Gut Microbiota as Well as Metabolomes of Wistar Rats Recover within Two Weeks after Doripenem Antibiotic Treatment

Abstract

An understanding of the changes in gut microbiome composition and its associated metabolic functions is important to assess the potential implications thereof on host health. Thus, to elucidate the connection between the gut microbiome and the fecal and plasma metabolomes, two poorly bioavailable carbapenem antibiotics (doripenem and meropenem), were administered in a 28-day oral study to male and female Wistar rats. Additionally, the recovery of the gut microbiome and metabolomes in doripenem-exposed rats were studied one and two weeks after antibiotic treatment (i.e., doripenem-recovery groups). The 16S bacterial community analysis revealed an altered microbial population in all antibiotic treatments and a recovery of bacterial diversity in the doripenem-recovery groups. A similar pattern was observed in the fecal metabolomes of treated animals. In the recovery group, particularly after one week, an over-compensation was observed in fecal metabolites, as they were significantly changed in the opposite direction compared to previously changed metabolites upon 28 days of antibiotic exposure. Key plasma metabolites known to be diagnostic of antibiotic-induced microbial shifts, including indole derivatives, hippuric acid, and bile acids were also affected by the two carbapenems. Moreover, a unique increase in the levels of indole-3-acetic acid in plasma following meropenem treatment was observed. As was observed for the fecal metabolome, an overcompensation of plasma metabolites was observed in the recovery group. The data from this study provides insights into the connectivity of the microbiome and fecal and plasma metabolomes and demonstrates restoration post-antibiotic treatment not only for the microbiome but also for the metabolomes. The importance of overcompensation reactions for health needs further studies.

Keywords: carbapenems; gut microbiome; gut microbiota and metabolome recovery; metabolomics; repeated dose oral study.

Figure 1

Schematic representation of the study design for plasma and fecal metabolome analyses and the corresponding gut (fecal) 16S bacterial profiling in a 28-day oral gavage study using meropenem- and doripenem- (administered at high and low doses) treated male and female young adult Wistar rats. Note: For doripenem, an additional one- and two-week recovery period, as indicated by the orange dashed boxes, was included to analyze the reversibility. The top row indicates sampling timeline.

Figure 2

Line plots representing food consumption (in grams) and body weight (in grams), in male (A,C) and female (B,D) animals, respectively, compared to the controls and different antibiotics treatment groups on days 0, 6, 13, and 27 and additionally on days 35 and 42/43 for the doripenem-recovery groups. The end of meropenem treatment and the start of doripenem-recovery groups have been highlighted with black arrows. Changes in body weight and food consumption were compared between control and treated groups and showed no significant differences (p-value < 0.05).

Figure 3

Shannon true diversity analysis of the antibiotic treatments, meropenem, doripenem, and controls for both male and female Wistar rats. The top row shows data for females (f) and the bottom row shows data for males (m). Doripenem treatment time points (days 1, 7, and 28) and recovery time points (days 35 and 42/43) are indicated with black and blue lines, respectively, on the x-axis. Whiskers denote standard deviations, and solid lines within the boxes indicate the group medians. The dots falling outside the boxes demonstrate the most extreme values.

Figure 4

Principal Coordinate Analysis (PCoA) of bacterial families from controls, meropenem, and doripenem antibiotic-treated rats at different time points. Rank-based clustering with Bray-Curtis distance matrix of samples from (A) males and (B) females are presented.

Figure 5

Stacked bar diagram showing relative abundances of the detected bacterial families in the different experimental groups. Inter-individual variability in the composition can be observed. Dose groups and sexes showed significant differences specifically in samples from days 1, 7, and 28 relative to the controls. Antibiotic-specific effects were observed on the intestinal community composition. For the doripenem group, treatment time points, i.e., days 1, 7, and 28, and recovery time points, i.e., 35 and 42/43, are highlighted with black and blue lines on the x-axis, respectively.

Figure 6

Stacked bar diagram showing relative abundances of bacterial families that were detected in the controls and doripenem-recovery groups. An indication of the recovery of abundant bacterial families one and two weeks post-doripenem treatment was observed and resulted in abundances comparable to the controls.

Figure 7

Differential abundances in dominant bacterial families one and two weeks post-doripenem-treated samples from the low- (LD) and high- (HD) dose groups relative to the controls for males (A) and females (B). Differential abundance analysis was performed using log2FC (log2 fold change) values.

Figure 8

Principal component analysis (PCA) of the fecal metabolome profiles of controls (red), meropenem- (blue), and doripenem- (green) treated rats. PCA for fecal metabolome profiles of (A) female and (B) male Wistar rats on days 7, 14, and 28 of the study and days 35 and 42/43 for the doripenem-recovery group. The data points are sized by the PC3 value. Different shapes of the data points represent respective sampling time points. Dose group-specific effects were marginal, so the groups were not separated based on the dose levels.

Figure 9

Principal component analysis (PCA) of the blood plasma metabolome profiles of controls (red), meropenem- (blue), and doripenem- (green) treated rats. PCA for plasma metabolome profiles of (A) female and (B) male Wistar rats on days 7, 14, and 28 of the study and on day 42/43 for the doripenem-recovery group. The data points are sized by the PC3 value. Different shapes of the data points represent respective sampling time points. Dose group-specific effects were marginal, so the groups were not separated based on the dose levels.

Figure 10

Schematic diagrams of the recovery processes of (A) gut or fecal microbiota and (B) plasma and feces metabolomes before and after doripenem antibiotic treatments. Baseline state indicates the original or ‘normal’ state of the gut microbiota or metabolomes prior to antibiotic administration, which becomes altered upon 28 days of antibiotic administration. Further, post-antibiotic treatment, a recovery or overcompensation takes place where the gut bacteria as well as the host metabolomes try to return to the ‘normal’ state.