Normalization of short-chain fatty acid concentration by bacterial count of stool samples improves discrimination between eubiotic and dysbiotic gut microbiota caused by Clostridioides difficile infection

Abstract

Short-chain fatty acids (SCFAs) represent a cornerstone of gut health, serving as critical mediators of immune modulation and overall host homeostasis. Patients with dysbiosis caused by Clostridioides difficile infection (CDI) typically exhibit lower SCFAs levels compared to healthy stool donors and, thus, the concentration of SCFAs has been proposed as a proxy marker of a healthy microbiota. However, there is no consistency in the methods used to quantify SCFAs in stool samples and usually, the results are normalized by the weight of the stool samples, which does not address differences in water and fiber content and ignores bacterial counts in the sample (the main component of stool that contributes to the composition of these metabolites in the sample). Here, we show that normalized SCFAs concentrations by the bacterial count improve discrimination between healthy and dysbiotic samples (patients with CDI), particularly when using acetate and propionate levels. After normalization, butyrate is the metabolite that best discriminates eubiotic and dysbiotic samples according to the area under the receiver operating characteristic (ROC) curve (AUC-ROC = 0.860, [95% CI: 0.786-0.934], p < .0001).

Keywords: Clostridioides difficile; Short-chain fatty acids (SCFAs); dysbiosis; fecal microbiota transplantation (FMT); gut; intestinal microbiota.

Figure 1.

Bacterial count (bacteria/µl) of stool samples. Dilutions 1:1,000; 1:5,000, and 1:10,000 were assessed (five technical replicates per dilution). R² average of 0.9979 (n = 6).

Figure 2.

Density plot of the bacterial counts (bacteria/µl) of the original sample. Data from healthy donors (n = 115) (blue) and dysbiotic patients with CDI (n = 40) (red).

Figure 3.

Quantification of the SCFA levels without normalization (µg/mL), normalized by fresh weight (µg/mg), and normalized by bacterial count of healthy donors (n = 115) (blue) and dysbiotic patients with CDI (n = 40) (red). a) acetate, b) propionate, and c) butyrate.

Figure 4.

Distribution of SCFAs levels normalized by bacterial count and ROC curve analysis for comparison of SCFAs levels in detecting healthy donors. a) acetate (purple), b) propionate (yellow), and c) butyrate (green); d) ROC curve for acetate (AUC-ROC = 0.696), propionate (AUC-ROC = 0.755) and butyrate (AUC-ROC = 0.860). In the density plots the dashed lines indicate the optimal cutoffs calculated in the ROC curve, in the ROC curve the dashed line is the random classifier. Healthy donors (n = 115) and dysbiotic patients with CDI (n = 40).