T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages

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Abstract

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.

Introduction

The microbiota of human intestine is a complex ecosystem and might have profound effects on health and disease through involvement in the nutrition, pathological process and immune function of the host Ducluzeau, 1988, Gibson and Roberfroid, 1995. The intestinal microbiata is a major source of stimulus in infancy, and the postnatal maturation of a balanced immune system requires constant microbial stimulation from the developing gut flora Holt et al., 1997, Laiho et al., 2002. Epidemiological studies suggest that the increase in prevalence of atopic diseases in western countries could be associated with improvements in public health and hygiene leading to reduced exposure to microbial stimulation in early life and, as a consequence, failure to develop tolerance to innocuous antigens (Bach, 2002). The study of the bacterial community structure and dynamics during the development of healthy infant gut flora may thus improve our understanding of the cause of atopic diseases and provide a basis for evaluating the effects of dietary treatments aimed at preventing allergy.

Current knowledge of gut microbial ecology and diversity in infants is largely based on the use of traditional culture techniques. It is clear that culture techniques have handicaps since certain bacteria are uncultivable and most media used for quantification of bacterial groups are nonspecific (Tannock, 1999). Alternative approaches based on 16S rRNA genes are now available for the study of complex bacterial ecosystems without the need for isolation and cultivation. The most established 16S rDNA-based technique relays on amplifying, cloning and sequencing 16S rRNA genes from bacterial DNA. Phylogenetic analysis of libraries of cloned 16S rRNA genes have been applied to investigate the diversity of human intestinal microbiota and revealed sequence information for both known and unknown bacterial species Hold et al., 2002, Suau et al., 1999. However, this technique is rather labor intensive and time consuming and therefore not suitable for comparative analysis of a large number of samples. For comparing infant fecal microbiota over time, more rapid and convenient methods are needed. One of these methods is terminal restriction fragment length polymorphism (T-RFLP) analysis. T-RFLP is a robust and reproducible method and has been used successfully for comparing microbial communities in soil samples (Lukow et al., 2000), marine sediment (Scala and Kerkhof, 2000) and intestinal samples from rat, chicken and pig Gong et al., 2002, Kaplan et al., 2001, Leser et al., 2000. It is currently one of the most rapid and powerful methods in microbial ecology for comparing the spatial and temporal changes in the bacterial community structure. However, interpreting large sets of data generated by T-RFLP analysis is difficult, and a statistical evaluation is needed. Principal component analysis (PCA) is a multivariate projection method and has been used in microbial ecology for comparison of complex communities Kaplan et al., 2001, Klamer et al., 2002.

The primary aim of the study was to clarify the suitability of T-RFLP combined with PCA to discover the changes during the development of infant fecal microbiota. To trace the changes in the microbiota, the predominant terminal restriction fragments (T-RFs) in the fecal communities were further identified by comparison with corresponding clones of 16S rRNA genes that were characterized by sequencing from the same samples. To the best of our knowledge, this is the first attempt to applied T-RFLP to compare the fecal microbiota in infants over time.

Section snippets

Subjects and sample collection

Two healthy infants, designated infants A and B, participated in this study. Both babies were vaginally delivered and were breastfed after birth. Infant A received only breast milk for the first 4 months. After 4 months, the formula milk replaced the breast milk and solid food was added gradually to the diet. Infant B was fed both breast and formula milk from the beginning of his life. The solid food was introduced to the diet after 4 months, but the breast-feeding was not stopped until the

Results

In this study, the development of fecal microbiota of two Swedish infants was followed by T-RFLP analysis of amplified 16S rRNA genes. To identify T-RFs in the fecal microbial communities, 16S rDNA clone libraries from the same samples were constructed and RFLP were applied in order to screen the clones and define the OTUs. Representative clones of each OTU were subjected to T-RFLP and the T-RFLP patterns of the clones were compared with those of the corresponding fecal microbial communities.

Discussion

In recent years, direct, DNA-based fingerprinting techniques such as T-RFLP and denaturing gradient gel electrophoresis (DGGE) have proven to be valuable tools in characterizing complex bacterial communities in the environment Muyzer et al., 1993, Zoetendal et al., 2002, Leser et al., 2000, Scala and Kerkhof, 2000. In this study, T-RFLP was used to monitor the development of fecal communities in the subjects, and the bacterial groups that corresponded to the predominant T-RFs were identified by

Acknowledgements

We thank Birgitta Sörenby for her excellent technical assistance. Fecal samples of infants were provided by Drs. Agnes Wold and Ingegerd Adlerberth. This study was financially supported by the European Commission (QLK4-2000-00538) and an unrestricted grant from Probi, Sweden.

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    Present address: Probi AB, Ideon gamma 1, Sölvegatan 41, SE-223 70 Lund, Sweden.

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