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      Rectal Swabs for Analysis of the Intestinal Microbiota

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          Abstract

          The composition of the gut microbiota is associated with various disease states, most notably inflammatory bowel disease, obesity and malnutrition. This underlines that analysis of intestinal microbiota is potentially an interesting target for clinical diagnostics. Currently, the most commonly used sample types are feces and mucosal biopsy specimens. Because sampling method, storage and processing of samples impact microbiota analysis, each sample type has its own limitations. An ideal sample type for use in routine diagnostics should be easy to obtain in a standardized fashion without perturbation of the microbiota. Rectal swabs may satisfy these criteria, but little is known about microbiota analysis on these sample types. In this study we investigated the characteristics and applicability of rectal swabs for gut microbiota profiling in a clinical routine setting in patients presenting with various gastro-intestinal disorders. We found that rectal swabs appeared to be a convenient means of sampling the human gut microbiota. Swabs can be performed on demand, whenever a patient presents; swab-derived microbiota profiles are reproducible, whether they are gathered at home by patients or by medical professionals in an outpatient setting and may be ideally suited for clinical diagnostics and large-scale studies.

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          Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples.

          Storage conditions are considered to be a critical component of DNA-based microbial community analysis methods. However, whether differences in short-term sample storage conditions impact the assessment of bacterial community composition and diversity requires systematic and quantitative assessment. Therefore, we used barcoded pyrosequencing of bacterial 16S rRNA genes to survey communities, harvested from a variety of habitats [soil, human gut (feces) and human skin] and subsequently stored at 20, 4, -20 and -80 degrees C for 3 and 14 days. Our results indicate that the phylogenetic structure and diversity of communities in individual samples were not significantly influenced by the storage temperature or the duration of storage. Likewise, the relative abundances of most taxa were largely unaffected by temperature even after 14 days of storage. Our results indicate that environmental factors and biases in molecular techniques likely confer greater amounts of variation to microbial communities than do differences in short-term storage conditions, including storage for up to 2 weeks at room temperature. These results suggest that many samples collected and stored under field conditions without refrigeration may be useful for microbial community analyses.
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            Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis.

            Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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              Storage conditions of intestinal microbiota matter in metagenomic analysis

              Background The structure and function of human gut microbiota is currently inferred from metagenomic and metatranscriptomic analyses. Recovery of intact DNA and RNA is therefore a critical step in these studies. Here, we evaluated how different storage conditions of fecal samples affect the quality of extracted nucleic acids and the stability of their microbial communities. Results We assessed the quality of genomic DNA and total RNA by microcapillary electrophoresis and analyzed the bacterial community structure by pyrosequencing the 16S rRNA gene. DNA and RNA started to fragment when samples were kept at room temperature for more than 24 h. The use of RNAse inhibitors diminished RNA degradation but this protection was not consistent among individuals. DNA and RNA degradation also occurred when frozen samples were defrosted for a short period (1 h) before nucleic acid extraction. The same conditions that affected DNA and RNA integrity also altered the relative abundance of most taxa in the bacterial community analysis. In this case, intra-individual variability of microbial diversity was larger than inter-individual one. Conclusions Though this preliminary work explored a very limited number of parameters, the results suggest that storage conditions of fecal samples affect the integrity of DNA and RNA and the composition of their microbial community. For optimal preservation, stool samples should be kept at room temperature and brought at the laboratory within 24 h after collection or be stored immediately at −20°C in a home freezer and transported afterwards in a freezer pack to ensure that they do not defrost at any time. Mixing the samples with RNAse inhibitors outside the laboratory is not recommended since proper homogenization of the stool is difficult to monitor.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                14 July 2014
                : 9
                : 7
                : e101344
                Affiliations
                [1 ]Department of Medical Microbiology and Infection control, VU University medical center, Amsterdam, the Netherlands
                [2 ]Department of Gastroenterology and Hepatology, VU University medical center, Amsterdam, the Netherlands
                [3 ]Department of Epidemiology and Biostatistics, VU University medical center, Amsterdam, the Netherlands
                [4 ]Department of Internal Medicine, Gastroenterology and Geriatrics, ORBIS medical center, Sittard-Geleen, the Netherlands
                [5 ]Department of medical microbiology, Maastricht University medical center, Maastricht, the Netherlands
                New York State Dept. Health, United States of America
                Author notes

                Competing Interests: AEB and PS have proprietary rights on the IS-pro platform technology. All other authors declare to have no competing interests. Potential competing interests do not alter the author's adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: AB AvB PS. Performed the experiments: AB AvB MG. Analyzed the data: AB AE JB. Contributed reagents/materials/analysis tools: MG. Wrote the paper: AB CV AvB PS AE.

                Article
                PONE-D-13-41399
                10.1371/journal.pone.0101344
                4096398
                25020051
                335ad7ae-f224-4036-b472-77d2a5955986
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 October 2013
                : 5 June 2014
                Page count
                Pages: 8
                Funding
                The authors have no funding or support to report.
                Categories
                Research Article
                Biology and Life Sciences
                Biotechnology
                Medical Devices and Equipment
                Microbiology
                Medical Microbiology
                Medicine and Health Sciences
                Clinical Medicine
                Diagnostic Medicine
                Clinical Laboratory Sciences
                Gastroenterology and Hepatology
                Bacterial and Foodborne Illness
                Infectious Diseases
                Pathology and Laboratory Medicine
                Anatomical Pathology
                Cytopathology
                Clinical Pathology
                Clinical Microbiology
                Molecular Pathology

                Uncategorized
                Uncategorized

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