Determination of the antimicrobial activity of D-limonene on eschericia coli by metabolomics approach

In this thesis study, the antimicrobial effects of D-limonene, a natural monoterpene, on Escherichia coli (E. coli) were investigated using an untargeted Gas Chromatography-Mass Spectrometry (GC-MS)-based metabolomics analysis method, and the potential effects of this active compound on microorganisms were evaluated. In the studies, the minimum inhibitory concentration (MIC) value of D-limonene was determined using a well diffusion test, and then it was applied to E. coli at a subinhibitory concentration of 4000 µL/L and incubated for 6, 12, and 24 hours. As part of the metabolomic analyses, metabolites were collected from intracellular and extracellular fractions of E. coli, derivatized, and analyzed by GC-MS. The obtained data were evaluated using the MS-DIAL software. Statistical evaluations were performed on the MetaboAnalyst platform, and metabolites showing significant differences were identified. According to the findings; a large number of metabolite signals (features) were obtained in the Kovats (EI) Library comparison of biological samples in MS-DIAL; however, these data were filtered according to quality criteria such as RT similarity, total match score and signal/noise ratio, and only 60 metabolites that were reliably identified were included in the statistical analyses. As a result of D-limonene application, increases were observed in metabolites such as L-(-)-malic acid, uracil, and 3-aminopropionitrile, while decreases were observed in metabolites such as citric acid, butanedioic acid (also known as succinate), cadaverine, and ornithine (p<0.05). In PCA and PLS-DA score analyses conducted to evaluate the overall variation between samples, significant differences were observed between the D-limonene-treated groups and the control groups. In heatmap analyses conducted to evaluate intergroup metabolite changes in more detail, significant differences in metabolite levels were observed, particularly in the extracellular fractions. According to enrichment analysis and pathway analysis results, D-limonene was found to have a significant effect on glycine-serine-threonine metabolism (p<0.001; effect value=0.52), glutathione metabolism (p<0.001; effect value=0.06), alanine, aspartate, and glutamate metabolism (p<0.001; effect value=0.52), and the TCA cycle (p<0.001; effect value=0.18). The study also included comparative analyses with gentamicin antibiotic, used as a positive control group, and it was observed that some of D-limonene's metabolic effects overlap with antibiotic-like mechanisms. These findings indicate that D-limonene does not merely act on the membrane surface but also targets cellular metabolism, thereby inducing systemic metabolic stress. In conclusion, the metabolomics approach supports the notion that D-limonene possesses an antibiotic-like multifaceted mechanism of action and may be considered as a potential alternative antimicrobial agent.