Perturbation and resilience of the gut microbiome up to 3 months after β-lactams exposure in healthy volunteers suggest an important role of microbial β-lactamases

Background Antibiotics notoriously perturb the gut microbiota. We treated healthy volunteers either with cefotaxime or ceftriaxone for 3 days, and collected in each subject 12 faecal samples up to day 90. Using untargeted and targeted phenotypic and genotypic approaches, we studied the changes in the bacterial, phage and fungal components of the microbiota as well as the metabolome and the β-lactamase activity of the stools. This allowed assessing their degrees of perturbation and resilience. Results While only two subjects had detectable concentrations of antibiotics in their faeces, suggesting important antibiotic degradation in the gut, the intravenous treatment perturbed very significantly the bacterial and phage microbiota, as well as the composition of the metabolome. In contrast, treatment impact was relatively low on the fungal microbiota. At the end of the surveillance period, we found evidence of resilience across the gut system since most components returned to a state like the initial one, even if the structure of the bacterial microbiota changed and the dynamics of the different components over time were rarely correlated. The observed richness of the antibiotic resistance genes repertoire was significantly reduced up to day 30, while a significant increase in the relative abundance of β-lactamase encoding genes was observed up to day 10, consistent with a concomitant increase in the β-lactamase activity of the microbiota. The level of β-lactamase activity at baseline was positively associated with the resilience of the metabolome content of the stools. Conclusions In healthy adults, antibiotics perturb many components of the microbiota, which return close to the baseline state within 30 days. These data suggest an important role of endogenous β-lactamase-producing anaerobes in protecting the functions of the microbiota by de-activating the antibiotics reaching the colon. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-023-01746-0.


Supplementary Table
. Effect of the antibiotics on each of the studied components, according to the treatment group in the 22 healthy volunteers included in the CEREMI trial.
For 'low dimensionality' systems, the log10 fold changes from baseline were used (except for variables relative to system's richness which were not transformed), whereas for 'high-dimensionality' systems we used the normalized distance from baseline.Baseline data were obtained from the day -1 sample.AUC D0D10 (respectively AUC D0D30) were normalized by the actual time observed between baseline and day 10 (respectively day 30).Data are presented as median (min; max).P-values refer to the result of non-parametric Wilcoxon test.Q-value corresponds to the test after Benjamini & Hochberg's correction.P-values and Q-values in bold represent values below 0.05.For 'low dimensionality' systems, the log10 fold changes from baseline were used (except for variables relative to system's richness which were not transformed), whereas for 'high-dimensionality' systems we used the normalized distance from baseline.Baseline data were obtained from the day -1 sample.AUC D0D10 (respectively AUCD0D30) were normalized by the actual time observed between baseline and day 10 (respectively day 30).Data are presented as median (min; max 0.0 0.9 0.0 0.1 0.9 0.9 0.6 0.8 0.8 0.9 0.8 0.7 0.9 0.9 0.0 0.9 0.8 0.5 0.8 1.0 0.9 0.7 Bile acids transformation capacity (log10) 0.9 0.8 0.6 0.8 0.6 0.8 0.9 0.8 0.8 0.9 0.6 0.8 1.0 0.7 0.3 0.9 1.0 0.9 1.0 1.0 1.0 0.9

Supplementary Table S3. Spearman's correlations of the maximal perturbations of studied gut microbiota and stool components in the 22 healthy volunteers included in the CEREMI trial.
). P-values refer to the result of non-parametric Wilcoxon test.Q-value corresponds to the test after Benjamini & Hochberg's correction.P-values and Q-values in bold represent values below 0.05.

Table S4 . Spearman's correlations of the maximal resiliences of studied gut microbiota and stool components in the 22 healthy volunteers included in the CEREMI trial.
P-values corresponds to the uncorrected test of the Spearman's correlation coefficient to 0. Q-value corresponds to the test of the Spearman's correlation coefficient to 0 after Benjamini & Hochberg's correction.P-values in bold represent values below 0.05.Conc., concentration; Cholesterol conv.rate, Cholesterol conversion rate; BA transf.Capacity, Bile acids transformation capacity.

Table S5 . Spearman's correlations between the baseline characteristics and the maximal perturbations of the studied gut microbiota and gut components in the 22 healthy volunteers included in the CEREMI trial.
P-values corresponds to the uncorrected test of the Spearman's correlation coefficient to 0. Q-value corresponds to the test of the Spearman's correlation coefficient to 0 after Benjamini & Hochberg's correction.P-values in bold represent values below 0.05.Conc., concentration; Cholesterol conv.rate, Cholesterol conversion rate; BA transf.Capacity, Bile acids transformation capacity.

Table S6 . Spearman's correlations between the baseline characteristics and the maximal resiliences of the studied gut microbiota and gut components in the 22 healthy volunteers included in the CEREMI trial.
P-values corresponds to the uncorrected test of the Spearman's correlation coefficient to 0. Q-value corresponds to the test of the Spearman's correlation coefficient to 0 after Benjamini & Hochberg's correction.P-values in bold represent values below 0.05.Conc., concentration; Cholesterol conv.rate, Cholesterol conversion rate; BA transf.Capacity, Bile acids transformation capacity.Humières, C. et al.Perturbation and resilience of the gut microbiome up to three months after β-lactams exposure in healthy volunteers suggest an important role of microbial β-lactamases

Table S7 . Baseline characteristics, maximal perturbations and maximal resiliences of the studied gut microbiota and gut components in the 22 healthy volunteers included in the CEREMI trial
Variables are colored according to their level relative to the median value, colored in white.For the baseline state of the microbiota, the green color is indicative of the high richness of the microbiota, while red indicates poor richness.For systems' perturbation and resilience, green color indicates low perturbation and high resilience, and red color indicates high perturbation and low resilience.Subjects 3 and 16 had detectable levels of antibiotics in faeces; together with subjects 22 and 28 they had no β-lactamase activity at baseline.Conc., concentration ; Cholesterol conv.rate, Cholesterol conversion rate ; BA transf.Capacity, Bile acids transformation capacity.