Sensitive and Simplified Detection of Antibiotic Influence on the Dynamic and Versatile Changes of Fecal Short-Chain Fatty Acids

Abstract

Short-chain fatty acids (SCFAs), produced by anaerobic fermentation of mainly indigestible dietary carbohydrates by gut microbiota, have a profound influence on intestinal function and host energy metabolism. Antibiotics may seriously disturb the balance of fecal SCFAs. To evaluate the impacts of antibiotics on fecal SCFAs produced by gut microbiota, a simple, reproducible and accurate gas chromatography (GC) method, which can simultaneously analyze seven SCFAs in fecal samples, was developed and validated. The ranges of detection and quantitation of the SCFAs reached 0.0868 ~ 0.393 and 0.261 ~ 1.18 μg·mL-1 respectively, in an optimized protocol for SCFAs extraction and analysis that used 10 mL 75% ethanol aqueous solution containing 1% HCl, without ultrasonication. The technique exhibited excellent intra-day (relative standard deviation (RSD) ≤ 2.54%) and inter-day (RSD ≤ 4.33%) precisions for all the SCFAs. Later, we administered broad-spectrum antibiotics, cefdinir or azithromycin to rats and analyzed the alterations in fecal SCFAs. The total amount, types and distribution of nearly all fecal SCFAs were significantly altered during the administration and even after withdrawal of the antibiotics in rats. The effects of cefdinir on the SCFAs were more pronounced than those of azithromycin. Our findings suggest SCFAs may serve as sensitive indicators to monitor the influences of antibiotics on SCFAs originated by intestinal bacteria. Our improved SCFAs analysis method is a potential platform for a standard clinical test of the effects of new antibiotics on SCFAs.

Fig 1. Optimization of the sample extraction method.

(A) ratio of ethanol to water; (B) the percentage of HCl; (C) ultrasonication time; and (D) extraction volume.

Fig 2. Representative gas chromatograms of samples.

(A) mixed standards solution; (B) the control fecal sample; (C) the fecal sample from the cefdinir-treated group on the first day. 1. acetic acid; 2. propionic acid; 3. isobutyric acid; 4. butyric acid; 5. isovaleric acid; 6. valeric acid; 7. hexanoic acid; IS-1. 2-ethyl butyric acid; IS-2. 2-ethyl hexanoic acid.

Fig 3. Bodyweight variation in the control, cefdinir- or azithromycin-treated groups.

ΔW-Con: weight gain in the control groups. ΔW-Cef: weight gain in the cefdinir-treated groups. ΔW-Azi: weight gain in the azithromycin-treated groups. All measurements were between day 0 and day 13.

Fig 4. The concentrations of seven SCFAs extracted from fecal samples as a function of time.

Antibiotics (cefdinir or azithromycin) were administered for the first 6 days. (A) acetic acid; (B) propionic acid; (C) isobutyric acid; (D) butyric acid; (E) isovaleric acid; (F) valeric acid; and (G) hexanoic acid. Arrow means administration of antibiotic.

Fig 5

The schematic diagram of AUC with a function of time (A), and AUC of SCFAs in fecal samples in the periods of antibiotic-treated and recovery (B). (a) acetic acid; (b) propionic acid; (c) isobutyric acid; (d) butyric acid; (e) isovaleric acid; (f) valeric acid; (g) hexanoic acid.

Fig 6. Relative proportions of seven SCFAs in fecal samples from the control, cefdinir-treated or azithromycin-treated groups during and post-antibiotic administration.

(The size of the pie charts represents the relative total quantity of SCFAs; “N” represents the control group. “C” represents the cefdinir group. “A” represents the azithromycin group).