title: The effect of sampling and storage on the fecal microbiota composition in healthy and diseased subjects.
abstract: Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80 °C, -20 °C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80 °C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p < 0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80 °C versus the other methods and -80 °C samples (p < 0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80 °C, but was higher in fecal swabs (p < 0.05). Storage up to 24 hours (at +4 °C or room temperature) or freezing at -20 °C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Division Gastroenterology-Hepatology, Maastricht University Medical Center+, Maastricht, The Netherlands; School of Nutrition and Translational Research in Metabolism (NUTRIM), Department of Medical Microbiology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Danyta I
authors.initials: DI
authors.lastname: Tedjo
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Division Gastroenterology-Hepatology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Daisy M A E
authors.initials: DM
authors.lastname: Jonkers
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Department of Medical Microbiology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Savelkoul
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Division Gastroenterology-Hepatology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Ad A
authors.initials: AA
authors.lastname: Masclee
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Department of Medical Microbiology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: van Best
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Division Gastroenterology-Hepatology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Marieke J
authors.initials: MJ
authors.lastname: Pierik
authors.affiliation: School of Nutrition and Translational Research in Metabolism (NUTRIM), Department of Medical Microbiology, Maastricht University Medical Center+, Maastricht, The Netherlands; School for Public Health and Primary Care (Caphri), Department of Epidemiology, Maastricht University, Maastricht, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: Penders
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification; D044822:Biodiversity; D003093:Colitis, Ulcerative / Q000382:microbiology; D003424:Crohn Disease / Q000382:microbiology; D005243:Feces / Q000382:microbiology*; D005260:Female; D006801:Humans; D043183:Irritable Bowel Syndrome / Q000382:microbiology; D008297:Male; D064307:Microbiota; D008875:Middle Aged; D013048:Specimen Handling / Q000379:methods*