Impact of Chronic Tetracycline Exposure on Human Intestinal Microbiota in a Continuous Flow Bioreactor Model

Abstract

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 10⁷ ± 0.3 × 10⁷ down to 2 × 10⁶ ± 0.8 × 10⁶ CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

Keywords: chronic exposure; continuous flow bioreactor model; human intestinal microbiota; tetracycline.

Figure 1

A laboratory continuous flow bioreactor (six reactors with 0, 0.015, 0.15, 1.5, 15, and 150 µg/mL tetracycline) (A); a schematic diagram and timeline (B); overview of measuring effects of tetracycline on human intestinal microbiota (C).

Figure 2

Comparison of total viable counts on BHI (A) and CDC (B) in controls (no treatment) and tetracycline-treated samples after 7 days. * indicates statistically significant differences from control (p < 0.05).

Figure 3

Bacterial communities change at the genus (A) and OTU levels (B) in controls (no treatment) and tetracycline-treated samples after 7 days. Heatmap (B) shows fold change of OTUs with >0.1% of relative abundance at control, and OTUs number/taxonomic group based on Mothur’s classification are represented for each row.

Figure 4

Changes in relative abundance of family (A), and genus (B), level and comparison of family Enterobacteriaceae (C), and genus Escherichia (D) in controls (no treatment) and tetracycline-treated samples after 7 days. * indicates statistically significant differences from control (p < 0.05).

Figure 5

Comparison of percentage of four tetracycline resistance genes (tetB, tetD, tetQ, and tetW) as quantified by qRT-PCR in the family Enterobacteriaceae (A–D) and the genus Escherichia (E–H) in controls (no treatment) and tetracycline-treated samples after 7 days. tetB: (A,E), tetD: (B,F), tetQ: (C,G), tetW: (D,E). Quantification by qRT-PCR was performed in triplicate. Error bars indicate standard deviations. * indicates statistically significant differences from control (p < 0.05).

Figure 6

Comparison of acetate (A), propionate (B), butyrate (C), and isobutyrate (D) in controls (no treatment) and tetracycline-treated samples after 7 days. * indicates statistically significant differences from control (p < 0.05).

Figure 7

Comparison of percentage of tetracycline resistance (MIC > 16 µg/mL) of family Enterobacteriaceae (A) and genus Escherichia (B) in controls (no treatment) and tetracycline-treated samples after 7 days. * indicates statistically significant differences from control (p < 0.05).