Escherichia coli Exopolysaccharides Induced by Ceftriaxone Regulated Human Gut Microbiota in vitro

Abstract

A stable intestinal microflora is an essential prerequisite for human health. This study investigated the interaction between Escherichia coli exopolysaccharides (named EPS-m2) and the human gut microbiota (HGM) in vitro. The EPS-m2 was produced by E. coli WM3064 when treated with ceftriaxone. The monosaccharide composition analysis revealed that EPS-m2 is composed of glucuronic acid, glucose, fucose, galactose/N-acetyl glucosamine, arabinose, xylose, and ribose with a molar ratio of approximately 77:44:29:28:2:1:1. The carbohydrates, protein, and uronic acids contents in EPS-m2 was 78.6 ± 0.1%, 4.38 ± 0.11%, and 3.86 ± 0.09%, respectively. In vitro batch fermentation experiments showed that 77% of EPS-m2 could be degraded by human fecal microbiota after 72 h of fermentation. In reverse, 16S rRNA gene sequencing analysis showed that EPS-m2 increased the abundance of Alistipes, Acinetobacter, Alloprevotella, Howardella, and Oxalobacter; GC detection illustrated that EPS-m2 enhanced the production of SCFAs. These findings indicated that EPS-m2 supplementation could regulate the HGM and might facilitate modulation of human health.

Keywords: 16S rRNA gene sequencing; EPS-m2; SCFA; ceftriaxone; gut microbiota.

FIGURE 1

Production and monosaccharide composition of EPS-m2. (A) Mucoid colonies of EPS-m2-producing bacterial on LB plates containing 0.3 mM DAP. (B) EPS-m2 yields in LB medium with different antibiotic agents were determined by the ratio of OD₅₃₀ to OD₆₀₀. (C) The monosaccharide composition of EPS-m2 was measured by using the DIONEX ICS 5000 system. The picture of “standard 20” represents the 10 standard monosaccharides. 1–10 represents the peaks of the standards L-fucose, L-rhamnose, L-arabinose, D-galactose/N-acetyl glucosamine, D-glucose, D-mannose, D-xylose, D-fructose, D-ribose, and D- glucuronic acid, respectively. “T” represents the retention time for absorption peaks. Amp, ampicillin; IMP, imipenem; CTRX, ceftriaxone. The concentrations of these three antibiotics used in this study were all 0.5 μg/mL.

FIGURE 2

Degradation of EPS-m2 by HGM in vitro. IntDen of each dot in TLC plates was measured using ImageJ software (https://imagej.nih.gov/ij; National Institutes of Health, Bethesda, MD, United States). The degradation rates of EPS-m2 by HGM were then calculated.

FIGURE 3

PCoA analysis of the microbial composition of samples. “Fecal” represents original fecal samples. “VIW” and “VIS” represent fermented samples adding EPS-m2 and starch, respectively. “24” and “48” represents the fermentation time of 24 h (A) and 48 h (B). VI represents the fermented samples using VI media.

FIGURE 4

Identification of significantly different bacteria using LEfSe. “Fecal” represents the volunteers’ original fecal samples. “VIW” and “VIS” represent fermented samples after adding EPS-m2 and starch, respectively. “24” and “48” represents the fermentation time.

FIGURE 5

Influence of EPS-m2 fermentation on SCFA production. The concentration of (A) acetic acid, (B) propionic acid, (C) butyric acid, and (D) and total SCFAs were measured using gas chromatography, and total SCFA represents the summary of acetic acid, propionic acid and butyric acid. “VIW,” “VIS,” and “VI” represent samples collected after EPS-m2 fermentation, starch fermentation, and no carbohydrate added, respectively. Significant differences between treatments were labeled using different normal letters. Triplicate measurements were performed for each sample in this study.