Antibiotic administration routes significantly influence the levels of antibiotic resistance in gut microbiota

Abstract

This study examined the impact of oral exposure to antibiotic-resistant bacteria and antibiotic administration methods on antibiotic resistance (AR) gene pools and the profile of resistant bacteria in host gastrointestinal (GI) tracts using C57BL/6J mice with natural gut microbiota. Mice inoculated with a mixture of tet(M)-carrying Enterococcus spp. or blaCMY-2-carrying Escherichia coli were treated with different doses of tetracycline hydrochloride (Tet) or ampicillin sodium (Amp) and delivered via either feed or intravenous (i.v.) injection. Quantitative PCR assessment of mouse fecal samples revealed that (i) AR gene pools were below the detection limit in mice without prior inoculation of AR gene carriers regardless of subsequent exposure to corresponding antibiotics; (ii) oral exposure to high doses of Tet and Amp in mice inoculated with AR gene carriers led to rapid enrichment of corresponding AR gene pools in feces; (iii) significantly less or delayed development of AR in the GI tract of the AR carrier-inoculated mice was observed when the same doses of antibiotics were administered via i.v. injection rather than oral administration; and (iv) antibiotic dosage, and maybe the excretion route, affected AR in the GI tract. The shift of dominant AR bacterial populations in the gut microbiota was consistent with the dynamics of AR gene pools. The emergence of endogenous resistant bacteria in the gut microbiota corresponding to drug exposure was also observed. Together, these data suggest that oral administration of antibiotics has a prominent effect on AR amplification and development in gut microbiota, which may be minimized by alternative drug administration approaches, as illustrated by i.v. injection in this study and proper drug selection.

Fig 1

Real-time PCR quantification of AR gene pools in mice upon corresponding antibiotic exposure. The detection limit of AR gene pools in this study is 10⁵ copies/g. If data were below the detection limit, they are presented as being at the detection limit. (A and C) tet(M) gene pool with Tet treatment at 50 (A) or 2 (C) mg/kg body weight/day. (E) bla_CMY-2 gene pool with Amp treatment at 30 mg/kg body weight/day. (B, D, and F) 16S rRNA gene pool with Tet at 50 (B) or 2 (D) mg/kg body weight/day or Amp at 30 (F) mg/kg body weight/day. Data presented include ART bacteria-inoculated experimental groups with corresponding oral Tet (Tet50-PO, Tet2-PO), Amp (Amp30-PO), i.v. Tet (Tet50-IV, Tet2-IV), i.v. Amp (Amp30-IV); ART bacteria-inoculated control groups with saline instead of antibiotic treatment (saline-POt50, saline-POt2, saline-POa30, and saline-IVa30); control groups without bacterial inoculation but treated with antibiotics (NI-Tet50-PO, NI-Tet2-PO, NI-Amp30-PO, NI-Amp30-IV). Data are not shown for control groups saline-IVt50, NI-Tet50-IV, saline-IVt2, and NI-Tet2-IV, which are similar to those for saline-POt50, NI-Tet50-PO, saline-POt2, and NI-Tet2-PO groups, respectively. The error bars represent standard deviations of the data from animal subjects used in the study. The vertical dashed lines indicate the last day of antibiotic administration.

Fig 2

Denaturing gradient gel electrophoresis analysis of predominant 16S rRNA genes in total fecal DNA extracts from inoculated mice administered with antibiotics. Mouse subjects were exposed to 50 mg/kg body weight/day Tet via the oral (A) and i.v. (B) route or to 30 mg/kg body weight/day Amp via the oral (C) and i.v. (D) route. In panels A and B, fecal DNA of a mouse subject before inoculation with the strain cocktail (lane 2); after inoculation but before Tet administration (lane 3); 1, 2, 3, 4, and 5 days with Tet exposure (lanes 4 to 8); and 8, 10, and 14 days (Tet was lifted; lanes 9 to 11) were used as the template to amplify the 16S rRNA gene V3 region. Marker (lanes 1 and 14) and inoculated strain clusters (lanes 12 and 13) were included. In panels C and D, fecal DNA from a mouse subject before cocktail strain inoculation (lane 2); after inoculation but before Amp administration (lane 3); 1, 2, 3, 4, and 5 days with Amp administration (lanes 4 to 8); and 7, 11, and 15 days (Amp was lifted; lanes 9 to 11) were used to amplify the 16S rRNA gene V3 region. Lane 1, DNA ladder; lane 12, inoculated strain cluster. Residential strain symbols: α, β, and γ, uncultured bacteria; δ, Escherichia coli; ε, Lactobacillus species; ζ, Enterococcus species. Inoculated strains: a, strain cluster 3; b, strain cluster 2; c, strain cluster 1; d, strain cluster 4; e, strain cluster 5.