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. 2023 Jan 5;11(1):135.
doi: 10.3390/microorganisms11010135.

Human Fecal Bile Acid Analysis after Investigational Microbiota-Based Live Biotherapeutic Delivery for Recurrent Clostridioides difficile Infection

Affiliations

Human Fecal Bile Acid Analysis after Investigational Microbiota-Based Live Biotherapeutic Delivery for Recurrent Clostridioides difficile Infection

Romeo Papazyan et al. Microorganisms. .

Abstract

Microbiome-based therapeutics are increasingly evaluated as a strategy to reduce recurrent Clostridioides difficile infection (rCDI), with proposed mechanisms including restoration of the microbiota and microbiota-mediated functions, such as bile acid (BA) metabolism. This study reports a quantitative and sensitive assay for targeted metabolomic assessment, and the application of the assay to profile BA composition in a Phase 2 trial of the investigational microbiota-based live biotherapeutic RBX2660 for reduction of rCDI. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 35 BAs from 113 participant stool samples from 27 RBX2660-treated rCDI participants in the double-blinded, placebo-controlled clinical trial. The results demonstrate a high-confidence assay as represented by sensitivity, linearity, accuracy, and precision. Furthermore, the assay enabled the observation of primary BAs as the dominant BA species at baseline in stool samples from clinical trial participants, consistent with the expected loss of commensals after broad-spectrum antibiotic treatment. After RBX2660 administration, there was a significant drop in primary BAs concurrent with increased secondary BAs that sustained through 24 months post-RBX2660. Taken together, we describe a robust assay that demonstrates altered BA metabolism in rCDI patients treated with RBX2660 administration.

Keywords: Clostridioides difficile; RBX2660; bile acids; dysbiosis; microbiota-based investigational therapeutic.

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Conflict of interest statement

R.P., N.F., K.S., and B.C.F. are employees of Ferring Pharmaceuticals. K.B. is an employee of Rebiotix Inc., a Ferring company. C.G. and W.D.S. are employed by BioRankings, which received fees for this analysis from Rebiotix Inc., a Ferring company.

Figures

Figure 1
Figure 1
Assay procedures and readouts. (A) Scheme of sample preparation for controls (yellow) and test samples (green) processed in parallel. (B) Representative chromatograms of two standards (CDCA and DCA) in blue and a blank buffer in red (top). Representative control sample in red showing natural levels of indicated BAs and control sample with spiked BAs in blue.
Figure 2
Figure 2
Assay quantification and sensitivity in fecal matrix. (A) Limit of detection (LoD) and limit of quantification (LoQ) to upper limit of quantification (ULoQ) of the indicated BA highlighting the sensitivity of our method. (B) Inter- and Intra-day runs show high precision (%CV) and high accuracy of quality control samples. 3 independent experiments were run with each BA using a 4-point concentration gradient. (C) Detection of fecal BA after 0, 1, and 2 cycles of freeze–thaws.
Figure 3
Figure 3
Large-scale BA changes detected in rCDI patients treated with RBX2660. Heatmap of fecal BA levels (ng/g) in wet fecal matter from PUNCH CD2 trial participants. Each row is a single patient sample, with rows grouped by timepoints (at baseline or time post baseline) of when the stool samples were collected. Annotations in boxes above the heatmap include primary (light gray) and secondary (dark gray) BA, conjugate (green) and deconjugated (white) BA, as well as 7-hydroxylation (7-OH, blue), 7-oxo (7=O, red), and 7-dehydroxylated (7-H, tan) BA.
Figure 4
Figure 4
Quantitative and specific BA changes. (A) Absolute fecal levels of the primary BAs TCA, GCA, CA known to promote C. difficile germination. (B) Absolute fecal levels of the secondary BAs LCA and DCA associated with suppression of C. difficile outgrowth. Samples from study participants were taken at baseline, week 1, week 4, and week 8 post RBX2660 treatment. Red line represents the geometric mean. Statistical significant was determined by a linear mixed model and the changes from baseline were significantly different for all BAs (p < 0.05).
Figure 5
Figure 5
BA compositions over time. All detected bile acids were categorized and grouped as either primary or secondary and conjugated or deconjugated to highlight the average changes to fecal BA composition over the course of the study in rCDI patients receiving RBX2660.

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