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

• Published in: BMC microbiology Vol. 14; no. 1; p. 112
• Main Authors: Santiago, Alba, Panda, Suchita, Mengels, Griet, Martinez, Xavier, Azpiroz, Fernando, Dore, Joel, Guarner, Francisco, Manichanh, Chaysavanh
• Format: Journal Article
• Language: English
• Published: London BioMed Central 01.05.2014; BioMed Central Ltd
• Subjects: Bacteria; Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Bifidobacterium; Biological Microscopy; Biomedical and Life Sciences; Biota; Cluster Analysis; Community composition; Deoxyribonucleic acid; Diarrhea; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Feces - microbiology; genomics and proteomics; Healthy Volunteers; Homogenization; Humans; Irritable bowel syndrome; Life Sciences; Methods; Microbial genetics; Microbiology; Mycology; Nucleic acids; Parasitology; Phylogenetics; Phylogeny; Physiological aspects; Research Article; Ribosomal RNA; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; Specimen Handling - methods; Specimen Handling - standards; Virology; Water content; Needs for standardisation; Bead-beating; Faecal sample collection; Diarrhoea; 16S ribosomal RNA; DIARRHEA; GREENGENES; DIVERSITY; RIBOSOMAL-RNA; TOOL
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/1471-2180-14-112

Background The 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. Results We 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. Conclusion The 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 .