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J Microbiol Methods. 2016 Sep 5;130:95-99. doi: 10.1016/j.mimet.2016.09.002. [Epub ahead of print]

Different next generation sequencing platforms produce different microbial profiles and diversity in cystic fibrosis sputum.

Hahn A1, Sanyal A2, Perez GF3, Colberg-Poley AM4, Campos J5, Rose MC6, Pérez-Losada M7.

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Abstract

BACKGROUND:

Cystic fibrosis (CF) is an autosomal recessive disease characterized by recurrent lung infections. Studies of the lung microbiome have shown an association between decreasing diversity and progressive disease. 454 pyrosequencing has frequently been used to study the lung microbiome in CF, but will no longer be supported. We sought to identify the benefits and drawbacks of using two state-of-the-art next generation sequencing (NGS) platforms, MiSeq and PacBio RSII, to characterize the CF lung microbiome. Each has its advantages and limitations.

METHODS:

Twelve samples of extracted bacterial DNA were sequenced on both MiSeq and PacBio NGS platforms. DNA was amplified for the V4 region of the 16S rRNA gene and libraries were sequenced on the MiSeq sequencing platform, while the full 16S rRNA gene was sequenced on the PacBio RSII sequencing platform. Raw FASTQ files generated by the MiSeq and PacBio platforms were processed in mothur v1.35.1.

RESULTS:

There was extreme discordance in alpha-diversity of the CF lung microbiome when using the two platforms. Because of its depth of coverage, sequencing of the 16S rRNA V4 gene region using MiSeq allowed for the observation of many more operational taxonomic units (OTUs) and higher Chao1 and Shannon indices than the PacBio RSII. Interestingly, several patients in our cohort had Escherichia, an unusual pathogen in CF. Also, likely because of its coverage of the complete 16S rRNA gene, only PacBio RSII was able to identify Burkholderia, an important CF pathogen.

CONCLUSION:

When comparing microbiome diversity in clinical samples from CF patients using 16S sequences, MiSeq and PacBio NGS platforms may generate different results in microbial community composition and structure. It may be necessary to use different platforms when trying to correctly identify dominant pathogens versus measuring alpha-diversity estimates, and it would be important to use the same platform for comparisons to minimize errors in interpretation.

Copyright © 2016 Elsevier B.V. All rights reserved.

KEYWORDS:

16S rRNA; Cystic fibrosis; Lung microbiome; MiSeq; Next generation sequencing; PacBio RSII

PMID: 27609714 DOI: 10.1016/j.mimet.2016.09.002

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82.

J Allergy Clin Immunol. 2016 Sep 5. pii: S0091-6749(16)30893-4. doi: 10.1016/j.jaci.2016.07.029. [Epub ahead of print]

Skin microbiome prior to development of atopic dermatitis: early colonization with commensal staphylococci at 2 months is associated with a lower risk of atopic dermatitis at 1 year.

Kennedy EA1, Connolly J2, O'B Hourihane J2, Fallon PG3, McLean WI4, Murray D2, Jo JH1, Segre JA5, Kong HH6, Irvine AD7.

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Abstract

BACKGROUND:

Disease flares of established atopic dermatitis (AD) are generally associated with a low-diversity skin microbiota and Staphylococcus aureus dominance. The temporal transition of the skin microbiome between early infancy and the dysbiosis of established AD is unknown.

METHODS:

We randomly selected 50 children from the Cork BASELINE longitudinal birth cohort for microbiome sampling at three times in the first six months of life, at four skin sites relevant to AD: the antecubital and popliteal fossae, nasal tip, and cheek. We identified ten infants who developed AD and compared them with ten randomly selected control infants with no AD. We performed bacterial 16S ribosomal RNA sequencing and analysis directly from clinical samples.

RESULTS:

Bacterial community structures and diversity shifted over time, suggesting that age strongly affects the skin microbiome in infants. Unlike established AD, these infantile AD patients did not have noticeably dysbiotic communities prior to or with disease and were not colonized by S. aureus. In comparing patients and controls, infants who had affected skin at month 12 had statistically significant differences in bacterial communities on the antecubital fossa at month 2 compared to infants who were unaffected at month 12. In particular, commensal staphylococci were significantly less abundant in infants affected at month 12, suggesting that this genus may protect against the later development of AD.

CONCLUSIONS:

This study suggests that 12-month-old infants with AD were not colonized with Staphylococcus aureus before developing AD. Additional studies are needed to confirm if colonization with commensal staphylococci modulates skin immunity and attenuates development of AD.

Copyright © 2016. Published by Elsevier Inc.

KEYWORDS:

16S sequencing; Atopic dermatitis; Longitudinal birth cohort; Skin; Staphylococcus aureus; microbiome

PMID: 27609659 DOI: 10.1016/j.jaci.2016.07.029

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Inflamm Bowel Dis. 2016 Oct;22(10):2328-40. doi: 10.1097/MIB.0000000000000914.

Antibiotic Treatment Induces Long-lasting Changes in the Fecal Microbiota that Protect Against Colitis.

Ward NL1, Phillips CD, Nguyen DD, Shanmugam NK, Song Y, Hodin R, Shi HN, Cherayil BJ, Goldstein AM.

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Abstract

BACKGROUND:

The interplay between host genetics, immunity, and microbiota is central to the pathogenesis of inflammatory bowel disease. Previous population-based studies suggested a link between antibiotic use and increased inflammatory bowel disease risk, but the mechanisms are unknown. The purpose of this study was to determine the long-term effects of antibiotic administration on microbiota composition, innate immunity, and susceptibility to colitis, as well as the mechanism by which antibiotics alter host colitogenicity.

METHODS:

Wild-type mice were given broad-spectrum antibiotics or no antibiotics for 2 weeks, and subsequent immunophenotyping and 16S rRNA gene sequencing-based analysis of the fecal microbiome were performed 6 weeks later. In a separate experiment, control and antibiotic-treated mice were given 7 days of dextran sulfate sodium, 6 weeks after completing antibiotic treatment, and the severity of colitis scored histologically. Fecal transfer was performed from control or antibiotic-treated mice to recipient mice whose endogenous microbiota had been cleared with antibiotics, and the susceptibility of the recipients to dextran sulfate sodium-induced colitis was analyzed. Naive CD4 T cells were transferred from control and antibiotic-treated mice to immunodeficient Rag-1 recipients and the severity of colitis compared.

RESULTS:

Antibiotics led to sustained dysbiosis and changes in T-cell subpopulations, including reductions in colonic lamina propria total T cells and CD4 T cells. Antibiotics conferred protection against dextran sulfate sodium colitis, and this effect was transferable by fecal transplant but not by naive T cells.

CONCLUSIONS:

Antibiotic exposure protects against colitis, and this effect is transferable with fecal microbiota from antibiotic-treated mice, supporting a protective effect of the microbial community.

PMID: 27607336 DOI: 10.1097/MIB.0000000000000914

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84.

Sci Rep. 2016 Sep 8;6:33142. doi: 10.1038/srep33142.

Deep sequencing reveals microbiota dysbiosis of tongue coat in patients with liver carcinoma.

Lu H1, Ren Z1,2, Li A1, Zhang H1, Jiang J1,2, Xu S2, Luo Q1, Zhou K1, Sun X3, Zheng S1,2, Li L1.

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Abstract

Liver carcinoma (LC) is a common malignancy worldwide, associated with high morbidity and mortality. Characterizing microbiome profiles of tongue coat may provide useful insights and potential diagnostic marker for LC patients. Herein, we are the first time to investigate tongue coat microbiome of LC patients with cirrhosis based on 16S ribosomal RNA (rRNA) gene sequencing. After strict inclusion and exclusion criteria, 35 early LC patients with cirrhosis and 25 matched healthy subjects were enrolled. Microbiome diversity of tongue coat in LC patients was significantly increased shown by Shannon, Simpson and Chao 1 indexes. Microbiome on tongue coat was significantly distinguished LC patients from healthy subjects by principal component analysis. Tongue coat microbial profiles represented 38 operational taxonomic units assigned to 23 different genera, distinguishing LC patients. Linear discriminant analysis (LDA) effect size (LEfSe) reveals significant microbial dysbiosis of tongue coats in LC patients. Strikingly, Oribacterium and Fusobacterium could distinguish LC patients from healthy subjects. LEfSe outputs show microbial gene functions related to categories of nickel/iron_transport, amino_acid_transport, energy produced system and metabolism between LC patients and healthy subjects. These findings firstly identify microbiota dysbiosis of tongue coat in LC patients, may providing novel and non-invasive potential diagnostic biomarker of LC.

PMID: 27605161 PMCID: PMC5015078 DOI: 10.1038/srep33142

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