea urchin Lytechinus variegatus gut ecosystem along with samples from its habitat by using NextGen amplicon sequencing and downstream bioinformatics analyses. The microbial communities of the gut tissue revealed a near-exclusive abundance of Campylobacteraceae, whereas the pharynx tissue consisted of Tenericutes, followed by Gamma-, Alpha- and Epsilonproteobacteria at approximately equal capacities. The gut digesta and egested fecal pellets exhibited a microbial profile comprised of Gammaproteobacteria, mainly Vibrio, and Bacteroidetes. Both the seagrass and surrounding sea water revealed Alpha- and Betaproteobacteria. Bray-Curtis distances of microbial communities indicated a clustering profile with low intrasample variation. Predictive metagenomics performed on the microbial communities revealed that the gut tissue had high relative abundances of metabolisms assigned to the KEGG-Level-2 designation of energy metabolisms compared to the gut digesta, which had higher carbohydrate, amino acid and lipid metabolisms. Overall, the results of this study elaborate the spatial distribution of microbial communities in the gut ecosystem of L. variegatus, and specifically a selective attribute for Campylobacteraceae in the gut tissue. Also, the predictive functional significance of bacterial communities in uniquely compartmentalized gut ecosystems of L. variegatus has been described.

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KEYWORDS:

16S rRNA; Gulf of Mexico; PICRUSt; bioinformatics; microbiota; next-generation sequencing