Absence of gut microbiota restoration following meropenem treatment in a systemic infection model

Abstract

Antibiotics have been shown to have unintended effects on the host by disrupting the beneficial microbiota. In this study, we explored and longitudinally monitored microbiota during and after antibiotic treatment for infection. We induced a systemic Escherichia coli infection in 45 BALB/c mice, with 25 as uninfected controls. The disease was treated with meropenem. The animals were evaluated longitudinally three times, including 3 hours, five days of treatment, and 30 days post-treatment. Intestinal, lung, blood, and peritoneal lavage samples were collected for analysis. Our findings show that meropenem effectively treated the lethal systemic infection caused by E. coli. Hematological parameters, crypt depth, and goblet cell numbers remained unchanged for 30 days post-antibiotic treatment. However, antibiotic treatment impaired the recovery of the gut microbiota, even 30 days post-treatment. This long-term meropenem impact on microbiota was observed in both infected and non-infected (saline-treated) groups. Alpha and beta diversity analyses did not indicate a microbiota recovery. Notably, Lactobacillus and Bacteroides decreased, while Clostridioides, Enterococcus, and Escherichia-Shigella increased. This study provides novel evidence that, even 30 days after meropenem treatment in a simulated E. coli infection, the intestinal microbiota does not recover, highlighting prolonged antibiotic-induced dysbiosis.

Fig. 1. Experimental design.

A An experimental approach was used to assess the impact of the antibiotic meropenem on animals infected with E. coli. B Survival analysis of animals infected with MDR E. coli, observed over a treatment period of 120 h (5 d) with meropenem and a subsequent follow-up of 720 h (30 d). C Evaluation of weight changes during the treatment period with three different doses of meropenem, extending up to 30 d post-treatment. ***p < 0.001 denotes statistical significance compared to the saline group. D Quantifying bacterial recovery from peritoneal lavage, lungs, and blood at 3 h post-infection, following 5 d treatment, and 30 d post-treatment with meropenem. The results are expressed as Log CFU mL^-1 in peritoneal lavage and blood and as Log CFU g^-1 in lung tissue. ****p < 0.00001 indicates statistical significance compared to the saline or saline + MER groups at the same time points, and ###p < 0.0001 indicates significance compared to the infected + MER group at the same time points. MER refers to meropenem.

Fig. 2. Blood and intestinal analysis.

A Principal Component Analysis (PCA) was conducted to examine the structure and variation within the dataset. The plot’s axes represent the principal components of the PCA, while the colors denote different groups within the data. The dispersion of points on the plot reveals distinct patterns, trends, or clusters. The group acronyms are defined as follows: S.T0, Saline in the 3 h period post-infection; I.T0 denotes Infected within 3 h of infection; SS.T1, Saline-Saline 5 d post-infection; SM.T1, Saline-Meropenem 5 d post-infection; IM.T1, Infection-Meropenem 5 d post-infection; IS.T1, Infection-Saline 5 d post-infection; SS.T2, Saline-Saline 30 d post-infection; SM.T2, Saline-Meropenem 30 d post-infection; IM.T2, Infection-Meropenem 30 d post-infection. B For intestinal analyses, the T-test was employed to examine the differences between the two groups in the 3 h period. One-way ANOVA was utilized to analyze the other periods separately, with Bonferroni post-hoc corrections applied. C Histological images of goblet cells and crypts in the small intestine. The black arrows indicate the goblet cells that were measured.

Fig. 3. Alpha and beta-diversity analyses upon meropenem treatment.

A Alpha-diversity measure using Simpson and Shannon at ASV level across all the samples. The samples are represented on the X-axis and their estimated diversity on the Y-axis. Each sample is colored based on treatment class. B Beta-diversity analysis of all groups using Principal Coordinate Analysis (PCoA) with Bray-Curtis distance and phylogenetic-based unweighted UniFrac distances. Beta-diversity analysis of all groups using PCoA with Bray-Curtis distance and weighted UniFrac distances. S.T0 saline in the 3 h period post infection, I.T0 infected within 3 h of infection, SS.T1 saline-saline 5 days post infection, SM.T1 saline-meropenem 5 days post infection, IM.T1 infection-meropenem 5 days post infection, IS.T1 infection-saline 5 days post infection, SS.T2 saline-saline 30 days post infection, SM.T2 saline-meropenem 30 days post infection, IM.T2 infection-meropenem 30 days post infection.

Fig. 4. Alterations in genera during the critical antibiotic period and longitudinally.

Certain genera exhibited changes between groups within the same period (A), while other genera demonstrated changes within the same group throughout different periods (B). All results presented in this figure indicated a difference of p ≤ 0.01, as summarized in S2 Table.

Fig. 5. Gut microbiota recovery following antibiotic treatment.

A comparison was made between the Infection and Antibiotic group and the total control group (Saline-Saline). Eighteen different genera (A and B) exhibited changes following infection and antibiotic use. All results presented in this figure indicated a difference of p ≤ 0.08, as summarized in S2 Table.

Fig. 6. Gut microbiota recovery in other comparisons following antibiotic use.

A A comparison was made between antibiotic use with and without infection (Infection + Meropenem vs. Saline + Meropenem). B The effect of the antibiotic alone was also analyzed by comparing the Saline + Meropenem group with the total control group (Saline-Saline). All results presented in this figure indicated a difference of p ≤ 0.01, as summarized in S3 Table.