IADR Abstract Archives
Targeted metabolomics to identify biofilm pathways of Enterococcus faecalis isolates
Objectives: Enterococcus faecalis biofilm formation is associated with endodontic treatment failure. Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide, nitrogen compound, and cellular amino acid processes were associated with differences in biofilm formation. We aimed to (i) further characterize E. faecalis biofilm formation pathways by semi-targeted and targeted nitrogen panel analysis of the strong (Ef 63) and weak (Ef 64) biofilm formers and (ii) validate the identified metabolic pathways using targeted pathway inhibitors.
Methods: Semi-targeted analysis of Ef 63, Ef 64 and control strain Ef 29212 was performed by selecting metabolites that were both part of the previously identified pathways and of a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p<0.05) were selected with fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis (PEA). The identified pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted pathway inhibitors.
Results: Metabolomics analysis showed upregulation of N-acetylglutamate, N-acetylaspartate, malonic semi-aldehyde, citrulline, and 3-phenyllactic acid and downregulation of choline, adenosine, glutamine, and octylamine in Ef 63 vs 64. PEA identified differential regulation of pyruvaldehyde degradation, methionine metabolism, glutathione metabolism, aspartate metabolism, and pyruvate metabolism. MBIC and CFU assays using pathway inhibitors, polydatin (glutathione metabolism), nitazoxanide (pyruvate metabolism), glutathione (pyruvaldehyde degradation), benzothiadiazole (methionine metabolism), and O-carboxymethyl hydroxylamine (aspartate metabolism) showed inhibitory effects of glutathione and O-carboxymethyl hydroxylamine against E. faecalis biofilm formation.
Conclusions: The study demonstrated the importance of pyruvaldehyde degradation and aspartate metabolism in E. faecalis biofilm formation. Targeted therapeutics development can thereby reduce the economic and healthcare burden of E. faecalis infections.
Methods: Semi-targeted analysis of Ef 63, Ef 64 and control strain Ef 29212 was performed by selecting metabolites that were both part of the previously identified pathways and of a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p<0.05) were selected with fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis (PEA). The identified pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted pathway inhibitors.
Results: Metabolomics analysis showed upregulation of N-acetylglutamate, N-acetylaspartate, malonic semi-aldehyde, citrulline, and 3-phenyllactic acid and downregulation of choline, adenosine, glutamine, and octylamine in Ef 63 vs 64. PEA identified differential regulation of pyruvaldehyde degradation, methionine metabolism, glutathione metabolism, aspartate metabolism, and pyruvate metabolism. MBIC and CFU assays using pathway inhibitors, polydatin (glutathione metabolism), nitazoxanide (pyruvate metabolism), glutathione (pyruvaldehyde degradation), benzothiadiazole (methionine metabolism), and O-carboxymethyl hydroxylamine (aspartate metabolism) showed inhibitory effects of glutathione and O-carboxymethyl hydroxylamine against E. faecalis biofilm formation.
Conclusions: The study demonstrated the importance of pyruvaldehyde degradation and aspartate metabolism in E. faecalis biofilm formation. Targeted therapeutics development can thereby reduce the economic and healthcare burden of E. faecalis infections.