Molecular fingerprinting of the fecal microbiota of children raised according to different lifestyles

Abstract

In this population-based study, 90 children from three European countries were examined to determine the impact of lifestyle on the fecal microbiota. The study was designed to assess the impact of two extreme lifestyles that we hypothesized could impact the microbial composition in the gut: i.e., an anthroposophic lifestyle (restricted use of antibiotics, greater consumption of fermented vegetables, etc.) versus living on a farm (greater consumption of farm milk, contact with animals, etc.). In previous studies, these lifestyles correlated with lower prevalence of allergies. Terminal restriction fragment length polymorphism (T-RFLP) was used to assess the bacterial composition in fecal samples since recent studies have shown that the majority of this community cannot be cultivated. The T-RFLP data were used to calculate richness and evenness of the fecal microbiota. Children that were attending Steiner schools (anthroposophic children) had a significantly higher diversity of microbes in their feces than farm children, who in turn also had lower diversity than the control groups. Specific primers were also used to focus on the Lactobacillus-like community (lactic acid bacteria [LAB]). Large differences were found in the LAB subpopulations in the sampled groups. In some children, the LAB subpopulation was dominated by a species that has not yet been cultivated.

FIG. 1.

PCA plot of the T-RFLP data for the fecal bacterial community compositions of 90 individuals. Each symbol contains the average of duplicate T-RFLP profiles based on TRF size and relative abundance data for a specific individual. Blue, farm children; green, farm reference children; red, Steiner schoolchildren; gray, Steiner reference children. The percentage of variation explained by each principal component (PC) is shown in parentheses.

FIG. 2.

Diversity of the fecal microbiota presented for groups of individuals according to the questionnaire data (see Table S1 in the supplemental material). Data represent the median values with the interquartile range. Simpson's index was calculated based on T-RFLP data. Asterisks indicate lifestyle factors for which significant differences were observed.

FIG. 3.

PCA plots of T-RFLP profiles using LAB group-specific primers for all individuals from whom samples were obtained. Farm children are shown in blue, and Steiner schoolchildren are shown in red. Both reference groups are shown in gray. Panels A and B are identical, except that different TRFs are highlighted. In panel A, data from individuals with dominant TRFs of 213 and 214 bp are highlighted as solid circles. In panel B, data from individuals with a dominant TRF of 250 bp are highlighted as solid circles. Each symbol represents the average of duplicate T-RFLP profiles, including TRF sizes and relative abundance values, for an individual. Larger circles correspond to higher abundances; crosses correspond to individuals that have an absence of these TRFs. The percentage of variation explained by each principal component is shown in brackets.