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. 2019 Oct:127:146-154.
doi: 10.1016/j.bone.2019.06.010. Epub 2019 Jun 14.

The microbial metagenome and bone tissue composition in mice with microbiome-induced reductions in bone strength

Jason D Guss  1 , Erik Taylor  2 , Zach Rouse  3 , Sebastian Roubert  2 , Catherine H Higgins  4 , Corinne J Thomas  5 , Shefford P Baker  3 , Deepak Vashishth  5 , Eve Donnelly  6 , M Kyla Shea  7 , Sarah L Booth  7 , Rodrigo C Bicalho  4 , Christopher J Hernandez  8
Affiliations

The microbial metagenome and bone tissue composition in mice with microbiome-induced reductions in bone strength

Jason D Guss et al. Bone. 2019 Oct.

Abstract

The genetic components of microbial species that inhabit the body are known collectively as the microbiome. Modifications to the microbiome have been implicated in disease processes throughout the body and have recently been shown to influence bone. Prior work has associated changes in the microbial taxonomy (phyla, class, species, etc.) in the gut with bone phenotypes but has provided limited information regarding mechanisms. With the goal of achieving a more mechanistic understanding of the effects of the microbiome on bone, we perform a metagenomic analysis of the gut microbiome that provides information on the functional capacity of the microbes (all microbial genes present) rather than only characterizing the microbial taxa. Male C57Bl/6 mice were subjected to disruption of the gut microbiota (ΔMicrobiome) using oral antibiotics (from 4 to 16 weeks of age) or remained untreated (n = 6-7/group). Disruption of the gut microbiome in this manner has been shown to lead to reductions in tissue mechanical properties and whole bone strength in adulthood with only minor changes in bone geometry and density. ΔMicrobiome led to modifications in the abundance of microbial genes responsible for the synthesis of the bacterial cell wall and capsule; bacterially synthesized carbohydrates; and bacterially synthesized vitamins (B and K) (p < 0.01). Follow up analysis focused on vitamin K, a factor that has previously been associated with bone health. The vitamin K content of the cecum, liver and kidneys was primarily microbe-derived forms of vitamin K (menaquinones) and was decreased by 32-66% in ∆Microbiome mice compared to untreated animals (p < 0.01). Bone mineral crystallinity determined using Raman spectroscopy was decreased in ∆Microbiome mice (p = 0.01). This study illustrates the use of metagenomic analysis to link the microbiome to bone phenotypes and provides preliminary findings implicating microbially synthesized vitamin-K as a regulator of bone matrix quality.

Keywords: Biomechanics; Bone matrix; Microbiome; Osteoimmunology; Osteoporosis; Raman spectroscopy.

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Conflict of interest statement

Disclosures

All authors state that they have no conflicts of interest.

Figures

Figure 1.
Figure 1.
(A) Disruption of the gut microbiome (ΔMicrobiome) results in reductions in tissue strength assessed through three point bending of the mouse femur (figure adapted from [8]). (B) A heatmap summarizing the metagenomic analysis of the fecal microbiota. Each column represents an individual animal (n=6 per group). (C) Principal coordinate analysis summarizes the differences in the functional capacity of the gut microbiota between the two groups (p = 0.003, R2 = 0.93, ANOSIM).
Figure 2.
Figure 2.
The relative abundance of genes associated with key pathways for (A) vitamin synthesis, (B) vitamin K synthesis (shown in the order of synthesis), (C) carbohydrates, and (D) bacterial cell wall and capsule components are altered in ΔMicrobiome mice (n=6/group). * - p < 0.002. Effect size is shown in Supplementary Figure 1.
Figure 3.
Figure 3.
Vitamin K content was altered by disruption of the gut microbiome in the (A) Cecum, (B) Liver and (C) Kidney. Each color in a column represents the average concentration of n=6 samples (MK11 and MK13 were nondetectable, wee Supplementary Table 1). * indicates p < 0.001. (D) Matrix bound osteocalcin showed a trend indicating reduced concentration in ΔMicrobiome mice (p=0.05).
Figure 4.
Figure 4.
(A) Five Raman point spectra were collected in each of the four anatomical quadrants of a tibial diaphysis cross section. The average of the five spectra within each quadrant was determined. ΔMicrobiome was associated with reduced (B) crystallinity and (C) no noticeable differences in mineral:matrix ratio after accounting for variation among quadrants (n=4 specimens/group, error bars indicate SD).
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