A double-blind, 377-subject randomized study identifies Ruminococcus, Coprococcus, Christensenella, and Collinsella as long-term potential key players in the modulation of the gut microbiome of lactose intolerant individuals by galacto-oligosaccharides

M A Azcarate-Peril, J Roach, A Marsh, William D Chey, William J Sandborn, Andrew J Ritter, Dennis A Savaiano, T R Klaenhammer, M A Azcarate-Peril, J Roach, A Marsh, William D Chey, William J Sandborn, Andrew J Ritter, Dennis A Savaiano, T R Klaenhammer

Abstract

Background. Our recent publication (Chey et al., Nutrients 2020) showed that a 30-day administration of pure galacto-oligosaccharides (GOS) significantly reduced symptoms and altered the fecal microbiome in patients with lactose intolerance (LI). Results. In this addendum, we performed an in-depth analysis of the fecal microbiome of the 377 LI patients randomized to one of two GOS doses (Low, 10-15 grams/day or High, 15-20 grams/day), or placebo in a multi-center, double-blinded, placebo-controlled trial. Sequencing of 16S rRNA amplicons was done on GOS or placebo groups at weeks zero (baseline), four (end of treatment), nine, 16 and 22. Taxa impacted by treatment and subsequent dairy consumption included lactose-fermenting species of Bifidobacterium, Lactobacillus, Lactococcus, and Streptococcus. Increased secondary fermentation microorganisms included Coprococcus and Ruminococcus species, Blautia producta, and Methanobrevibacterium. Finally, tertiary fermenters that use acetate to generate butyrate were also increased, including Faecalibacterium prausnitzii, Roseburia faecis, and C. eutactus. Conclusions. Results confirmed and expanded data on GOS microbiome modulation in LI individuals. Microbiome analysis at 16 and 22 weeks after treatment further suggested relatively long-term benefits when individuals continued consumption of dairy products.

Trial registration: ClinicalTrials.gov NCT02673749.

Keywords: Bifidobacterium; GOS; Prebiotics; galacto-oligosaccharides; human lactase; lactose intolerance; microbiome modulation; short chain fatty acids.

Conflict of interest statement

AJR was the Co-Founder, President and Chief Executive Officer of Ritter Pharmaceuticals, Inc. (acquired by Qualigen Therapeutics) from 2007 to 2020.

Figures

Figure 1.
Figure 1.
(a) Phylum composition of the gut microbiome of individuals that received either placebo or GOS treatments (Low or High GOS). The most abundant taxa are indicated , . (b) Faith Phylogenetic Diversity (PD) averages between treatments and (c) between treatments at different times. (d) Pairwise PERMANOVA comparisons between Unifrac Unweighted distances by treatment and times. *Corrected p (q)<0.05
Figure 1.
Figure 1.
(a) Phylum composition of the gut microbiome of individuals that received either placebo or GOS treatments (Low or High GOS). The most abundant taxa are indicated , . (b) Faith Phylogenetic Diversity (PD) averages between treatments and (c) between treatments at different times. (d) Pairwise PERMANOVA comparisons between Unifrac Unweighted distances by treatment and times. *Corrected p (q)<0.05
Figure 2.
Figure 2.
(a) Representation of taxa at genus level differentially represented in at least one group and one time point (FDR corrected Kruskal-Wallis P <.05). The heatmap was generated using log2-transformed data in the Heat Map with Dendrogram app within OriginPro 9.7.5.184. (b) Relative abundance by treatment of Coprococcus catus, Bifidobacterium, Faecalibacterium prausnitzii and Methanobrevibacterium over time (in weeks). *Kruskall-Wallis FDR-corrected p < .05
Figure 2.
Figure 2.
(a) Representation of taxa at genus level differentially represented in at least one group and one time point (FDR corrected Kruskal-Wallis P <.05). The heatmap was generated using log2-transformed data in the Heat Map with Dendrogram app within OriginPro 9.7.5.184. (b) Relative abundance by treatment of Coprococcus catus, Bifidobacterium, Faecalibacterium prausnitzii and Methanobrevibacterium over time (in weeks). *Kruskall-Wallis FDR-corrected p < .05
Figure 3.
Figure 3.
(a) Abundance of the phylum Actinobacteria, family Bifidobacteriaceae, and genus Bifidobacterium determined by high-throughput (HT) qPCR by treatment and time at weeks 0 (baseline), 4 (end of GOS treatment), and 9 (end of trial, lactose challenge). (b) Heat map showing abundance of Bifidobacterium species by treatment and time. (c) Abundance of Lactobacillus casei determined by HT qPCR. *p < .05
Figure 3.
Figure 3.
(a) Abundance of the phylum Actinobacteria, family Bifidobacteriaceae, and genus Bifidobacterium determined by high-throughput (HT) qPCR by treatment and time at weeks 0 (baseline), 4 (end of GOS treatment), and 9 (end of trial, lactose challenge). (b) Heat map showing abundance of Bifidobacterium species by treatment and time. (c) Abundance of Lactobacillus casei determined by HT qPCR. *p < .05
Figure 4.
Figure 4.
Analysis of alpha diversity (Faith Phylogenetic Diversity values) by (a) age, (b) age by treatment, and (c) age by treatment and time. H and p values from Kruskall-Wallis analysis indicating the impact significance of the factor considered on diversity are denoted the bottom of each figure. (d) Adonis (PERMANOVA) R2 values representing effect size of patients’ characteristics and treatment on microbiome composition
Figure 4.
Figure 4.
Analysis of alpha diversity (Faith Phylogenetic Diversity values) by (a) age, (b) age by treatment, and (c) age by treatment and time. H and p values from Kruskall-Wallis analysis indicating the impact significance of the factor considered on diversity are denoted the bottom of each figure. (d) Adonis (PERMANOVA) R2 values representing effect size of patients’ characteristics and treatment on microbiome composition
Figure 5.
Figure 5.
Predicted functionality of the GOS-enhanced microbiota. (a) The cellobiose and glutamate bacterial transporters were overrepresented in the prebiotic group. (b) Pathway KO00511 (Other glycan degradation), with genes overrepresented in the GOS group in the N-glycan biosynthesis pathway. (c) Genes responsible for initiation of fatty acid biosynthesis overrepresented in the GOS groups. (d) Summary of the gut metabolic processes and potentially responsible microorganisms that participate in biotransformation of GOS. Blue boxes indicate organisms known to carry out the enzymatic process, while green boxes are potential new players in the intestinal cross feeding of GOS. Depiction was based on our results and published research studies.
Figure 5.
Figure 5.
Predicted functionality of the GOS-enhanced microbiota. (a) The cellobiose and glutamate bacterial transporters were overrepresented in the prebiotic group. (b) Pathway KO00511 (Other glycan degradation), with genes overrepresented in the GOS group in the N-glycan biosynthesis pathway. (c) Genes responsible for initiation of fatty acid biosynthesis overrepresented in the GOS groups. (d) Summary of the gut metabolic processes and potentially responsible microorganisms that participate in biotransformation of GOS. Blue boxes indicate organisms known to carry out the enzymatic process, while green boxes are potential new players in the intestinal cross feeding of GOS. Depiction was based on our results and published research studies.

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