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In vitro enrofloxacin binding in human fecal slurries
Youngbeom Ahn1, Sean W Linder, Brian T Veach
1Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079-9502, USA.
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.
Insights
Veterinary fluoroquinolone enrofloxacin binds to fecal matter, reducing its potential activity in the human gut. This study assessed enrofloxacin binding in fecal suspensions to understand antimicrobial residue behavior.
Area of Science:
- Environmental Science
- Microbiology
- Pharmacology
Background:
- Antibiotic inactivation studies typically use concentrations relevant to therapeutic doses.
- Limited data exists on the inactivation of antibiotic residues from animal-derived foods in the human gut.
Purpose of the Study:
- To investigate the binding of veterinary fluoroquinolone enrofloxacin to fecal matter.
- To determine the microbiological activity of enrofloxacin residues in the human colon.
Main Methods:
- Enrofloxacin binding was measured in fecal suspensions (10-50% w/v) using analytical chemistry and microbiological assays.
- Fecal samples underwent physicochemical and molecular characterization.
- Bacterial composition of human intestinal microbiota was analyzed using DGGE and pyrosequencing.
Main Results:
- Enrofloxacin binding to fecal contents occurred rapidly (within 10 min) and remained constant.
- Analytical chemistry assays showed enrofloxacin binding of approximately 50-56% in diluted fecal samples.
- Both assay methods demonstrated a consistent correlation.
Conclusions:
- Enrofloxacin exhibits significant binding to fecal matter, suggesting reduced bioavailability and activity in the human colon.
- The study provides crucial methodological insights for regulatory authorities evaluating in vitro antimicrobial residue testing.
