In vitro test systems to determine tetracycline residue binding to human feces

Abstract

The use of antimicrobials, such as tetracycline, in food-producing animals may result in antimicrobial drug residues (ADR) in edible tissues from treated animals and contribute to the emergence of antibiotic resistant bacteria. The Veterinary International Conference on Harmonization (VICH) document (VICH GL36(R)/FDA-CVM Guidance for Industry#159) provides guidance on evaluating the safety of veterinary ADR in the human foods as related to effects on the human intestinal microbiome. One recognized research gap is a need for additional data and testing requirements to determine the fraction of an oral dose of ADR available to intestinal microorganisms. In the present study, we address this need by examining the binding of tetracycline to human feces using chemical and microbiological assays. High-performance liquid chromatography and liquid chromatography mass spectrometry assays showed that 25% (w/v) diluted steam sterilized feces dosed with 0.15 and 1.5 μg/ml tetracycline had binding of 58.2 ± 10.8% and 56.9 ± 9.1%, respectively. Tetracycline binding to fecal slurries gave similar results. Microbiological assays with two reference bacterial strains validated the results of the chemical assays. Based on data from chemical and microbiological assays methods, the fraction of dose available to microorganisms was 0.418 and 0.431 of the 0.15 and 1.5 μg/ml tetracycline treatments, respectively. This study also proposes factors to be considered when designing and conducting experiments to determine the percent of an antimicrobial agents that is available to microorganisms in the gastrointestinal tract.

Keywords: Binding; Human fecal slurries; In vitro test systems; Tetracycline.

Fig. 1.

VICH Guideline 36 formula approach equation for determining a microbiological ADI.

Fig. 2.

Overview of the in vitro test systems approach to determine the fraction of oral dose available to microorganisms. This process is composed of three steps: Experimental conditions and setup (Step 1), in vitro chemical assay (Step 2) and in vitro microbiological assay (Step 3). In this study, the fraction of the dose available for colonic microorganisms was calculated by an in vitro chemical assay (Step 2) and confirmed by an in vitro microbiological assay (Step 3).

Fig. 3.

The abiotic transformation of tetracycline in 0.1% formic acid and 3% (w/v) human fecal supernatant for 9 days.

Fig. 4.

Comparison of the percent of tetracycline binding to steam sterilized fecal slurries and non-sterilized fecal slurries after 2 h incubation in BHI medium with selected tetracycline concentrations (15, 20, 30, and 50 μg/ml). Graphs represent averages of triplicate samples; selected tetracycline concentrations and error bars represent the standard deviations.

Fig. 5.

The percent of tetracycline binding under different fecal slurry concentrations over 2 h incubation in BHI medium with selected tetracycline concentrations (0.15, 1.5, 15 and 150 μg/ml). Fecal samples were collected from three healthy volunteers. Graphs represent averages of triplicate tests from three human fecal slurries; selected tetracycline concentrations and error bars represent the standard deviations.

Fig. 6.

Time course for determining the amount of tetracycline remaining in the supernatant in steam sterilized 25% fecal slurries at 15 μg/ml (A) and 150 μg/ml (B) tetracycline concentrations. Graphs represent averages of triplicate samples; selected tetracycline concentrations and error bars represent the standard deviations.

Fig. 7.

Comparison of the tetracycline concentration to binding under steam sterilized 25% fecal slurries after 2 h incubation in BHI medium with selected tetracycline concentrations (15, 20, 30, 50, 70, 100 and 150 μg/ml). Graphs represent averages of triplicate samples and error bars represent the standard deviations.

Fig. 8.

In Vitro fecal slurry test system-chemical assays. The percent of tetracycline binding to 0.15 μg/ml (A) and 1.5 μg/ml (B) tetracycline over 2 h incubation in steam sterilized 25% (w/v) fecal slurries. Graphs represent averages of triplicate samples and error bars represent the standard deviations.

Fig. 9.

Procedure of appropriate methods to determine fraction of oral dose available to microorganisms (N = test number).