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AIM: (i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. METHODOLOGY: 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 and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. RESULTS: Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. CONCLUSIONS: The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.
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Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography-mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup. Key features Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run. Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model. Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.