Antibiotic Treatment Induces Long-Lasting Effects on Gut Microbiota and the Enteric Nervous System in Mice

Abstract

The side effects of antibiotic treatment directly correlate with intestinal dysbiosis. However, a balanced gut microbiota supports the integrity of the enteric nervous system (ENS), which controls gastrointestinal neuromuscular functions. In this study, we investigated the long-term effects of antibiotic-induced microbial dysbiosis on the ENS and the impact of the spontaneous re-establishment of the gut microbiota on gastrointestinal functions. C57BL/6J mice were treated daily for two weeks with antibiotics. After 0-6 weeks of antibiotics wash-out, we determined (a) gut microbiota composition, (b) gastrointestinal motility, (c) integrity of the ENS, (d) neurochemical code, and (e) inflammation. Two weeks of antibiotic treatment significantly altered gut microbial composition; the genera Clostridium, Lachnoclostridium, and Akkermansia did not regain their relative abundance following six weeks of antibiotic discontinuation. Mice treated with antibiotics experienced delayed gastrointestinal transit and altered expression of neuronal markers. The anomalies of the ENS persisted for up to 4 weeks after the antibiotic interruption; the expression of neuronal HuC/D, glial-derived neurotrophic factor (Gdnf), and nerve growth factor (Ngf) mRNA transcripts did not recover. In this study, we strengthened the idea that antibiotic-induced gastrointestinal dysmotility directly correlates with gut dysbiosis as well as structural and functional damage to the ENS.

Keywords: antibiotic; enteric nervous system; gastrointestinal motility disorder; gut microbiota; inflammation.

Figure 1

Mice were treated with vehicle or antibiotic mixture (abx) and sacrificed 2 weeks (wks) later; otherwise, mice were treated with abx and sacrificed 2, 4, or 6 weeks (wks) post antibiotic wash-out (w/o). The total bacterial load was estimated from the DNA isolated from the cecal content with qPCR using primers for the V2 and V6 regions of bacterial 16S genes. Dark circle: V2, vehicle; grey circle: V6 vehicle; dark square: V2 abx; grey square: V6 abx; dark up-triangle: V2 2 wks wash-out; grey up-triangle: V6 2 wks wash-out; dark down-triangle: V2 4wks wash-out; grey down-triangle: V6 4 wks wash-out; dark rhombus: V2 6 wks wash-out; grey rhombus: V6 6 wks wash-out (a). DNA was sequenced using Illumina MiSeq chemistry; operational taxonomic unit (OTU) relative abundance is reported. Light grey: vehicle; dark grey: abx treatement; intermediate grey: wash-out. (b). Taxonomical differences among samples were determined using principal coordinate analysis (PCoA) (c). The average relative abundance percentage for the 11 most abundant genera is displayed (d). Data are reported as mean ± SD; number of mice for each experimental group = 10 mice per group; ^a denotes p < 0.02 vs. V2 copy number in vehicle-treated mice; ^b denotes p < 0.02 vs. V6 copy number in vehicle-treated mice; ^c denotes p < 0.05 vs. vehicle-treated mice.

Figure 2

Mice were treated with vehicle or antibiotic mixture (abx) and sacrificed 2 weeks (wks) later; otherwise, mice were treated with abx and sacrificed 2, 4, or 6 weeks (wks) post antibiotic wash-out (w/o). Gastrointestinal dysmotility was determined by administering the animals with non-absorbable FITC-labelled dextran by oral gavage. Animals were sacrificed 60 min later. Gastric emptying was calculated as the percentage of dextran retained in the stomach with respect to the total amount of fluorescence in the gastrointestinal tract. Light grey: vehicle; dark grey: abx; intermediate grey: wash-out. (a). The transit of the fluorescent probe in the ileum is reported as the calculated geometric centre, which is the centre of the distribution of fluorescent dextran in the ileum (b). The number of faecal pellets expelled in 1 h. Circle: vehicle treated mice; square: abx; up-triangle: 2 wks wash-out; down-triangle: 4 wks wash-out; rhombus: 6 wks wash-out. (c). Time (seconds, s) required for the expulsion of a glass bead inserted into the rectum (d). Data are reported as mean ± SD. number of mice for each experimental group = 9–16 mice per group. ^c denotes p < 0.05 vs. vehicle-treated mice.

Figure 3

Mice were treated with vehicle or antibiotic mixture (abx) and sacrificed 2 weeks (wks) later; otherwise, mice were treated with abx and sacrificed 2, 4, or 6 weeks (wks) post antibiotic wash-out (w/o). Immunofluorescence on whole-mount preparations of the distal ileum for βIII-tubulin (neuronal markers) was performed. Representative images of 4 independent experiments; 10 independent fields per animal were examined. Scale bars: 75 µm (a). Western blot analysis of HuC/D and S-100β expression on protein extracts obtained from the LMMP. β-actin was used as a loading control. Representative images of 3 independent experiments are provided (b). Protein signals of HuC/D and S-100β were determined through densitometric analysis of Western blots reported in (b). Data are reported as mean ± SD. number of mice for each experimental group = 3 mice per group. ^c denotes p < 0.05 vs. vehicle-treated mice. Light grey: vehicle; dark grey: abx; intermediate grey: wash-out (c).

Figure 4

Mice were treated with vehicle or antibiotic mixture (abx) and sacrificed 2 weeks (wks) later; otherwise, mice were treated with abx and sacrificed 2, 4, or 6 weeks (wks) post antibiotic wash-out (w/o). Immunofluorescence on whole mount preparations of the distal ileum for substance P was performed. Representative images of 4 independent experiments; n = 3 mice per group; 10 independent fields per animal were examined. Scale bars: 75 µm (a). Western blot analysis of nNOS levels in total protein extracts obtained from the longitudinal muscle myenteric plexus (LMMP). β-actin was used as a loading control. Representative images of 3 independent experiments are shown (b). Protein signals of nNOS were determined through densitometric analysis of Western blotting reported in (b). Data are reported as mean ± SD. number of mice for each experimental group = 3 mice per group. ^c denotes p < 0.05 vs. vehicle-treated mice. Light grey: vehicle; dark grey: abx; intermediate grey: wash-out (c).

Figure 5

Mice were treated with vehicle or antibiotic mixture (abx) and sacrificed 2 weeks (wks) later; otherwise, mice were treated with abx and sacrificed 2, 4, or 6 weeks (wks) post antibiotic wash-out (w/o). The longitudinal muscle myenteric plexus (LMMP) was obtained at each time point, subjected to RNA extraction, and quantitative PCR of the neurotrophic factors: glial-derived neurotrophic factor (Gdnf) (a), nerve growth factor (Ngf) (b), ciliary neurotrophic factor (Cntf) (c), neurotrophin 5 (Ntf5) (d), neurotrophin 3 (Ntf3) (e), and brain-derived neurotrophic factor (Bdnf) (f). Light grey: vehicle; dark grey: abx; intermediate grey: wash-out. Data are reported as mean ± SD of the calculated fold changes over mRNA levels detected in vehicle-treated mice using the ΔC_T method. Number of mice for each experimental group = 6 mice per group. ^c denotes p < 0.05 vs. vehicle-treated mice.

Figure 6

Mice were treated with vehicle or antibiotic mixture (abx) and sacrificed 2 weeks (wks) later; otherwise, mice were treated with abx and sacrificed 2, 4, or 6 weeks (wks) post antibiotic wash-out (w/o). The longitudinal muscle myenteric plexus (LMMP) was obtained at each time point. The pro-inflammatory cytokines tumour necrosis factor (TNF)-α (a), interferon (IFN)-γ (b), and interleukin (IL)-1β (c) were determined using commercially available ELISA kits. Light grey: vehicle; dark grey: abx; intermediate grey: wash-out. Data were normalized by total protein content measured using the bicinchoninic acid method and are reported as mean ± SD. Number of mice for each experimental group = 7 mice per group. ^c denotes p < 0.05 vs. vehicle-treated mice.

Figure 7

Experimental design of the study. Mice were administered a mixture of antibiotics every 12 h for 2 weeks (in green; wks, −2÷ 0). Animals were sacrificed at the end of the antibiotic treatment (abx) or 2, 4, or 6 weeks post antibiotic discontinuation (in orange; wash-out, w/o). Control animals (in blue; vehicle) received the same volume of the vehicle for the same period; since data obtained from the control animals at the different time points are comparable, the results of the controls are organized and reported as a single animal control group. Asterisks indicate the time of collection of the cecal content and the time of the sacrifice. Abbreviations: wks, weeks; abx, antibiotics; w/0, wash-out. * denotes sacrifice of the animals.