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Background: Urinary levels of small molecules generated by the gut microbiota (GMB) constitute potential biomarkers for the state of the GMB. Such metabolites include numerous small phenolic molecules linked to anaerobic bacteria, particularly Clostridium species. Due to multiple technical challenges, however, the relationship between these chemicals and the GMB remains poorly characterized. Improved, high-performance liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis can now reliably separate and quantify low levels of multiple small phenolic molecules and their structural isomers. Methods: CF-1 (female mice) were treated over 2 consecutive days with either i) vehicle, ii) one of 2 different antibiotic regimens (clindamycin or piperacillin/tazobactam) known to inhibit intestinal anaerobes and promote colonization by Clostridium difficile and other pathogens or iii) an antibiotic (aztreonam) that suppresses facultative Gram-negative bacteria but not enterococci or anaerobes and does not promote pathogen colonization Urine collected 24 hours after the last treatment was analyzed by LC-MS. Results: We identified over 25 compounds, many of which had not been previously reported in mouse urine. Eleven small phenolic molecules showed significant antibiotic-related changes. Urinary levels of the hydroxyphenylpropionic acids were suppressed by clindamycin and piperacillin/tazobactam treatment, but were elevated in aztreonam-treated mice. In addition, aztreonam treatment was associated with elevated levels of the dihydroxyhydrocinnamic acids. Conclusions: Profiles of differential changes in urinary small phenolic molecules may provide an index of anaerobic bacterial species in the GMB and could prove useful in monitoring susceptibility to overgrowth of pathogens such as C. difficile .

pathogens and immunity

Identification and Quantification by Targeted Metabolomics of Antibiotic-Responsive Urinary Small Phenolic Molecules Derived from the Intestinal Microbiota in Mice