In vitro enrofloxacin binding in human fecal slurries

Abstract

Most antibiotic inactivation studies have been conducted through in vitro incubations of human use aminoglycosides, beta-lactams, and fluoroquinolones, usually at fecal concentrations expected with therapeutic dose regimens in humans and animals. Less is known about the inactivation of these molecules when ingested at concentrations consistent with residue levels present in animal-derived foods from antibiotic treated animals. In this investigation, we used the fluoroquinolone, enrofloxacin which is specifically marketed for veterinary medicine as test compound. Fecal suspensions at 10%, 25%, and 50% (w/v) were subjected to physicochemical and molecular characterization and used in the drug binding studies. The fecal binding of enrofloxacin added at concentrations of 0.06, 0.1, 1, 5, 15, 50, and 150 mg/L was determined in various fecal slurry suspensions using analytical chemistry and microbiological assay methods. There was consistent correlation between both assay methods. By the analytical chemistry assay, the 10%, 25% and 50% diluted autoclaved fecal samples dosed with enrofloxacin showed binding of 50±4.6%, 54±6.5% and 56±6.8% of the enrofloxacin, respectively. Binding of enrofloxacin to fecal contents occurred rapidly within 10 min and remained constant over the incubation period. Denaturing gradient gel electrophoreses and pyrosequencing analysis showed varied profiles of the bacterial composition of the human intestinal microbiota for fecal samples from different individuals. This study provided information on methodological questions that have concerned regulatory authorities on in vitro testing to determine if concentrations of veterinary antimicrobial agent residues entering the human colon remain microbiologically active.

Fig. 1.

Conceptual decision tree approaches to derive a microbiological acceptable daily intake (ADI) adapted from VICH Guideline 36.

Fig. 2.

VICH Guideline 36 formula approach equation for determining an ADI.

Fig. 3.

DGGE profiles of 16S rDNA genes from four healthy volunteer fecal samples (A). Measurements were performed in triplicate at different amplifications from the same fecal sample. Relative abundances of dominant bacterial taxa in different fecal slurry concentrations from Person A (B). Relative abundances were estimated from the proportional abundances of classified sequences.

Fig. 4.

Enrofloxacin binding to various fecal slurry concentrations. Measurements were performed in triplicate at selected enrofloxacin concentrations (0.06, 0.1 and 1 mg/L) by LC/MS after 8 h incubation. Fecal samples were collected from one volunteer on three separate occasions. Data represents a 8 h sample, since there was no statistically significant difference in binding between 2 h and 24 h.

Fig. 5.

Time course for determining the amount of enrofloxacin remaining in the supernatant in steam sterilized and non-sterilized 25% fecal slurries at 50 mg/L enrofloxacin concentrations. Symbols represent averages of triplicate tests from four human fecal slurries and error bars represent the standard deviation.