Lactobacillus rhamnosus GG Intake Modifies Preschool Children's Intestinal Microbiota, Alleviates Penicillin-Associated Changes, and Reduces Antibiotic Use

Abstract

Antibiotic use is considered among the most severe causes of disturbance to children's developing intestinal microbiota, and frequently causes adverse gastrointestinal effects ranging from mild and transient diarrhoea to life-threatening infections. Probiotics are commonly advocated to help in preventing antibiotic-associated gastrointestinal symptoms. However, it is currently unknown whether probiotics alleviate the antibiotic-associated changes in children's microbiota. Furthermore, it is not known how long-term probiotic consumption influences the developing microbiota of children. We analysed the influence of long-term Lactobacillus rhamnosus GG intake on preschool children's antibiotic use, and antibiotic-associated gastrointestinal complaints in a double blind, randomized placebo-controlled trial with 231 children aged 2-7. In addition, we analysed the effect of L. rhanmosus GG on the intestinal microbiota in a subset of 88 children. The results show that long-term L. rhamnosus GG supplementation has an influence on the composition of the intestinal microbiota in children, causing an increase in the abundance of Prevotella, Lactococcus, and Ruminococcus, and a decrease in Escherichia. The treatment appeared to prevent some of the changes in the microbiota associated with penicillin use, but not those associated with macrolide use. The treatment, however, did reduce the frequency of gastrointestinal complaints after a macrolide course. Finally, the treatment appeared to prevent certain bacterial infections for up to 3 years after the trial, as indicated by reduced antibiotic use.

Trial registration: ClinicalTrials.gov NCT01014676.

Fig 1. Flow-chart of the study cohort.

Fig 2. Incidence of antibiotic use (%) in the placebo and L. rhamnosus GG groups.

The dashed line shows the end of the intervention. The p-values indicate the significance of the difference (indicated by the arrows at different time points, based on survival regression models) in antibiotic use between the groups at the end of the intervention, 1 year after the intervention, 2 years after the intervention, and 2.7 years after the intervention.

Fig 3. Cumulative number of antibiotic courses per person in the placebo and L. rhamnosus GG groups.

The dashed line shows the end of the intervention. The p-values indicate the significance of the difference (indicated by the arrows at different time points, based on negative binomial models) in antibiotic use between the groups at the end of the intervention, 1 year after the intervention, 2 years after the intervention, and 2.7 years after the intervention.

Fig 4. Frequency of gastrointestinal complaints per day per person during the intervention.

The frequency of complaints was calculated separately for the following periods: during the month before any antibiotic course, during the week before any antibiotic course, during a penicillin or a macrolide course, and during the month after a penicillin or a macrolide course. Some children received several courses and therefore contributed to the calculation of penicillin and macrolide-associated symptoms. Significant differences based on non-parametric Kruskal-Wallis (when comparing groups) and paired-sample Wilcoxon (when comparing different time periods in the same individuals) -tests are shown.

Fig 5. Microbiota diversity and stability by treatment group.

Diversity after the intervention was calculated as the Inverse Simpson index (panel A), and microbiota stability as the Pearson correlation between the pre- and post-intervention sample (panel B). Significant differences based on analysis of variance are shown.

Fig 6. Microbiota composition by treatment group.

Composition in the placebo group (panel A) and L. rhamnosus GG group (panel B). The component scores were calculated using principal coordinates analysis of the Pearson correlation distances in the species-level data. All samples were included in the same analysis, and the groups are shown in different panels for clarity.

Fig 7. Bacterial groups associated with the treatments.

Genus-level bacterial groups significantly associated with at least one of the treatments (L. rhamnosus GG or antibiotics), clustered based on their response profile to the L. rhamnosus GG and antibiotics. The average total abundances (± standard error) of the clusters in the different treatment groups are shown in the barplots. Significance of the difference from the control group (based on negative binomial models) are indicated by the asterisks: * p<0.05; ** p<0.01; *** p<0.001.