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Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota

Journal article
Authors Fei Sjöberg
Forough Nowrouzian
I. Rangel
Charles Hannoun
Edward R.B. Moore
Ingegerd Adlerberth
Agnes E Wold
Published in Journal of Microbiological Methods
Volume 94
Issue 1
Pages 37-46
ISSN 0167-7012
Publication year 2013
Published at Institute of Biomedicine, Department of Infectious Medicine
Pages 37-46
Language en
Links dx.doi.org/10.1016/j.mimet.2013.04....
Keywords T-RFLP, Quantitative culture, Clone, Infantile gut, Microbiota diversity, 16S RNA, 16s ribosomal-rna, gastrointestinal-tract, atopic eczema, intestinal, microbiota, reduced diversity, fecal microbiota, translocation, communities, assignment, sequences
Subject categories Microbiology

Abstract

The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >10(6) CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition, but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.

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