Processing faecal samples: a step forward for standards in microbial community analysis

BMC Microbiology

Research Article

Joel Dore¹  Griet Mengels²  Xavier Martinez²  Suchita Panda²  Alba Santiago²  Francisco Guarner³  Fernando Azpiroz³  Chaysavanh Manichanh³ 

[1]INRA, Institut National de la Recherche Agronomique, 78350, Jouy en Josas, France

[2]Vall d’Hebron Research Institute, Digestive System Research Unit, Passeig de la Vall d’Hebron 119-129, 08035, Barcelona, Spain

[3]Vall d’Hebron Research Institute, Digestive System Research Unit, Passeig de la Vall d’Hebron 119-129, 08035, Barcelona, Spain

[4]Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas, CIBERehd, Madrid, Spain

关键词: 16S ribosomal RNA;    Faecal sample collection;    Needs for standardisation;    Diarrhoea;    Bead-beating;   

DOI  :  10.1186/1471-2180-14-112

 received in 2014-02-13, accepted in 2014-04-25,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe microbial community analysis of stools requires optimised and standardised protocols for their collection, homogenisation, microbial disruption and nucleic acid extraction. Here we examined whether different layers of the stool are equally representative of the microbiome. We also studied the effect of stool water content, which typically increases in diarrhoeic samples, and of a microbial disruption method on DNA integrity and, therefore, on providing an unbiased microbial composition analysis.ResultsWe collected faecal samples from healthy subjects and performed microbial composition analysis by pyrosequencing the V4 region of the 16S rRNA gene. To examine the effect of stool structure, we compared the inner and outer layers of the samples (N = 8). Both layers presented minor differences in microbial composition and abundance at the species level. These differences did not significantly bias the microbial community specific to an individual. To evaluate the effect of stool water content and bead-beating, we used various volumes of a water-based salt solution and beads of distinct weights before nucleic acid extraction (N = 4). The different proportions of water did not affect the UniFrac-based clustering of samples from the same subject However, the use or omission of a bead-beating step produced different proportions of Gram-positive and Gram-negative bacteria and significant changes in the UniFrac-based clustering of the samples.ConclusionThe degree of hydration and homogenisation of faecal samples do not significantly alter their microbial community composition. However, the use of bead-beating is critical for the proper detection of Gram-positive bacteria such as Blautia and Bifidobacterium.

【 授权许可】

Unknown   
© Santiago et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.