identify plasma metabolites related to microbiome changes due to antibiotic treatment, broad-spectrum antibiotics belonging to the class of aminoglycosides (neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were applied. These were administered orally for 28 days to male rats, and blood was sampled for metabolic profiling after 7, 14 and 28 days. Whereas aminoglycosides are poorly bioavailable after oral administration, the other two classes can be absorbed from the gut [9–13]. For each class of antibiotics specific plasma metabolome patterns could be established in the MetaMap®Tox database and first key metabolites (hippuric acid, indole-3-acetic acid, indole-3-propionic acid, 3-indoxylsulfate and glycerol) could be identified, indicating a change of the gut microbiome. However, for the tetracycline and fluoroquinolone antibiotics, a possible influence of systemic toxicity had to be taken into account when evaluating their effects on the plasma metabolome due to their bioavailability.
With this study design it could be shown that gut microbial changes lead to changes in the plasma metabolome, therefore further experimental studies were set up to elucidate this interaction. Consequently, three antibiotics (vancomycin, streptomycin and roxithromycin) from different classes were administered orally to rats with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum) analyzing the same set of metabolites as before in plasma (Chapter 3). After antibiotic treatment, metabolite changes observed at different dose levels and in both sexes were evaluated. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not in the different gut tissues. The most relevant changes in metabolite values were comparable in feces and cecum content and also among sexes. The metabolite profile showed compound specific effects on the microbiome, in line with the
activity spectra of the antibiotics tested. Vancomycin showed the largest effects in the feces and cecum metabolome, followed by roxithromycin and then by streptomycin for which changes were modest. For all antibiotics the largest changes were observed for the classes of lipids, bile acids, amino acids and amino acid related metabolites. In general, it has to be noted that analysis was targeted. An untargeted analysis or a broader range of metabolites, e.g. including the analysis of short chain fatty acids (SCFAs), may have revealed significant differences between matrices and possibly even sexes.
With our targeted analysis, we identified feces as the best matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Thus, it offers the advantage of being a non-invasive sampling method, also enabling a longitudinal study design. However, this matrix only partially reflects the metabolite profile of the gut content, since the upper part, i.e. the small intestine, shows distinct differences in the composition of the microbial composition [14] which may considerably differ regarding the metabolite profile. For our metabolome studies, it requires overnight-fasted animals for the blood sampling and then the animals do not have content in the small intestine, so this matrix was not included in the studies.
In Chapter 4, we applied lincomycin and clindamycin, another class of antibiotics known to show no or low systemic toxicity after oral administration, and modulated microbial communities of Wistar rats to gain a comprehensive understanding of the implications of microbiome alterations. A metabolomics approach and taxonomic profiling were applied to characterize the effects of these antibiotics on the functionality and composition of the microbiome and to identify microbiome-related metabolites. After treatment, the diversity of the microbial community was drastically reduced. Whereas other phyla disappeared, the abundance of Firmicutes and Proteobacteria was highly increased. Again, most changes in plasma and feces metabolites were observed for metabolites belonging to the class of complex lipids, fatty acids and related metabolites as well as amino acids and related compounds. The three analyzed primary bile acids (taurocholic acid, glycochenodeoxycholic acid and cholic acid) were markedly affected and displayed diverging results. In both plasma and feces taurocholic acid was highly upregulated upon treatment, whereas glycochenodeoxycholic acid was downregulated. Interestingly, cholic acid was upregulated in feces, however, it was downregulated in plasma. Taking into account that quantitatively the most pronounced changes were related to bile acids, it can be stated that the gut microbiome plays a very important role in the composition of the bile acid pool because secondary bile acids are exclusively formed by bacterial enzymes. Since a change of the gut microbiota can impact the bile acid pool, we used six antibiotics from five different classes (lincosamides, glycopeptides, macrolides, fluoroquinolones, aminoglycosides) and modulated microbial communities of Wistar rats to elucidate changes in the bile acid metabolism related to gut microbial changes (Chapter 5). The results showed that changes in the gut microbial community affected the bile acid pool in plasma and feces of the host, and that bile acid profiling can be indicative for an alteration of the gut microbiome. After treatment, significant changes of primary and secondary bile acids in
both matrices of treated animals could be observed. For cholic acid and taurocholic acid, as discussed in Chapter 4, there was an increase of taurine-conjugated primary bile acids in both plasma and feces. Contrary, cholic acid and most of the analyzed secondary bile acids were found to be significantly downregulated in plasma whereas cholic acid accumulated in the feces. Although different classes of antibiotics with different activity spectra against gut microbes were applied, the overall effect on the bile acid pool tended to be similar in both matrices. However, streptomycin showed less significant changes, probably because it is not active against obligate anaerobes. Since the bile acid-liver-gut microbiota axis plays an important role in the host’s health, e.g. via activation or inactivation of nuclear receptors in the liver or intestine, an alteration of the bile acid pool might have implications for toxicological evaluations regarding the gut-liver axis, the immune system and other body functions known to be influenced by the gut microbiota and needs to be further investigated.