Metabolomics Analysis Identifies Intestinal Microbiota-Derived Biomarkers of Colonization Resistance in Clindamycin-Treated Mice

Abstract

BackgroundThe intestinal microbiota protect the host against enteric pathogens through a defense mechanism termed colonization resistance. Antibiotics excreted into the intestinal tract may disrupt colonization resistance and alter normal metabolic functions of the microbiota. We used a mouse model to test the hypothesis that alterations in levels of bacterial metabolites in fecal specimens could provide useful biomarkers indicating disrupted or intact colonization resistance after antibiotic treatment.MethodsTo assess in vivo colonization resistance, mice were challenged with oral vancomycin-resistant Enterococcus or Clostridium difficile spores at varying time points after treatment with the lincosamide antibiotic clindamycin. For concurrent groups of antibiotic-treated mice, stool samples were analyzed using quantitative real-time polymerase chain reaction to assess changes in the microbiota and using non-targeted metabolic profiling. To assess whether the findings were applicable to another antibiotic class that suppresses intestinal anaerobes, similar experiments were conducted with piperacillin/tazobactam.ResultsColonization resistance began to recover within 5 days and was intact by 12 days after clindamycin treatment, coinciding with the recovery bacteria from the families Lachnospiraceae and Ruminococcaceae, both part of the phylum Firmicutes. Clindamycin treatment caused marked changes in metabolites present in fecal specimens. Of 484 compounds analyzed, 146 (30%) exhibited a significant increase or decrease in concentration during clindamycin treatment followed by recovery to baseline that coincided with restoration of in vivo colonization resistance. Identified as potential biomarkers of colonization resistance, these compounds included intermediates in carbohydrate or protein metabolism that increased (pentitols, gamma-glutamyl amino acids and inositol metabolites) or decreased (pentoses, dipeptides) with clindamycin treatment. Piperacillin/tazobactam treatment caused similar alterations in the intestinal microbiota and fecal metabolites.ConclusionsRecovery of colonization resistance after antibiotic treatment coincided with restoration of several fecal bacterial metabolites. These metabolites could provide useful biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.

Targeted Metabolomics Analysis Identifies Intestinal Microbiota-Derived Urinary Biomarkers of Colonization Resistance in Antibiotic-Treated Mice

“…Dietary tryptophan is metabolized to indole in the colon by tryptophanase produced by enteric bacteria, with subsequent conversion by other bacterial enzymes to indole-3-propionic acid (4). Our finding of increased N-acetyltryptophan and decreased indole-3-propionic acid concentrations in urine of piperacillin-tazobactam-treated mice is consistent with previously reported data for stool of antibiotic-treated mice and for serum of germfree mice (4,9,15). Finally, dietary lignans are plant-derived polyphenols that are metabolized by anaerobic intestinal bacteria to produce the enterolignans enterodiol and enterolactone (18)(19).…”

“…Our findings are consistent with data from previous studies in demonstrating that piperacillin-tazobactam treatment causes a disruption of colonization resistance, whereas aztreonam, an agent lacking activity against anaerobes, does not (7)(8)(9). The rapid restoration of colonization resistance against C. difficile by 6 to 11 days after treatment highlights the resilience of the microbiota.…”

“…The biomarkers identified are biologically plausible, and our findings for these compounds are consistent with data from previous studies with germfree mice and antibiotic-treated mice or humans (4,9,(15)(16)(17)(18)(19)(20). Cinnamoylglycine is a glycine conjugate of cinnamic acid that was previously shown to be abundant in the serum of conventional mice but present in minimal concentrations in the serum of germfree mice (4).…”

“…Antibiotics that are excreted into the intestinal tract may suppress the microbiota and disrupt bacterial metabolic functions and colonization resistance (5-9). We demonstrated previously that clindamycin or piperacillintazobactam treatment of mice resulted in the disruption of colonization resistance and marked changes in bacterial metabolites present in fecal specimens based on nontargeted metabolic profiling (9). Of 484 compounds analyzed, 146 (30%) exhibited a significant increase or decrease in concentrations during antibiotic treatment, followed by recovery to baseline that coincided with the restoration of in vivo colonization resistance.…”

Gleaning Insights from Fecal Microbiota Transplantation and Probiotic Studies for the Rational Design of Combination Microbial Therapies

“…Intestinal extracts from antibiotic-treated mice contain higher levels of primary bile salts than do extracts from untreated mice (121,133,134). Spores incubated with intestinal extracts from antibiotic-treated mice also germinate better than do spores incubated with untreated mouse extracts (133).…”