Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study

Abstract

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridiumsensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota. Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

Keywords: Bifidobacterium breve; coeliac disease; gluten free diet; intestinal microbiota; next generation sequencing; probiotic; qPCR.

Figure 1

Summary of inclusion/exclusion criteria adopted for samples selection. Samples who showed the right DNA quantification level have been sequenced. * Inclusion criteria are summarized in Klemenak et al. [23]. ** Analysis was performed at the beginning of the study (T0) and after 3 months of treatment (T1).

Figure 2

Hierarchically clustered heat map: Sample groups are reported in column, while phyla are reported in row.

Figure 3

The Firmicutes/Bacteroidetes ratio.

Figure 4

Relative abundance of the three phyla that show a statistical significance difference among CD, Control and Probiotic T1. CD group is composed of Probiotic T0, Placebo T0 and Placebo T1 samples. The * indicates p < 0.01. Supporting information on relative abundance and p-values is found in Tables S2 and S3.

Figure 5

Significant differences in phyla relative abundance among Control, Probiotic T0 and Probiotic T1 groups. The * indicates p < 0.01. Supporting information on relative abundance and p-values is found in Tables S4 and S5.

Figure 6

Statistical significant differences in families relative abundance among Control, Probiotic T0 and Probiotic T1 groups. The * indicates p < 0.01. Supporting information on relative abundance and p-values is found in Tables S6 and S7.

Figure 7

Alpha diversity indices among the studied groups.

Figure 8

Box plots showing qPCR analysis of Lactobacillus group expressed in log CFU per gram of faecal sample relative to CD group and Control group. CD group is composed of Probiotic T0, Placebo T0 and Placebo T1 samples. Statistical difference between the two groups (p-values of < 0.01).

Figure 9

Box plots showing qPCR analysis of Bacteroides fragilis group expressed in log CFU per gram of faecal sample relative to CD group and Control group. CD group is composed of Probiotic T0, Placebo T0 and Placebo T1 samples. Statistical difference between the two groups (p-values of < 0.01).

Figure 10

Box plots showing qPCR analysis of total enterobacteria expressed in log CFU per gram of faecal sample relative to CD group and Control group. CD group is composed of Probiotic T0, Placebo T0 and Placebo T1 samples. Statistical difference between the two groups (p-values of < 0.01).