Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants

Abstract

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut.

Figure 1

Significantly diverse bacterial community dynamics across cow's milk allergy and its treatment. (a) Shannon diversity, (b) Pielou's evenness and (c) bacterial load in fecal samples from each healthy (n=20) or age-matched pre-treatment cow's milk allergic (CMA, n=18–19) patients at diagnosis. (d) Family level differential abundance across healthy, CMA pre-treatment and treated groups, as computed by MetagenomeSeq. Families depicted are those determined to be differentially abundant. (e) Generalized linear model fitting of patient demographic information across relative abundance of family Lachnospiraceae. Parallel x axis represents the relative contribution value of every factor, as predicted by the GLM model. *P<0.05, **P<0.0001, by two-sided t-test or by GLM model.

Figure 2

Microbial community dynamics of fecal samples from cow's milk allergic infants (CMA) before and after treatment. (a) Butyrate (n-butyric acid) concentration in fecal samples from healthy patients (n=20), or from CMA patients before (CMA, n=19) and after treatment (post-EHCF, n=7; post-EHCF+LGG, n=12). (b) Differential features (genera) selection analysis across healthy, CMA pre-treatment and treated groups (EHCF and EHCF+LGG). Abundance matrix was processed using Kruskal–Wallis H-test (post hoc tests=Tukey Kramer, multiple test correction=Bonferroni) with hierarchical clustering of both rows (genera; y axis; clusters are represented by color bars) and columns (samples; x axis; clustering is performed with ‘average' linkage, using Bray–Curtis' distance for genera and ‘correlation' for samples). The heatmap key shows percent relative abundance. Oligotyping analysis reveals strain-level differential selection in (c) Roseburia, (d) Coprococcus and (d) Blautia enriched across CMA and EHCF+LGG samples. Samples from EHCF+LGG-treated infants determined to be tolerant after double blind placebo-controlled oral food challenge analysis are labeled as ‘T'. **P< 0.05, ***P < 0.001, by Kruskal–Wallis H-test.