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. 2020 Mar 14;21(6):1986.
doi: 10.3390/ijms21061986.

Isolation and Quantification of Uremic Toxin Precursor-Generating Gut Bacteria in Chronic Kidney Disease Patients

Tessa Gryp  1   2   3 , Geert R B Huys  3 , Marie Joossens  3 , Wim Van Biesen  1 , Griet Glorieux  1 , Mario Vaneechoutte  2
Affiliations

Isolation and Quantification of Uremic Toxin Precursor-Generating Gut Bacteria in Chronic Kidney Disease Patients

Tessa Gryp et al. Int J Mol Sci. .

Abstract

In chronic kidney disease (CKD), impaired kidney function results in accumulation of uremic toxins, which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as p-cresyl sulfate, indoxyl sulfate and indole-3-acetic acid, originate from phenolic and indolic compounds, which are end products of gut bacterial metabolization of aromatic amino acids (AAA). This study investigates gut microbial composition at different CKD stages by isolating, identifying and quantifying PBUT precursor-generating bacteria. Fecal DNA extracts from 14 controls and 138 CKD patients were used to quantify total bacterial number and 11 bacterial taxa with qPCR. Moreover, isolated bacteria from CKD 1 and CKD 5 fecal samples were cultured in broth medium supplemented with AAA under aerobic and anaerobic conditions, and classified as PBUT precursor-generators based on their generation capacity of phenolic and indolic compounds, measured with U(H)PLC. In total, 148 different fecal bacterial species were isolated, of which 92 were PBUT precursor-generators. These bacterial species can be a potential target for reducing PBUT plasma levels in CKD. qPCR indicated lower abundance of short chain fatty acid-generating bacteria, Bifidobacterium spp. and Streptococcus spp., and higher Enterobacteriaceae and E. coli with impaired kidney function, confirming an altered gut microbial composition in CKD.

Keywords: aromatic amino acids; bacterial culture; bacterial metabolization; chronic kidney disease; fecal bacteria; indole; indole-3-acetic acid; p-cresol; phenol; protein-bound uremic toxins; qPCR.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Bacterial gene copies per gram feces per chronic kidney disease (CKD) stage: (a) Total bacterial 16S rRNA; (b) Clostridioides difficile; (c) Streptococcus spp.; (d) Bacteroides spp.; (e) Enterobacteriaceae; (f) Escherichia coli; (g) Butyricicoccus spp.; (h) Faecalibacterium prausnitzii; (i) Roseburia spp.; (j) Akkermansia muciniphila; (k) Bifidobacterium spp.; (l) Lactobacillus spp. *: p ≤ 0.05; **: p ≤ 0.001; rs: Spearman’s rank test; gray square: correlation between respective bacteria versus CKD stages 1–5 and control.
Figure 2
Figure 2
Bacterial gene copies per gram feces for the control, CKD and dialysis group: (a) Total bacterial 16S rRNA; (b) Clostridioides difficile; (c) Streptococcus spp.; (d) Bacteroides spp.; (e) Enterobacteriaceae; (f) Escherichia coli; (g) Butyricicoccus spp.; (h) Faecalibacterium prausnitzii; (i) Roseburia spp.; (j) Akkermansia muciniphila; (k) Bifidobacterium spp.; (l) Lactobacillus spp. *: p ≤ 0.05; ** p ≤ 0.001.
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