Antibiotics in early life alter the murine colonic microbiome and adiposity

Abstract

Antibiotics administered in low doses have been widely used as growth promoters in the agricultural industry since the 1950s, yet the mechanisms for this effect are unclear. Because antimicrobial agents of different classes and varying activity are effective across several vertebrate species, we proposed that such subtherapeutic administration alters the population structure of the gut microbiome as well as its metabolic capabilities. We generated a model of adiposity by giving subtherapeutic antibiotic therapy to young mice and evaluated changes in the composition and capabilities of the gut microbiome. Administration of subtherapeutic antibiotic therapy increased adiposity in young mice and increased hormone levels related to metabolism. We observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol. In this model, we demonstrate the alteration of early-life murine metabolic homeostasis through antibiotic manipulation.

Figure 1. Weight and body composition of control and STAT mice

a, Weight gain did not differ between control and STAT mice (n = 10 mice per group). b, Representative DEXA show per cent body fat in control (22.9%; top) and STAT (32.0%; bottom) mice. c, Total fat mass was significantly increased (P < 0.05) in all STAT groups compared to controls. d, Per cent body fat was significantly increased in all STAT groups (all P < 0.05) except vancomycin. e, Lean mass was lower in STAT mice, but not significantly different from controls. Data are presented as mean ± s.e.m. For all figures: all, all antibiotics; C, controls; Ct, chlortetracycline; P, penicillin; P+V, penicillin plus vancomycin; V, vancomycin.

Figure 2. Bone development and serum GIP measurements

a, After 3 weeks of STAT, bone mineral density was significantly increased in each group (n = 10 mice per group) compared to controls (P < 0.05) but did not persist at 6 weeks. b, Serum GIP levels measured at death were significantly increased in the vancomycin, penicillin plus vancomycin, and chlortetracycline groups and in the aggregate antibiotic group compared to controls (P < 0.05). Data are presented as mean ± s.e.m. Box plots show median ± interquartile range (IQR) and 95% ranges (whiskers).

Figure 3. Changes in the faecal gut microbiome after 50 days of STAT

a, There were no significant differences in microbial census between the STAT and control groups (n = 10 mice per group) evaluated by qPCR with universal primers for 16S rRNA and internal transcribed spacer (ITS). b, By 454-pyrosequencing, Firmicutes were shown to be increased in the STAT mice at multiple taxonomic levels. (Controls n = 10, penicillin n = 9, vancomycin n = 10, penicillin plus vancomycin n = 8, chlortetracycline n = 10; *P < 0.05.) Data are presented as mean ± s.e.m. c, Heat map of specimens showing relative abundance of bacteria present at >1% at the family taxonomic level. Hierarchical clustering based on Euclidean distance identified nonrandom branch distributions of control and STAT mice (P < 0.05). Lachno., Lachnospiraceae; Porphyr., Porphyromonadaceae.

Figure 4. Caecal SCFA production after STAT exposure

a, Quantitative PCR was performed for butyryl CoA transferase (BCoAT) and formyltetrahydrofolate synthetase (FTHFS) at experiment weeks 3 and 6 on STAT and control groups (n = 10 mice per group). At 3 weeks, BCoAT was diminished in two STAT groups and the aggregate group, a difference that persisted only in the chlortetracycline group. FTHFS copies do not show a consistent pattern. *P < 0.05, **P < 0.01, ***P < 0.001 comparing STAT to controls; hash symbol indicates significant difference between 3 and 6 weeks. b, SCFA concentration analysed by gas chromatography (GC) shows increases in SCFAs in each of the STAT groups compared to controls. c, The ratio of butyrate relative to acetate is significantly higher in the STAT mice than controls. Data are presented as mean ± s.e.m.

Figure 5. Differentially regulated genes related to hepatic lipogenesis, identified through microarray and quantitative PCR analyses

a, Microarray analysis of liver specimens surveyed for differences in >45,000 genes; 397 genes were significantly up- or downregulated. Heat maps generated by gene set enrichment analysis (GSEA) identify differences between the STAT and control mice (n = 6 mice per group), including pathways related to fatty acid metabolism and lipid metabolic processes. b, Mapping of metabolic genes detected by microarray onto specific pathways, including those related to lipogenesis and triglyceride synthesis, show consistent changes with STAT. Data are presented as mean ± s.e.m. White bars, controls; black bars, STAT; *P < 0.05, **P < 0.01. TG, triglycerides; VLDL, very low density lipoproteins.